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Processing and presentation of epitopes by
dendritic cells and macrophages in HIV
infection
Jens Dinter, Berlin 2015
Processing and presentation of epitopes by
dendritic cells and macrophages in HIV infection
vorgelegt von Dipl.-Ing.
Jens Dinter geb. in Berlin
von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften - Dr. rer. nat. -
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Professor Dr. Juri Rappsilber Gutachter: Professor Dr. Roland Lauster Gutachter: Professor Dr. Jens Kurreck Gutachterin: Professor Dr. Sylvie Le Gall Tag der wissenschaftlichen Aussprache: 27. März 2015
Berlin 2015
Abstract
Human immunodeficiency virus (HIV)-1 infection causes a chronic infection associated with a
progressive CD4+ T cell depletion and dysregulation of the immune system. HIV-1 infects
several CD4-expressing immune cell subsets, including CD4+ T cells, monocytes, dendritic cells
(DCs) and macrophages (Møs). Recognition and killing of these cell subsets by HIV-specific
CD8+ T cells (CTLs) requires the presentation of viral epitopes by MHC I. Whether all HIV-
infectable cell subsets equally process and present the same HIV-1 epitopes remains open, but
is critical for the identification of protective HIV-specific CTL responses that should recognize
and kill all HIV-infectable cell subsets and should be part of a HIV-1 vaccine.
In this study we focus on the processing and presentation of HIV-1 epitopes by DCs and Møs,
which are important in priming and activation of anti-viral immune responses, but also
targeted by HIV-1. We show that proteolytic activities and expression levels of cytosolic
proteases involved in antigen processing significantly differ between monocyte-derived DCs
and Møs matured with TLR ligands or upon HIV-1 infection. These differences affect the
amount and the kinetics of epitope production and lead to differences in CTL responses, which
may contribute to the unequal capacity of HIV-specific CTLs to control viral load.
HIV-1 can enter DCs and Møs by fusion at the plasma membrane for delivery into the cytosol
or by internalization into endo-lysosomal vesicles. How trafficking of HIV-1 through various cell
compartments affects the degradation patterns of HIV-1 peptides and the recognition of
infected cells by immune cells is not understood. Using DC and Mø cell extracts as a source of
proteases from various cell compartments to degrade long HIV-1 peptides, we identify by mass
spectrometry cell-specific and compartment-specific degradation patterns. We show a
preferential production and a superior intracellular stability of peptides containing
immunodominant epitopes corresponding to stronger CTL responses, which may contribute to
the establishment of immunodominance patterns during HIV-1 infection.
During disease progression constant immune pressure on immunodominant epitopes results in
the appearance of frequently detected HLA-associated escape mutations to avoid CTL
recognition. We show for the first time that processing of escape mutations in cross-
presentation-competent cell compartments reduces intracellular stability and impairs epitope
production, which leads to less epitope available for presentation and thus immune escape.
Together these results demonstrate the impact of protein degradation patterns in subcellular
compartments on epitope production for CTL recognition and their contribution to
immunodominance patterns and immune escape during HIV-1 infection.
Zusammenfassung
Eine Infektion durch das humane Immundefizienz-Virus (HIV)-1 führt zu einer chronischen
Infektion einhergehend mit einer kontinuierlichen Reduktion der CD4+ T-Zellen und einer
Fehlregulation des Immunsystems. HIV-1 infiziert mehrere CD4-exprimierende Immunzellen,
unter anderem CD4+ T-Zellen, Monozyten, Dendritische Zellen und Makrophagen. Die
Erkennung und Lyse von HIV-infizierten Zellen durch CD8+ T-Zellen erfordert die Präsentation
viraler Epitope auf MHC I Molekülen. Es ist bisher nicht geklärt, ob alle HIV-infizierten Zellen
gleichermaßen diese viralen Epitope prozessieren und präsentieren, obwohl dies ein wichtiger
Aspekt in der Identifizierung von protektiven CD8+ T-Zellantworten ist, die alle infizierten
Zellen erkennen und lysieren sollen und deswegen Bestandteil eines HIV-Vaccines sein sollten.
Der Fokus dieser Arbeit liegt in der Prozessierung und Präsentation von HIV-1 Epitopen durch
Dendritische Zellen und Makrophagen. Diese Zellen spielen eine wichtige Rolle in der
Aktivierung der Immunantwort gegen HIV-1, werden allerdings auch durch HIV-1 infiziert. Wir
zeigen, dass wichtige Proteasen, die in der Epitop-Prozessierung involviert sind, ihre
Aktivitäten und Expressionslevel durch TLR-Aktivierung oder HIV-1 Infektion von Dendritischen
Zellen und Makrophagen verändern. Diese Veränderungen führen zu unterschiedlichen
Produktionen von HIV-1 Epitopen und dadurch zu unterschiedlichen CD8+ T-Zellantworten.
Dies könnte dazu führen, dass CD8+ T-Zellen HIV-infizierte Dendritische Zellen und
Makrophagen nicht gleichermaßen erkennen und lysieren können und somit nicht die
Ausbreitung der Infektion verhindern können.
HIV-1 kann durch direkte Fusion mit der Plasmamembran oder durch die Aufnahme in Endo-
und Lysosomen in Dendritische Zellen und Makrophagen gelangen. Wie HIV-1 Peptide in
diesen verschiedenen Zellkompartimenten abgebaut werden, ist bisher nicht bekannt. Wir
zeigen mit Hilfe von massenspektrometrischen Analysen, dass Proteasen aus verschiedenen
Zellkompartimenten HIV-1 Peptide unterschiedlich abbauen, wobei Unterschiede zwischen
Zelltypen und Zellkompartimenten zu erkennen sind. Der Abbau von Peptiden bevorzugt die
Produktion immundominanter Epitope unabhängig von dem Zelltyp oder dem
Zellkompartiment und führt dadurch zu einer erhöhten CD8+ T-Zellantwort spezifisch für diese
Epitope. Dies könnte zu der Etablierung immundominater CD8+ T-Zellantworten während einer
HIV-1 Infektion beitragen.
Die konstante Erkennung immundominanter Epitope durch HIV-spezifische CD8+ T-Zellen, führt
zum Auftreten von Mutationen, die dazu führen, dass CD8+ T-Zellen diese Epitope nicht mehr
erkennen können und diese somit der Immunantwort entkommen. Wir zeigen hier zum ersten
Mal, dass die Prozessierung dieser Mutationen in Endo- und Lysosomen zu einer reduzierten
Epitop-Produktion und Epitop-Stabilität führen. Dies führt wiederum dazu, dass weniger Epitop
zur Präsentation zur Verfügung steht und dies eine CD8+ T-Zellantwort verhindert.
Diese Ergebnisse demonstrieren, dass die Prozessierung von HIV-1 Proteinen in verschiedenen
Zellkompartimenten auf die Etablierung von immundominanten CD8+ T-Zellantworten sowie
auf das Entkommen dieser immundominanten Epitope nach Einbau von Mutationen einen
wichtigen Einfluss hat.
Publications
Dinter J., Duong E., Lai N. Y., Berberich M. J., Kourjian G., Bracho-Sanchez E., Chu D., Su H.,
Zhang S. C., Le Gall S. Variable processing and cross-presentation of HIV by dendritic cells and
macrophages shapes CTL immunodominance and immune escape; PLoS Pathogens 2015 Mar
17; 11(3):e1004725
Dinter J.*, Gourdain P.*, Lai N. Y.*, Duong E., Bracho-Sanchez E., Rucevic M., Liebesny P. H.,
Xu Y., Shimada M., Ghebremichael M., Kavanagh D. G. and Le Gall S. Different antigen-
processing activities in dendritic cells, macrophages, and monocytes lead to uneven
production of HIV epitopes and affect CTL recognition; Journal of Immunology 2014 Nov 1; 193
(9):4322-34
Rucevic M., Boucau J., Dinter J., Kourjian G., Le Gall S. Mechanisms of HIV protein degradation
into epitopes: implications for vaccine design; Viruses 2014 Aug 21; 6(8):3271-92
Mattison C., Dinter J., Berberich M., Chung S.-Y., Reed S., Le Gall S., Grimm C. In-vitro
evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides;
Food Science & Nutrition 2015, in press
Conference contributions
8th International Workshop on Antigen Processing and Presentation in Philadelphia, USA (June
2014) Poster: “Differential cross-presentation of HIV epitopes by dendritic cells and
macrophages identifies variable intracellular degradation rates of HIV peptides”
Ragon Symposium on Humanized Mouse HIV Vaccine Initiatives in Boston, USA (February
2014) Talk: “Processing of peptide antigens by human antigen presenting cells”
13th Annual Meeting of the Federation of Clinical Immunology Societies (FOCIS) in Boston, USA
(June 2013) Talk: “Variable Cross-Presentation of HIV Epitopes by Dendritic Cells and
Macrophages to CD8 T Cells Uncovers Differences in Intracellular Peptide Degradation Rates”
Keystone Symposium: Understanding dendritic cell biology to advance disease therapies in
Keystone, USA (March 2013) Poster: “Variable cross-presentation of HIV epitopes by dendritic
cells and macrophages to CD8 T cells uncovers differences in peptide degradation rates”
AIDS Vaccine 2012 in Boston, USA (September 2012) Poster: “Variable processing and
presentation of HIV epitopes in dendritic cells and macrophages to CD8 T cells”
99th Annual Meeting of the American Association of Immunologists in Boston, USA (May 2012)
Poster: “Variable processing and presentation of HIV epitopes in HIV-infectable cell subsets”
Table of Contents
List of Figures .......................................................................................................................... 4
List of Supplemental Figures .............................................................................................. 6
1. Introduction ..................................................................................................................... 7
1.1. HIV-1 infection ...................................................................................................................... 7
1.2. HIV-1-infectable cell subsets ........................................................................................... 8
1.3. Processing of HIV-1 peptides ........................................................................................ 10
1.3.1. The direct presentation pathway ...................................................................................... 11
1.3.2. The cross-presentation pathways .................................................................................... 13
1.4. Immune response to HIV-1 infection ......................................................................... 16
1.5. Immunodominance patterns in HIV-1 infection .................................................... 17
1.6. Viral escape mutations .................................................................................................... 19
1.7. HIV-1 vaccine design ........................................................................................................ 19
2. Motivation ...................................................................................................................... 21
3. Materials and Methods .............................................................................................. 23
3.1. Materials .............................................................................................................................. 23
3.1.1. Instruments ................................................................................................................................ 23
3.1.2. Chemicals, Solutions ............................................................................................................... 23
3.1.3. Buffers, Media ............................................................................................................................ 24
3.2. Study participants ............................................................................................................. 24
3.3. Cell culture .......................................................................................................................... 25
3.3.1. Generation of monocyte-derived DCs and Møs ........................................................... 25
3.3.2. Culture of epitope-specific CD8+ T cell clones ............................................................. 25
3.4. Analysis of the antigen processing machinery in DCs and Møs ........................ 25
3.4.1. Preparation of cell extracts .................................................................................................. 25
3.4.2. Fluorescent measurement of hydrolytic activities .................................................... 26
3.4.3. Analysis of the antigen processing machinery by Western blot .......................... 28
3.5. In vitro processing of HIV peptides ............................................................................ 29
3.5.1. In vitro peptide degradation assay ................................................................................... 30
3.5.2. Analysis of the antigenicity of the degradation products ....................................... 30
3.5.3. Identification and semi-quantification of degradation products by MS ........... 30
3.5.4. Intracellular peptide stability assay ................................................................................. 31
3.5.5. Quantification of epitope degradation by reversed-phase HPLC analysis ....... 31
Table of Contents 2
3.6. Cross-presentation of exogenous HIV proteins by DCs and Møs ..................... 33
3.6.1. ELISPOT cross-presentation assay ................................................................................... 33
3.6.2. P24 uptake assay ..................................................................................................................... 33
3.7. Statistical analysis ............................................................................................................ 34
4. Results ............................................................................................................................. 35
4.1. Hydrolytic activities of cytosolic proteases involved in antigen processing in
DCs and Møs .................................................................................................................................... 35
4.1.1. Proteasomal hydrolytic activities in DCs and Møs change differently upon
maturation with TLR4 or TLR7/8 ligands ..................................................................................... 35
4.1.2. Hydrolytic activities of cytosolic aminopeptidases and TPP II are comparable
in DCs and Møs, whereas TOP activities are significantly higher in Møs .......................... 36
4.1.3. TLR-mediated maturation of DCs and Møs results in early changes of
proteasomal activities and reflect changes detected upon HIV infection ........................ 38
4.2. Expression of cytosolic and ER-resident proteases involved in antigen
processing in DCs and Møs ........................................................................................................ 40
4.2.1. Higher expression levels of the catalytical and structural subunits of the
constitutive and immunoproteasome in Møs compared with DCs ..................................... 40
4.2.2. Møs have higher expression levels of cytosolic TOP and ER-resident ERAP1
and ERAP2 compared to DCs .............................................................................................................. 42
4.3. HIV-1 epitope processing by cytosolic peptidases in DCs and Møs................. 43
4.3.1. Proteasomal activities in cell extracts of DCs and Møs reflect those measured
in intact, live cells ..................................................................................................................................... 43
4.3.2. The kinetics and amounts of optimal HIV-1 epitopes produced in cytosolic
extracts of DCs and Møs vary among epitopes ............................................................................ 44
4.4. Processing of HIV-1 peptides by cytosolic and endo-lysosomal peptidases in
DCs and Møs .................................................................................................................................... 50
4.4.1. Lysosomal activities in live intact DCs and Møs correlate with activities
measured in cell extracts ...................................................................................................................... 50
4.4.2. Preferential production of immunodominant epitopes KF11 and TW10 in
cytosolic and endo-lysosomal cell extracts ................................................................................... 52
4.5. Cross-presentation of HIV-1 proteins by DCs and Møs ........................................ 56
4.5.1. Cross-presentation of the immunodominant Gag p24 B57-TW10 and B57-
KF11 epitopes is more efficient than that of subdominant B57-ISW9 epitope.............. 56
4.5.2. Protease activities in cross-presentation competent cell compartments
differently affect processing of HIV-1 epitopes in DCs and Møs .......................................... 58
Table of Contents 3
4.5.3. Low degradation of RK9-containing fragments results in high amounts of
HLA-A03 RK9 epitope cross-presented by DCs and Møs ........................................................ 60
4.6. Intracellular stability of HIV-1 epitopes in cytosolic and lysosomal cell
extracts ............................................................................................................................................. 62
4.6.1. Intracellular epitope stability of HIV-1 epitopes in cytosolic and lysosomal
cell extracts follows CTL responses hierarchy ............................................................................. 62
4.7. The impact of HIV-1 escape mutations on epitope production and peptide
stability in the cross-presentation pathway ....................................................................... 64
4.7.1. Frequent escape mutations in immunodominant epitopes reduce epitope
production and peptide stability in the cross-presentation pathway ................................ 64
5. Discussion ...................................................................................................................... 67
5.1. Antigen processing in HIV-infectable cell subsets ................................................ 67
5.2. Viral entry and intracellular trafficking of antigens ............................................ 69
5.3. Viral recognition and cell activation upon HIV-1 infection ............................... 70
5.4. Epitope processing in different subcellular compartments .............................. 72
5.5. Cross-presentation contributes to immunodominance patterns .................... 74
5.6. The impact of viral escape mutants on cross-presentation............................... 75
5.7. Impact of antigen processing and cross-presentation on vaccine design .... 76
6. References ..................................................................................................................... 80
7. Supplemental Figures .............................................................................................. 100
List of Figures 4
List of Figures
Figure 1. Overview of the HIV-1 replication cycle. ........................................................................ 8
Figure 2. Trafficking of antigens for processing and presentation by MHC I or MHC II molecules.
............................................................................................................................................ 11
Figure 3. Regulation of the pH during maturation of early endosomes to lysosomes. ............... 14
Figure 4. HIV-1 infection and disease progression. ..................................................................... 16
Figure 5. Representation of the fluorescence based protease activity assay. ............................ 26
Figure 6. Schematic representation of the in vitro degradation assay. ...................................... 29
Figure 7. Defining the intracellular stability of peptides by RP-HPLC quantification. ................. 32
Figure 8. Proteasomal activities in monocyte-derived DCs and Møs change differently upon
maturation with TLR4 or TLR7/8 ligands. ........................................................................... 36
Figure 9. DCs and Møs have comparable aminopeptidases and TPP II activities, whereas TOP
activities are higher in Møs. ............................................................................................... 37
Figure 10. Changes in antigen processing activities in maturing monocyte-derived DCs reflect
changes observed upon HIV infection. ............................................................................... 39
Figure 11. Higher expression levels of proteins involved in antigen processing in Møs compared
to DCs. ................................................................................................................................ 40
Figure 12. Higher expression levels of catalytically active and structural proteasome subunits in
Møs compared to DCs. ....................................................................................................... 41
Figure 13. Comparable expression levels of cytosolic post-proteasomal activities in DCs and
Møs. .................................................................................................................................... 42
Figure 14. Changes in proteasomal activities in cell extracts reflect those observed in live, intact
DCs and Møs. ...................................................................................................................... 44
Figure 15. Mø cell extracts produce HLA-A11-ATK9 epitope more efficiently than DCs. ............ 45
Figure 16. Mø cell extracts produce more A11-ATK9 epitope and epitope precursors compared
to DC cell extracts. .............................................................................................................. 46
Figure 17. Higher amounts of HLA-A11 ATK9 produced by Mø cell extracts resulted in higher
lysis of target cells by ATK9-specific CTLs. .......................................................................... 47
Figure 18. Extracts from DCs and Møs generate similar amounts of Nef-derived antigenic
peptides at similar rates. .................................................................................................... 49
Figure 19. Lysosomal activities in DC and Mø cell extracts reflect activities in live, intact cells
and can be activated at different pH values....................................................................... 51
List of Figures 5
Figure 20. The immunodominant epitope B57-KF11 is more efficiently produced in cross-
presentation-competent compartments than subdominant epitope B57-ISW9. ............... 53
Figure 21. Major cleavage sites within p24-35mer destroy the B57-ISW9 epitope. ................... 54
Figure 22. Slow degradation of TW10-containing peptides results in high amounts of B57-TW10
available for direct or cross-presentation. ......................................................................... 55
Figure 23. The immunodominant HLA-B57-restricted TW10 and KF11 epitopes are more
efficiently cross-presented than subdominant ISW9 epitope. ............................................ 57
Figure 24. The amount of exogenous p24 protein internalized by DCs and Møs affects the
amount of epitope cross-presented to CTLs. ...................................................................... 58
Figure 25. Inhibition of proteasomes increases B57-KF11 and B57-ISW9 epitope cross-
presentation by DCs but not Møs. ...................................................................................... 59
Figure 26. The RK9-containing fragment shows minor cleavage sites by cytosolic or endo-
lysosomal proteases. .......................................................................................................... 60
Figure 27. Limited degradation of RK9-containing fragments results in cross-presentation of
high amounts of HLA-A03 RK9 epitope. ............................................................................. 61
Figure 28. Variable stability of B57-KF11 and A03-RK9 in cytosolic and lysosomal cell extracts of
immature DCs and Møs. ..................................................................................................... 62
Figure 29. The intracellular stability of HIV-1 epitopes in the cytosol and lysosomes follows CTL
responses hierarchy. ........................................................................................................... 63
Figure 30. Escape mutations in immunodominant epitopes reduce epitope production in the
cross-presentation-competent cell compartments. ........................................................... 65
Figure 31. Reduced intracellular stability of TW10 escape mutants in cytosolic and lysosomal
cell extracts. ........................................................................................................................ 66
List of Supplemental Figures 6
List of Supplemental Figures
Supplemental Figure 1. Phenotypic analysis of DCs and Møs cell populations......................... 100
Supplemental Figure 2. Infection of immature DCs and Møs with VSVg ΔEnv NL4.3. .............. 101
Supplemental Figure 3. Degradation patterns of a HIV-1 RT 16-mer fragment in cytosolic
extracts of immature and mature DCs and Møs. ............................................................. 102
Supplemental Figure 4. Degradation of a HIV-1 Gag p24 35mer in DC cell extracts at pH4.0,
pH5.5 and pH7.4. .............................................................................................................. 103
Supplemental Figure 5. Variable production of 16 HIV-1 epitopes in cytosolic and endo-
lysosomal cell extracts of DCs and Møs. ........................................................................... 104
Supplemental Figure 6. Minor cleavage sites within p24-31mer results in less degradation of
B57-TW10-containing peptides. ....................................................................................... 105
Introduction 7
1. Introduction
1.1. HIV-1 infection
According to estimates by WHO and UNAIDS, 35 million people were living with human
immunodeficiency virus (HIV)-1 globally at the end of 2013. That same year, approximately 2.1
million people became newly infected, and 1.5 million died of AIDS-related causes.
HIV-1 infects several CD4-expressing immune cell subsets, including CD4+ T cells, monocytes,
Møs and DCs. HIV-1 enters cells by specific interactions of the virus and the receptor CD4 and
co-receptors CCR5 and CXCR4 on the target cell. After binding to co-receptors, viral and
cellular membranes fuse and the viral core is released into the cytoplasm of the cell, where
viral RNA is retro-transcribed and provirus is integrated into the cellular genome. Both viral-
and host-cell machineries are necessary for transcription of the viral genes using the
integrated provirus as template. Upon viral protein synthesis, the virus assembles and budding
of new virions occurs (Figure 1). Alternatively, viral fusion via the endosomal pathway has been
shown to constitute a possible mode of virus entry and may depend on the cell type and the
rate of endocytosis (1, 2).
The clinical course of HIV-1 infection in the absence of treatment shows an initial high peak of
plasma viral loads within the first 6 weeks of transmission, which spontaneously decreases as
HIV-1-specific immune responses develop. Already at this early time point of infection, HIV-1
disseminates to the lymphoid organs, and establishes viral reservoirs and latency throughout
the body. In the majority of HIV-1 infected individuals, the immune system ultimately fails to
control the infection, resulting in loss of CD4+ T cells, increasing plasma viral loads and
development of AIDS.
Multiple drugs have been developed to inhibit HIV-1 viral replication and to reverse the decline
in CD4+ T cell counts in HIV-1 infected individuals. Several steps in the HIV-1 replication cycle
are targeted by this highly active antiretroviral therapy (HAART). The three major groups of
drugs being used in clinics are inhibitors of the reverse transcriptase (nucleoside/nucleotide,
NRTI, and non-nucleoside, nNRTI), integrase inhibitors and protease inhibitors (Figure 1, shown
in green). In the majority of HIV-1 infected individuals treated with antiretroviral drugs plasma
HIV-1 viral loads drop significantly and CD4+ T cell counts increase, which can be maintained
for years. However, HAART does not fully restore the immune system and is associated with
considerable side effects and the development of resistances. Therefore, the design of an
effective HIV-1 vaccine is still the only way to control viral infection in the absence of HAART.
Introduction 8
Figure 1. Overview of the HIV-1 replication cycle.
HIV-1 binds to CD4 and the appropriate co-receptor (CCR5 or CXCR4) resulting in fusion of the
viral envelope and the cellular membrane and the release of viral nucleocapsid into the
cytoplasm. Following the uncoating viral RNA is reverse transcribed by the reverse
transcriptase, integrates into the cellular DNA by the integrase, transcribed by the cellular RNA
polymerase II and the resulting mRNA is exported to the cytoplasm and translated into viral
proteins. Viral proteins and genomic RNA are transported to the cellular membrane and
assembled. Immature virions are released. Polypeptide precursors are processed by the viral
Protease to produce mature viral particles. Several host derived restriction factors can inhibit
HIV-1 replication at different stages of the replication cycle (shown in red). HIV-1 has evolved to
counteract these innate antiviral factors by expression of viral proteins: the viral capsid for
TRIM5α, Vpu/Nef/Env for tetherin, Vif for APOBEC-3G, and Vpx for SAMHD1 (shown in blue).
The major families of antiretroviral drugs and the step during the replication cycle that they
block are indicated (shown in green) (3).
1.2. HIV-1-infectable cell subsets
CD4+ T cells, Møs and DCs patrolling the mucosal surface are the first immune cells to
encounter HIV-1. CD4+ T cells can be divided into several subpopulations with diverse cytokine
secretion patterns, which recruit and activate other immune cells including B cells, CD8+ T cells
and Møs (4). CD4+ T cell subpopulations reside within the gastrointestinal tract, the lymph
nodes, other lymphatic tissues and the blood. During the acute phase of HIV-1 infection a
dramatic loss of CD4+ CCR5+ T cells in the gastrointestinal tract can be detected (5), while the
chronic phase of infection, characterized by a massive production of proinflammatory
cytokines, is characterized by a gradual loss of peripheral CD4+ T cells. Despite being a main
target by HIV-1, previous studies showed that HIV-1 Gag-specific CD4+ T cell responses
Introduction 9
increased after acute HIV-1 infection in individuals who control HIV-1 infection (6) and were
significantly associated with control of viral replication (7, 8). However, it has been shown that
these HIV-specific CD4+ T cells are preferentially infected, rather than CD4+ T cells specific for
other antigens (9).
DCs represent an important subset in HIV-1 infection, as they affect viral transmission and
presentation of HIV-1 antigens. DCs can modulate antiviral immune responses by presentation
of MHC I and MHC II epitopes to CD8+ and CD4+ T cells and secret pro-inflammatory cytokines
and interferons to alter T cell proliferation and differentiation. DCs express high levels of the
HIV-1 entry receptors CCR5 and CXCR4, as well as low levels of CD4, allowing gp120 binding
and attachment of HIV-1 virions. Moreover, specific DC subsets express several receptors that
can bind HIV-1 gp120 (10), such as Langerin on Langerhans cells in the skin, DC
immunoreceptor (DCIR) on conventional DCs in the lamina propria, or DC-specific intercellular
adhesion molecule-grabbing nonintegrin (DC-SIGN) and the mannose receptor on dermal DCs.
DCs express high levels of restriction factors, such as SAMHD1, thus only a small proportion of
cells is productively infected (11). Besides productive infection, replication and formation of
new virions for infection of CD4+ T cells (termed cis-infection) DCs can promote infection to
CD4+ T cells in trans. C-type lectins expressed by DCs capture HIV-1 and migrate to CD4+ T cell
enriched lymphoid tissues, where HIV-1 trans-infection of active CD4+ T cells occurs and
facilitates viral dissemination. This direct non-replicative trans-infection across the infectious
synapse allows virions to be rapidly transferred when DCs and CD4+ T cells are in close contact
at the infectious synapse.
Møs can take up viral particles or virions for processing and presentation by MHC II to CD4+ T
cells or cross-presentation by MHC I to CD8+ T cells, which may be crucial for activation of
antiviral immune responses. A significant proportion of Møs at the mucosal surface is
productively infected with HIV-1 (12) and infection results in only limited virus production. In
contrast to CD4+ T cells, Møs are more resistant to the cytopathic effects of HIV-1 and better
able to evade the host immune response. The two major cellular reservoirs for HIV-1 are
therefore latently infected resting CD4+ T cells and Møs. Especially long-lived Møs accumulate
replication-competent HIV-1 for prolonged periods, even in patients receiving HAART (13).
Monocytes are composed of several subpopulations with the majority of monocytes in healthy
individuals expressing CD14 but not CD16. During infection the number of circulating
CD14+CD16+ monocytes increases dramatically. This CD16+ monocytes subset is preferentially
targeted by HIV-1 (14). Relative to CD4+ T cells, CD14+ monocytes are more resistant to
productive HIV-1 infection as a result of differential expression levels of several restriction
Introduction 10
factors, including SAMHD1 or APOBEC-3G (15). In contrast, CD16+ monocytes express less
APOBEC-3G in line with higher infectivity of this subset.
Understanding which HIV-1 epitopes are presented by all infectable cell subsets is essential to
evaluate the capacity of immunogens to generate T cell responses specific for those epitopes
in order to recognize and clear all infected cells.
1.3. Processing of HIV-1 peptides
All nucleated cells express MHC I molecules on their surface, while professional antigen-
presenting cells, including DCs, Møs and B cells, additionally express MHC II molecules on their
surface. The recognition and killing of HIV-infected cells requires the presentation of adequate
amounts of peptide-loaded MHC I molecules to HIV-specific CTLs. Therefore, it is important to
analyze the capacity of all HIV-infectable cell subsets to process and present MHC I-restricted
epitopes. Viral epitopes originate from HIV-1 proteins undergoing intracellular processing.
Endogenous antigens within the cytoplasm are mainly processed by the proteasome before
transportation into the endoplasmic reticulum (ER) via the transporter associated with antigen
processing (TAP). In the ER peptides can be further trimmed before loading onto MHC I
molecules for presentation to CD8+ T cells. MHC I peptides are usually 8-11 amino acids long
and confined by binding groove interactions at both the N- and C-termini.
In contrast, exogenous antigens are internalized into the endosomal/lysosomal compartment,
where antigens are processed by different cathepsins and other proteases. Peptides of usually
12-15 amino acids in length can then be loaded onto MHC II molecules (and even overlay the
MHC II binding groove) and presented to CD4+ T cells. Given the important role of CD4+ T cells
in providing help for CD8+ T cells and B cells (16) or even direct antiviral effects (17) it is crucial
to understand the processing of MHC II peptides in the endo-lysosomal compartments.
Despite the differences in the source of antigen and the processing pathways, exogenous
antigens can be translocated into the cytosol for further processing and presentation onto
MHC I, a process known as cross-presentation (Figure 2).
Introduction 11
Figure 2. Trafficking of antigens for processing and presentation by MHC I or MHC II
molecules.
Cytosolic proteins, such as viral or endogenous proteins, alternative transcripts or defective
ribosomal products (DRiPs), are processed primarily by the proteasome into shorter peptides.
These peptides can be further trimmed by cytosolic post-proteasomal peptidases before
transportation into the endoplasmic reticulum (ER) by the transporter associated with antigen
processing (TAP) for subsequent trimming and assembly by MHC I molecules. In professional
antigen-presenting cells (APCs) exogenous antigens can be internalized into endosomes and
lysosomes for processing by various cathepsins and presentation by MHC II to CD4+ T cells.
Alternatively, exogenous antigens can be translocated into the cytoplasm for further processing
by cytosolic proteases and MHC I loading in the ER, a phenomenon known as cross-
presentation. Loading of MHC II occurs in mature or late endosomal compartments, specialized
in processing and loading onto MHC II molecules.
1.3.1. The direct presentation pathway
In the cytosolic antigen-processing pathway proteins destined for presentation on MHC I are
primarily degraded by the proteasome (18-20). Different sources of antigen can be degraded,
such as cryptic transcriptional products (21), defective ribosomal products (DRiPs) (22, 23),
endogenous mature proteins, or exogenous antigens preprocessed in endo- and lysosomes.
The 26S proteasome is composed of a core cylinder, the 20S proteasome, which is capped at
each end by a 19S complex. The 20S proteasome consists of a barrel-shaped core structure
containing four stacked rings of seven subunits each. The outer rings are composed of alpha-
subunits and the middle two beta-subunits, three of which beta1 (chymotrypsin-like), beta2
(tryptic-like), and beta5 (caspase-like) constitute the active proteolytic components. The 19S
component has deubiquitinase and an unfoldase activity that allows the targeted proteins to
Introduction 12
enter the channel in the center of the barrel where the beta-subunit active sites reside (24).
The hydrolytic activities within the 26S proteasome cleave after hydrophobic, acidic, or basic
residues, respectively and therefore cleave the protein into many diverse oligopeptides,
although the precise location of the cleavages vary widely with each molecule degraded.
Whereas 26S proteasomes degrade mostly polyubiquitinated substrates, 20S proteasomes
capped with regulatory PA28/11S lids are able to degrade non-polyubiquitinated substrates.
Therefore, hydrolysis of proteins by proteasomes is the key step in the generation of most
antigenic peptides (24).
HIV-1 infection triggers the massive production of proinflammatory cytokines and type I and II
interferons (25, 26). Upon interferon-gamma, TNF-alpha or LPS stimulation, the three catalytic
beta-subunits of the constitutive proteasome are replaced by immunoproteasome subunits,
beta1i, beta2i, and beta5i, which have been shown to differ in cleavage specificities compared
to the constitutive subunits (27). Immunoproteasomes can also be constitutively expressed in
lymphoid tissues. Immunoproteasomes exhibit increased rates of substrate turnover with
enhanced cleavage after hydrophobic and basic amino acids, correlating to the preferred C-
terminal residues of many MHC I ligands (28, 29) and generate more N-terminally extended
peptides (30). The presence of both proteasomes and immunoproteasomes in the same cell is
associated with a greater variety of peptides available for presentation (31). Even though
constitutive and immunoproteasomes generate peptides with appropriate C-terminals they
actually destroy more epitopes than they generate (28, 32).
N-terminally extended peptides produced by the proteasomes require trimming by cytosolic
post-proteasomal peptidases, such as leucine aminopeptidase, tripeptidyl peptidase II (TPPII),
thimet oligopeptidases (TOP), puromycin sensitive aminopeptidase (PSA) and several others
(33) before transportation into the ER. Cytosolic peptidases cannot unfold proteins and
therefore degrade post-proteasomal degradation products. TPPII has been shown to generate
epitopes in a proteasome-independent manner by removing three residues at a time from the
N-terminus of peptides of more than 15 residues in lengths (34). Despite the contribution of
several cytosolic aminopeptidases to the generation of individual epitopes, these peptidases
also have an overall destructive effect on epitope generation (35, 36). A small fraction of the
peptides produced by proteasomes and post-proteasomal peptidases escape destruction in
the cytosol and are transported by TAP into the ER for loading onto MHC I. Thus, peptide
stability is an important parameter that has been shown to contribute to the amount of
epitope available for presentation (37, 38).
TAP transports peptides that range in length from about 7 to more than 20 amino acids and
can therefore import both mature epitopes and longer precursors into the ER (39). TAP
Introduction 13
deficient cells showed decreased MHC I surface expression levels indicating the importance of
TAP in antigen presentation and MHC surface expression. In the ER, ER-resident
aminopeptidases, such as ERAP1 and ERAP2 are able to remove from the N-terminus of
peptides nearly all amino acids except those followed by proline prior to MHC I loading and
presentation (40-43). Thus, N-terminal trimming occurs in both, the cytoplasm and the ER.
Peptide-MHC (pMHC) class I complexes are then transporter to the cell surface for
presentation and activation of CD8+ T cells with a cognate receptor.
Together, differences in the antigen-processing machinery among HIV-infectable cell subsets
may affect the timing and the amount of epitopes presented to HIV-specific CTLs, which will
affect the capacity of CTLs to recognize and kill all infected cells.
1.3.2. The cross-presentation pathways
Cross-presentation has been demonstrated to play an important role in the induction of
immune responses against viruses that do not infect APCs, viruses that impair direct
presentation of antigens upon infection or viruses that exhibit strict tissue tropism. HIV-1 can
enter cells by direct fusion at the plasma membrane or by endocytosis into the endocytic
system. In addition, vaccines, including viral vectors, peptides bound to nanoparticles or viral
particles, enter DCs by endocytosis. Whether processing of antigens by the cross-presentation
pathway versus the direct presentation pathway results in the presentation of the same
epitopes with comparable amounts and kinetics is still unknown. This aspect is important for
the design of a HIV vaccine, as processing of vaccines in the cross-presentation pathway should
result in the presentation of epitopes that match those presented by all HIV-infectable cell
subsets. In general, the cross-presentation pathway can be differentiated into two main
pathways: The cytosolic pathway and the vacuolar pathway.
1.3.2.1. The cytosolic cross-presentation pathway
Exogenous antigens, such as proteins, apoptotic cells or antibody-coated viruses can be
internalized by multiple mechanisms, including receptor-mediated endocytosis via clathrin-
coated vesicles, macropinocytosis, or phagocytosis. In the endocytic system antigens undergo
gradual proteolytic degradation by various cathepsins and other proteases including insulin-
regulated aminopeptidase (IRAP) (44, 45) along their journey from early endosomes and
phagosomes to lysosomes. In the cytosolic cross-presentation pathway, antigens are
translocated from endosomes or phagosomes into the cytosol for further degradation by the
proteasome (46, 47). After proteasomal degradation, antigen-derived peptides are transported
by TAP into the ER for further trimming and loading onto MHC I.
Introduction 14
Considering that DCs pick up antigen in the peripheral tissue and migrate for several hours to
lymph nodes (48), cross-priming of naïve CD8+ T cells in lymph nodes requires continuous
cross-presentation of epitopes. Thus prolonged antigen stability and low degradation is an
important prerequisite. It has been shown that limited antigen degradation correlates with
efficient cross-presentation (49, 50), which has been supported by findings showing that
artificially delivered antigens to highly degradative, late endocytic compartments by
lipososomes are presented on MHC II more efficiently than on MHC I (51).
Most lysosomal proteases have an optimal proteolytic activity between pH5.5 and 6.5 (52).
After internalization of exogenous antigens phagosomes fuse first with early and late
endosomes to acquire the lysosomal proteases and the proteins responsible for acidification
(Figure 3).
Figure 3. Regulation of the pH during maturation of early endosomes to lysosomes.
Exogenous antigens are internalized into early endosomes, which display a high pH and a low
degradation environment. These are probably the compartments that allow antigen escape to
the cytosol and most likely the site of peptide loading onto MHC I in the vacuolar pathway. As
endosomes and phagosomes mature, the pH drops and the levels of proteolysis increase. The
compartments become incompetent for cross-presentation, but enable peptide loading onto
MHC II.
In DCs several mechanisms have been described to preserve and retain antigen in the
endocytic system. First, DCs express lower levels of most lysosomal proteases than Møs (49)
and are less recruited to phagosomes (53). As a result, DCs degrade internalized antigens at
much lower rates than other phagocytes. Second, it has been shown that the pH in
phagosomes and endosomes in DCs is less acidic than in Møs, which results in lower activation
Introduction 15
of lysosomal proteases thus increasing cross-presentation (54). In DCs phagosomal pH is
approximately pH7.5 in contrast to Møs, which rapidly acidify their phagosomes, reaching
values of 4.5-5 following phagocytosis of inert latex beads (55). In this acidic and highly
proteolytic environment it is very unlikely that peptides are loaded onto MHC I. Endosome
acidification is mediated by the vacuolar ATPase (V-ATPase), which transports protons from
the cytosol into the endosomes. In DCs low levels of V-ATPase activity due to its incomplete
assembly (56), results in a neutral endosomal pH, which prevents rapid antigen degradation
and enhanced cross-presentation. In addition, high levels of activity of NADPH oxidase 2
(NOX2) in DCs, which mediates the generation of reactive oxygen species (ROS) causes the
consumption of protons and thereby the alkalinization of the endocytic compartments (49,
55). Taken together, prolonged MHC I-restricted presentation requires antigen transport into
specialized cell compartments, where high pH and low endocytic protease activity protects
antigens from extensive degradation and allows continuous processing of peptides for loading
onto MHC I.
1.3.2.2. The vacuolar pathway
In the vacuolar pathway, which is also termed TAP-independent cross-presentation pathway,
internalized antigens remain in endosomes and lysosomes, where they are degraded by
endocytic proteases such as cathepsin S. After degradation antigenic peptides are loaded onto
MHC I, which recycles from the cell membrane (57, 58) (Figure 3). The acidic environment in
these endosomes might allow peptides to dissociate from the MHC I molecules, enabling the
peptides processed in the endosomes to bind to MHC I molecules. Within endosomes, IRAP
has been shown to be involved in the trimming of antigens before loading onto MHC I (44).
IRAP displays a broader pH optimum compared to the ER-resident aminopeptidase ERAP,
allowing IRAP activity at a slightly acidic pH (59). IRAP activity might ensure that antigen-
derived peptides are trimmed to their optimal size for loading onto MHC I molecules.
This indicates that trafficking of an antigen through subcellular compartments harboring
different proteases may affect the degradation patterns and thus the amount of epitope
available for presentation.
Introduction 16
1.4. Immune response to HIV-1 infection
Figure 4. HIV-1 infection and disease progression.
HIV-1 infection leads to high levels of HIV-1 replication within the first 6 weeks of infection.
These high viral loads result in the generation of HIV-1-specific CD4+ and CD8+ T cell responses
within 4-6 weeks of infection. Within 1-3 months post infection the appearance of HIV-specific
antibodies indicates sero-conversion. During the subsequent asymptomatic phase of infection
blood CD4+ T cell counts decline slowly, even though successful HAART treatment can partially
reverse this loss. The onset of AIDS is characterized by a progressive loss in CD4+ T cells and
high plasma virus loads. Adapted from (60).
The majority of individuals infected with HIV-1 develop an acute viral syndrome within the first
three weeks of infection, characterized by fever, lymphadenopathy, and cutaneous rash in the
presence of very high levels of HIV-1 replication (61). These high viral loads result in the
generation of innate and adaptive immune responses, including the appearance of HIV-1-
specific CD4+ and CD8+ T cell responses within 4-6 weeks of HIV-1 infection and HIV-1-specific
antibodies, indicating sero-conversion, typically within 1-3 months after HIV-1 infection.
Subsequent to the development of HIV-specific immune responses, HIV-1 RNA levels decline
and eventually stabilize at the so-called viral set point. This viral set point is a strong predictor
for HIV-1 disease progression (62). The time between HIV-1 infection and the establishment of
the viral set point, usually between 3-6 months, is termed primary HIV-1 infection. With
reduction in plasma viral load, CD4+ and CD8+ T cell counts start to normalize, but neither
return to pre-infection levels as CD4+ T cell counts remain reduced and CD8+ T cell counts
remain elevated. However, HIV-specific immune responses fail to eliminate the virus leading to
a chronic infection in most individuals during an asymptomatic period that may persist for
months or years. During the chronic phase of infection, the blood CD4+ T cell counts decline
slowly, even though this loss can be partially reversed by successful HAART, until progressive
CD4+ T cell decline results in the onset of AIDS (Figure 4). The appearance of HIV-1-specific
CD8+ T cell responses has been shown to be temporally associated with the initial decline of
Introduction 17
HIV-1 viral load during primary infection, the resolution of clinical symptoms of the acute viral
syndrome and the rapid selection of viral CTL escape variants. These findings demonstrate an
important role of HIV-specific CD8+ T cells in the control of HIV-1 replication and indicate that
HIV-specific CD8+ T cells will most likely be important for HIV-1 vaccines, if we can identify
protective immune responses that can recognize and kill all HIV-infectable cell subsets.
1.5. Immunodominance patterns in HIV-1 infection
A central feature of many anti-viral T cell responses is the phenomenon of immunodominance.
Despite the expression of 3-6 different MHC I molecules in APCs and the potential generation
of thousands of distinct viral peptides, a large proportion of the anti-viral CTL responses tend
to be dominated by certain class I alleles presenting only a small number of viral peptides.
These CTL responses can be ordered into highly reproducible hierarchies as shown for HIV-1
(62-64), vaccinia virus (65), cytomegalovirus (CMV) (66) and West Nile virus (67).
Immunodominance is generally defined by ELISPOT assays as the most frequent responses in a
population sharing the same HLA allele or in an individual, as the response that induces the
strongest production of IFN-gamma (68, 69). In acute HIV-1 infection a predictable hierarchy of
narrow CD8+ T cell responses can be detected with consistent recognition of certain epitopes
and lack of recognition of others, whereas a broader range of responses has been identified in
chronic infection (63, 70, 71). Previous studies indicated that the immunodominance patterns
of HIV-specific CTL responses developed in primary HIV-1 infection affect the initial control
over viral replication (63, 64, 72) and strongly associate with the subsequent set point of viral
replication (62). However, rising HIV-1 replication in the chronic phase of infection is usually
paralleled by a disintegration of the original CD8+ T cell immunodominance patterns (73-75).
Moreover, other studies showed that immunodominant CTL responses were not always
associated with protection whereas some subdominant responses were associated with lower
viral load (76). Therefore, an effective HIV-1 vaccine should aim to break immunodominance
patterns observed in HIV-1 infection rather than mimicking them. This requires a better
understanding of the mechanisms that contribute to immunodominance. Several factors have
been implicated in shaping immunodominance patterns. Immunodominance can be associated
with the kinetics of protein production, as viral proteins expressed early after infection may
offer an earlier source of epitopes than viral proteins expressed at a later stage. However, in
HIV-1 infection, proteins synthesized later during the viral replication cycle, such as Gag, have
been shown to contribute to dominant CTL responses, although other studies showed that Gag
from incoming virions can be degraded prior to viral protein synthesis and thus lead to Gag-
specific CTL responses (77). Moreover, previous studies with HIV-1 have shown that the
Introduction 18
efficiency of HIV-epitope processing contributes to immunodominance. Degradation of long
peptides in PBMC extracts and purified proteasomes led to the preferential production of
dominant epitopes and its precursors whereas subdominant epitopes were degraded (78, 79).
This preferential processing has been shown to be sequence-dependent, which suggest that
the capacity of proteins to generate epitopes is governed by specific motifs. More recent
studies reported that the absence of proteasomal components is detrimental on the pool of
peptides generated and led to different production of viral peptide pools in comparison to WT
mice during LCMV infection (80). Furthermore, mice deficient in proteasomal catalytic
subunits, LMP2 and MECL1, showed reduced anti-viral CTL responses to immunodominant
influenza epitopes (81), whereas in LMP7-/- mice the overall presentation of this
immunodominant epitope was not affected. These findings indicated a direct impact of the
processing of peptides by proteasomes on the generation of immunodominant epitopes. If
processing of antigens in the cross-presentation pathway contributes to immunodominance
remains open.
TAP-dependent transport of peptides into the ER also plays an important role in
immunodominance. Peptides missing the correct C-terminal amino acid or being longer than
the preferred length for TAP binding are not transported into the ER lumen. Moreover,
mutations within epitopes or flanking sequences have been demonstrated to affect binding
affinity to TAP leading to an overall reduced anti-viral CTL response to peptides that would
have been normally immunodominant (78). Furthermore, studies in ERAP-1-/- mice have shown
the generation of different LCMV-specific CTL responses compared to WT mice infected with
the same virus. The production of ERAP-1 dependent epitopes was impaired, whereas ERAP-1-
independent epitopes or those usually destroyed by ERAP-1 increased in presentation to CTLs,
which affected immunodominance hierachies (82). Similar results for immunodominant HIV-
derived epitopes were observed in other studies (78).
The expression of specific MHC I molecule combinations and their level of expression
determine which epitopes will be recognized by specific CTLs and therefore contribute to
immunodominance. In HIV-1 infection, immunodominant responses are preferentially
restricted by HLA B alleles, especially HLA-B27 or HLA-B57 (64, 83). In this case CTL responses
restricted by other alleles than HLA-B27 or -B57 are always subdominant even though they
would be dominant in persons lacking HLA-B27 or -B57 (62, 84). Moreover, peptides with
highest binding affinity for a given HLA allele may favor peptide loading and a longer display of
epitopes at the cell surface and thus an earlier and prolonged interaction with CD8+ T cells (85,
86). Binding affinity of the peptide-MHC complex for the TCR may affect interaction with CD8+
T cells and contributes to immunodominance patterns. Previous studies demonstrated that
Introduction 19
high affinity of epitope-MHC I complexes for the TCR may prolong the interaction with
epitope-specific CTLs and contribute to immunodominance (86, 87). The number of T cell
precursors and the ability of these cells to proliferate and persist over time may also
contribute to immunodominance as shown previously (88).
1.6. Viral escape mutations
An important factor limiting the success of HIV-specific CTL responses to clear infection is the
capacity of HIV-1 to mutate targeted epitopes to evade CTL recognition (89). Viral escape
mutations occur during the acute and chronic phase of HIV-1 infection (90, 91). Escape
mutations can affect several steps during epitope production and presentation to CTLs.
Impaired production of epitope-containing peptides by the proteasome or by cytosolic
aminopeptidases may lead to less antigen available for presentation (92-94), while reduced
binding of the mutant to MHC I, or altered recognition by the TCR (95) affects epitope
presentation and recognition by CTLs. Other steps during the antigen processing pathway can
also be affected, such as reduced binding of peptides to TAP for transportation into the ER or
impaired trimming of precursors into antigenic peptides for loading onto MHC I molecules in
the ER (78, 93). Whether processing of escape mutants in the cross-presentation pathway
contributes to immune escape is not known.
While CTL escape mutants enable the evasion of host immune responses and have been
associated with a shift in immunodominance patterns and even the loss of HIV-1 control (89)
the requirement to preserve the viral fitness may limit the appearance of these mutants.
Indeed, previous studies showed that some CTL escape mutations diminished HIV-1 replication
and reverted back to WT sequences upon transmission to an HLA-mismatched individual (96,
97), while other studies demonstrated that the replication capacity of viruses containing CTL
escape mutations that compromised viral fitness could be restored by a compensatory
mutation within the epitope or in flanking sequences (98). These findings suggest that escape
mutants that disrupt functionally important regions of the virus may not sustain and that a
barrier for CTL escape may therefore contribute to containment. This may suggest that
simultaneous delivery of immunogens containing wild-type sequences and escape variants
may represent an effective approach to increase immune pressure on the virus, thus pushing
the virus to mutants that affect viral fitness (99, 100).
1.7. HIV-1 vaccine design
Although treatment of infected individuals with HAART provides successful viral control, HIV-1
remains persistent and is not eradicated. Moreover, HAART does not fully restore the immune
Introduction 20
system and is associated with considerable side effects, thus remaining a life-long
commitment. Therefore, the design of an effective HIV-1 vaccine is still the only way to control
viral infection in the absence of HAART. An effective vaccine against HIV-1 should ideally elicit
humoral and adaptive immunity, such as efficient antibody responses to prevent or limit HIV-1
entry into cells and a potent T cell response that will allow clearance of infected cells (101).
Challenges for the design of a HIV-1 vaccine include the absence of protective immunity in HIV-
1 infected patients, the genetic diversity of HIV-1, the ability of HIV-1 to form viral reservoirs
that cannot be eliminated with HAART, and the lack of B cells making specific neutralizing
antibodies.
HIV-1 infection leads to a predictable hierarchy of immunodominant and subdominant T cell
responses for each HLA allele. Since HIV-1 is not spontaneously cleared by the immune
responses developed during infection it is likely that a vaccine eliciting the same
immunodominance patterns as in natural HIV-1 infection will most likely not be successful at
preventing or clearing HIV-1 infection. Thus, an alternative approach may aim to break natural
immunodominance patterns during vaccination. Besides immunogen design, it is important to
identify specific APCs that can internalize the immunogen for efficient processing and
presentation. Another important aspect in vaccine design is the choice of vectors and
adjuvants, which can alter conditions in which peptides are processed.
Regardless of the immunogen and adjuvant used for vaccination, the HIV-1 immunogen has to
be processed in APCs for priming of immune responses. Therefore, it is important to better
understand how HIV-1 immunogens are degraded into peptides within different cell subsets
and subcellular compartments used for antigen trafficking to evaluate their capacity to
generate peptides compatible with loading onto a variety of MHC I or MHC II molecules.
Motivation 21
2. Motivation
HIV-specific CD8+ T cells play a major role in reducing viral load in acute HIV-1 infection and in
the spontaneous control of HIV-1 infection, and therefore will most likely become a critical
part of an effective HIV-1 vaccine. However multiple lines of evidence indicate that not all CTL
responses have equivalent capacity to kill HIV-infected cells and reduce viral load. Therefore it
is critical to define criteria leading to an efficient CTL response against all HIV-infectable cell
subsets.
1) Are all HIV-infectable cell subsets able to equally process and present the same epitopes?
Several CD4-expressing cell subsets, including CD4+ T cells, monocytes, Møs and DCs, are
permissive to HIV-1 infection. The identification of epitopes presented by all infectable cell
subsets is crucial for the identification and priming of protective CTL responses that should
recognize and kill all infected cell subsets. Whether all HIV-infectable cell subsets process and
present equivalent amounts of epitopes remains open. Differences in the kinetics or amounts
of epitopes displayed by any of these cell subsets may impair CTL recognition hence allow viral
spreading.
1.1) How does the antigen processing machinery in DCs and Møs affect the processing and
presentation of HIV-1 peptides?
1.2) Do DCs and Møs similarly process and present epitopes, which match those presented by
all other HIV-infectable cell subsets?
1.3) How does maturation of both cell subsets with TLR ligands, which are often used as
adjuvants during vaccination or present during HIV-1 infection, affect protease activities and
processing of HIV-1 peptides?
2) Does processing in the cross-presentation pathway versus the direct presentation
pathway lead to similar epitope presentation?
DCs and Møs have the ability to process and present MHC I epitopes from endogenous and
exogenous antigens via two main pathways: the cytosolic/direct pathway and the cross-
presentation pathway. Here we analyze the impact of antigen trafficking through different cell
compartments containing different proteases on the degradation patterns of HIV-1 peptides.
2.1) How does trafficking of antigens/immunogens through different cell compartments affect
its degradation patterns, the amount and the timing of epitope presentation?
2.2) Does processing of antigens/immunogens in the cross-presentation pathway result in
similar epitope presentation compared to the direct presentation pathway?
Motivation 22
3) What is the impact of cross-presentation on HIV-1 immunodominance patterns and
immune escape?
Multiple studies have shown that a narrow repertoire of epitopes is targeted during HIV-1
infection, which results in the formation of immunodominance patterns. The appearance of
escape mutations during disease progression has been shown to result in viral evasion from
the host’s immune responses. Escape mutations in immunodominant and subdominant
epitopes result in a shift in immunodominance patterns and can lead to loss of viral control.
HIV-1 can enter DCs and Møs by fusion at the plasma membrane for delivery into the cytosol,
or by internalization into endolysosomal vesicles. Therefore, the focus of this study is to
analyze the impact of the cross-presentation pathway on immunodominance patterns and
immune escape.
3.1) What is the contribution of cross-presentation to the establishment of immunodominance
patterns during HIV-1 infection?
3.2) Do escape mutants affect the processing of epitopes in cell compartments involved in
cross-presentation?
4) What are the implications for immunogen design?
An effective vaccine against HIV-1 should ideally elicit humoral and adaptive immunity, such as
efficient antibody responses to prevent or limit HIV-1 entry into cells and potent CD8+ T cells
for clearance of infected cells as well as CD4+ T cell help required for priming. Vaccines,
including peptides, viral vectors or peptides bound to nanoparticles, enter DCs by receptor-
dependent or -independent endocytosis. To evaluate the capacity of proteins/immunogens to
generate peptides compatible with loading onto a variety of MHC I or MHC II molecules
independent of a given HLA, it is necessary to better understand how HIV proteins are
degraded into peptides within different cell subsets and subcellular compartments used for
antigen/immunogen trafficking.
Materials & Methods 23
3. Materials and Methods
3.1. Materials
3.1.1. Instruments
Instruments
Centrifuge Sorvall Legend XTR Thermo Scientific, USA
CO2 incubator Galaxy 170R Eppendorf, Germany
CTL-ImmunoSpot S6 Macro Analyzer CTL, USA
ELISA Sunrise microplate reader Tecan, Switzerland
Flow cytometer, FACSCalibur BD Biosciences, USA
Flow cytometer, LSR II BD Biosciences, USA
HPLC system Waters, USA
Labquake Tube Shaker Thermo Scientific, USA
LTQ Orbitrap Mass Spectrometer Thermo Scientific, USA
Microcentrifuge 5417R Eppendorf, Germany
Microscope BH2 Olympus, USA
Microscope EVOS XL Life Technologies, USA
Nano-HPLC Eksigent, USA
NucleoCounter NC-200 Chemometec, Denmark
Odyssey CLx Infrared Imaging System LI-COR Biosciences, USA
PowerPac Universal Power Supply Bio-Rad, USA
Trans-Blot SD Semi-Dry Transfer Cell Bio-Rad, USA
VICTOR X5 Multilabel Plate Reader PerkinElmer, USA
Vortex-Genie 2 Scientific Industries, USA
XCell SureLock Mini-Cell Electrophoresis System Life Technologies, USA
3.1.2. Chemicals, Solutions
Chemicals, Solutions
Acetonitrile, 99.8%, for HPLC Fisher Scientific, USA
Acetonitrile, Optima LC/MS Fisher Scientific, USA
Adenosine 5’-triphosphate disodium salt hydrate (102) Sigma-Aldrich, USA
Materials & Methods 24
Chemicals, Solutions
Digitonin Sigma-Aldrich, USA
Dimethyl Sulfoxide (DMSO) Fisher Scientific, USA
DL-Dithiothreitol (DTT) Sigma-Aldrich, USA
Ethylenediaminetetraacetic acid disodium salt solution (EDTA) Sigma-Aldrich, USA
Fetal Bovine Serum, Heat inactivated (FBS) Sigma-Aldrich, USA
Formic acid, 99+% Thermo Scientific, USA
Glycerol Sigma-Aldrich, USA
Human Serum AB Gemini Bio-Products, USA
Hydrochloric acid, HCl Fisher Scientific, USA
Magnesium chloride, MgCl2 Sigma-Aldrich, USA
Penicillin-Streptomycin Solution, 50X Corning, USA
Potassium phosphate monobasic, KH2PO4 Sigma-Aldrich, USA
Potassium acetate, C2H3KO2 Sigma-Aldrich, USA
Sodium chloride, NaCl Sigma-Aldrich, USA
Trichloroacetic acid, ≥ 99.0% Sigma-Aldrich, USA
Trifluoroacetic acid, 99% Sigma-Aldrich, USA
Water, Optima LC/MS Fisher Scientific, USA
3.1.3. Buffers, Media
Buffers, Media
AIM-V media Invitrogen, USA
Dulbecco’s Phosphate Buffered Saline (PBS) Sigma-Aldrich, USA
Hank’s balanced salt solution Sigma-Aldrich, USA
HEPES, 1M solution Corning, USA
RPMI-1640 Medium, without L-glutamine Sigma-Aldrich, USA
Trizma hydrochloride buffer solution Sigma-Aldrich, USA
3.2. Study participants
HLA-typed blood donors were recruited at the Massachusetts General Hospital (MGH) in
Boston. All participants provided written informed consent for participation in the study.
Anonymous buffy coats were purchased from the MGH. This study was approved for use by
the Partners Human Research Committee under protocol 2005P001218 (Boston, MA).
Materials & Methods 25
3.3. Cell culture
3.3.1. Generation of monocyte-derived DCs and Møs
Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from HLA-typed
blood donors or anonymous buffy coats by Ficoll-Hypaque (Sigma-Aldrich, USA) density
centrifugation. Monocytes were enriched from PBMCs using CD14+ magnetic isolation kits
according to the manufacturer’s instructions (StemCell, Canada). Briefly, PBMCs were
incubated with human anti-CD14 antibodies followed by incubation with dextran-coated
magnetic particles in PBS, 2% human serum AB and 2mM EDTA. Labeled CD14+ monocytes
were subsequently isolated by magnetic separation. The purity of CD14+ monocytes was
>98%. DCs were differentiated from monocytes during a 6-day culture at 106 cells/mL in AIM-V
medium, supplemented with 1% HEPES, 1% human serum AB, 20ng/mL IL-4 (CellGenix,
Germany) and 10ng/mL GM-CSF (CellGenix, Germany). On day 2 and 4, fresh IL-4 and GM-CSF
were added. Møs were differentiated from monocytes during a 6-day culture at 106 cells/mL
in ultra low attachment plates (Corning, USA) in AIM-V medium supplemented with 1% HEPES
and 10% human serum AB. Where indicated, maturation of DCs and Møs was induced by TLR
ligand stimulation with 2µg/mL LPS (TLR4 ligand), 1µg/mL CL097, or 1µg/mL R848 (TLR 7/8
ligands) (Invivogen, USA) for 2 days.
To assess the purity of DCs and Møs populations, cells were stained using human anti-CD11c,
anti-CD14, and anti-BDCA1 antibodies (BD Biosciences, USA) and analyzed by flow cytometry
(Supplemental Figure 1).
3.3.2. Culture of epitope-specific CD8+ T cell clones
HLA-B57-restricted-ISW9-, B57-KF11-, B57-TW10-, A03-RK9-, B35-QY9- and A11-ATK9-specific
CTL clones were maintained in the presence of 50U/mL IL-2, using 0.1µg/mL CD3-specific
monoclonal antibody 12F6, and irradiated feeder cells as stimulus for T cell proliferation.
EBV-immortalized B cell lines were maintained in RPMI medium, 10% FBS, 1% HEPES, 2mM L-
glutamine, 50U/mL penicillin, and 50μg/mL streptomycin (CellGro, USA).
3.4. Analysis of the antigen processing machinery in DCs and Møs
3.4.1. Preparation of cell extracts
Cells were permeabilized using digitonin to effectively release the contents of the cytosol and
endo-lysosomes. DCs and Møs were permeabilized in ice-cold lysis buffer containing 0.125%
digitonin (50mM HEPES, 50mM potassium acetate, 5mM magnesium chloride, 1mM DTT, 1mM
ATP, 0.5mM EDTA, 10% Glycerol, pH 7.4), followed by 13,000 rpm centrifugation for 15
Materials & Methods 26
minutes at 4ºC to remove cell debris. The protein concentration was measured by Bio-Rad
protein assay according to the manufacturer’s instructions (Bio-Rad, USA).
3.4.2. Fluorescent measurement of hydrolytic activities
The hydrolytic activities of multiple cytosolic and endo-lysosomal proteases were measured
using fluorogenic peptide substrates. These substrates consist of three to four amino acid
residues attached to a fluorogenic reporter group at the C terminus (Figure 5A). The hydrolytic
activity of the protease cleaves the bond between an amino acid and the reporter group,
resulting in release of a highly fluorescent product, such as 7-amino-4-methylcoumarin (AMC).
The increase in fluorescence upon substrate cleavage can be measured on a fluorescence
plate reader over time and the maximum slope of the curve correlates to the hydrolytic
activity of the protease (Figure 5B). To check the specificity of the reaction, cells or cell
extracts were incubated with specific protease inhibitors before addition of the fluorogenic
substrates. Substrate incubated in the absence of cells or cell extracts were used to measure
the background of the reaction.
Figure 5. Representation of the fluorescence based protease activity assay.
A. Protease-specific substrates consist of a peptide residue linked to a fluorogenic reporter
group at the C terminus. Upon cleavage between the peptide residue and the fluorogenic
reporter group a highly fluorescent product is released.
B. Proteasomal tryptic activity was measured in immature DCs using Boc-LRR-AMC as a
fluorogenic substrate. Increase in fluorescence, which is proportional to the hydrolytic tryptic
activity, was measured with a fluorescence plate reader every 5 minutes. Cells pretreated with
MG132, a proteasome inhibitor, were used to check the specificity of the reaction. To measure
the background of the reaction, fluorogenic substrate was added to PBS without cells.
Materials & Methods 27
To measure cytosolic protease activities in live, intact cells, 2x104 DCs or Møs were plated per
well of a black 96-well plate in PBS/0.0025% digitonin in the presence or absence of relevant
inhibitors and incubated for 30 minutes before addition of the fluorogenic substrates.
Addition of low concentrations of digitonin slightly permeabilized the cell membrane and
enabled diffusion of the fluorogenic substrates into the cytosol. To measure proteolytic
activities of endo-lysosomal proteases, cells were incubated with the relevant substrates in
the absence of digitonin to ensure that the fluorogenic substrate is only taken up into
endosomes and lysosomes.
Cytosolic protease activities in whole cell extracts were measured using equivalent amounts of
extracts as determined by actin normalization in reaction buffer at pH7.4 (50mM HEPES,
50mM potassium acetate, 5mM magnesium chloride, 1mM DTT, 1mM ATP, 0.5mM EDTA). To
measure hydrolytic activities of endo-lysosomal proteases in whole cell extracts, the same
amount of cell extracts was used in reaction buffer at pH4.0, pH5.5 and pH7.4, respectively
(50mM sodium chloride, 50mM potassium phosphate, 2mM DTT, 2mM EDTA). The rate of
fluorescence emission, which is proportional to the kinetics of proteolytic activities, was
measured every 5 minutes at 37°C in a Victor-3 Plate Reader (Perkin Elmer, USA) (103).
Table 1. List of protease specific fluorogenic peptide substrates used to analyze hydrolytic
activities of cytosolic and endo-lysosomal peptidases.
Peptidase Substrate Concentration (µM) Company
Cells Extracts
Proteasome
Caspase-like Z-LLE-AMC 50µM 75μM EMD Millipore
Tryptic Boc-LRR-AMC 50μM 2.5μM Bachem
Chymotryptic Suc-LLVY-AMC 50μM 100μM Bachem
Post-proteasomal peptidases
Aminopeptidases H-Leu-AMC 5μM 12.5μM Bachem
TOP Mca-PLGPK-DNP 20μM 5μM Bachem
TPPII H-AAF-AMC 100μM 100μM Bachem
Endo-lysosomal peptidases
Cathepsin S Z-VVR-AMC 50µM 10μM Enzo Life Sciences
OmniCathepsins (Cathepsin B, L, S)
Z-FR-AMC 50µM 50μM Enzo Life Sciences
Cathepsin B Z-RR-AMC - 50μM Bachem
Cathepsin D&E Mca-GKPILFFRLK-Dnp
- 10µM Enzo Life Sciences
Materials & Methods 28
Table 2. List of protease specific inhibitors used to check the specificity of reactions.
Peptidase Inhibitor Concentration (µM) Company
Cells Extracts
Proteasome
Caspase-like
MG132 10µM 10µM Enzo Life Sciences Tryptic
Chymotryptic
Post-proteasomal peptidases
Aminopeptidases Bestatin 12μM - Sigma-Aldrich
TOP N-[(RS)-1-Carboxy -3-phenyl-propyl] -AAF-4-Abz-OH
10μM - Bachem
TPPII Butabindide 10μM - Tocris
Endo-lysosomal peptidases
Cathepsin S Z-FL-COCHO 10µM 10µM Calbiochem
OmniCathepsins (Cathepsin B, L, S)
E64 50µM 50µM Enzo Life Sciences
Cathepsin B Z-RLV-azagly-IV-OMe
- 10μM Bachem
Cathepsin D&E Pepstatin A - 100µM Enzo Life Sciences
3.4.3. Analysis of the antigen processing machinery by Western blot
Proteins in cell extracts were separated on a 4-12% Bis-Tris gradient gel (Invitrogen, USA)
according to their molecular weight and transferred to PVDF membranes (Millipore, USA). To
minimize non-specific binding of the primary antibodies, membranes were blocked with
Odyssey Blocking Buffer (Li-cor Biosciences, USA), and subsequently probed with antibodies
specific for β-actin, the subunit of the proteasome lid S1, the cytosolic leucine aminopeptidase
LAP (Abcam, USA), the proteasome activator subunit PA28α, the proteasome core subunit α7,
the catalytically active subunits of the constitutive and immunoproteasome β1, β2, β5, β1i, β5i
(Enzo Life Sciences, USA), the chaperone Hsp90, the cytosolic peptidase TOP, the ER-resident
aminopeptidases ERAP1 (Santa Cruz, USA), ERAP2 (R&D Systems, USA), and the cytosolic
peptidase TPPII (ProteinTech, USA). After overnight incubation with the primary antibodies,
membranes were washed multiple times and incubated with IRDye secondary antibodies.
Protein bands were visualized by dual infrared fluorophore Odyssey Infrared Imaging System,
and densitometric quantification was performed (Li-cor Biosciences, USA) (104). Equivalent
amounts of cell extracts, as determined by actin normalization, were used to measure
peptidase activities in cell extracts and to degrade HIV peptides using an in vitro degradation
assay.
Materials & Methods 29
3.5. In vitro processing of HIV peptides
To analyze and compare the degradation of peptides into epitopes in different subcellular
compartments, we used a previously developed in vitro degradation assay (105, 106). This
assay uses whole cell extracts of DCs and Møs as a source of peptidases from different cell
compartments to degrade synthetic HIV-1 peptides. In order to activate compartment-specific
proteases the pH value of the degradation buffer was adjusted to pH7.4, pH5.5 or pH4.0. At
these pH values proteases specific for the cytosol, endosomes, and lysosomes were activated
(Figure 3), which allowed us to analyze the processing of HIV peptides in these cell
compartments from the same cell (105). Degradation products at different time points were
identified by mass spectrometry (MS) and their antigenicity was tested by a 51Cr release assay.
To test the intracellular stability of optimal MHC I epitopes, synthetic peptides were incubated
in whole cell extracts. At different time points during the degradation the amount of
remaining epitope was quantified by RP-HPLC analysis (37) (Figure 6).
Figure 6. Schematic representation of the in vitro degradation assay.
DC or Mø cell extracts were used as a source of proteases to degrade synthetic HIV peptides or
optimal MHC I epitopes. Degradation products were identified by mass spectrometry and their
antigenicity was tested by a 51Cr release assay. To determine the intracellular stability of an
epitope the amount of remaining epitope at different time points during degradation was
quantified by RP-HPLC analysis.
Materials & Methods 30
3.5.1. In vitro peptide degradation assay
Highly purified peptides (>98% pure) (MGH peptide core facility or from Bio-Synthesis, USA)
were degraded in whole cell extracts at 37ºC in degradation buffer (50mM Tris-HCl, 137mM
potassium acetate, 1mM magnesium chloride, 1mM ATP; pH7.4, pH5.5, or pH4.0). At different
time points during degradation, aliquots of the reaction were taken and the reaction was
stopped by 5% formic acid to inactivate proteases. Peptide fragments present in the digestion
mix were purified by 5% trichloroacetic acid precipitation for 10 minutes on ice and then
centrifuged at 14,000 rpm for 30 minutes. Degradation samples were stored at -80ºC for
antigenicity assays and MS analyses.
3.5.2. Analysis of the antigenicity of the degradation products
Peptide fragments present in the digestion mix were purified by 5% trichloroacetic acid
precipitation and diluted in RPMI without serum. The pH was re-adjusted to pH7.4 with
NaOH. Chromium-51 (51Cr)-labeled HLA-matched B cells were pulsed with the purified
digestion products (diluted to 0.4μg/mL for 5-ATK9-2 and 0.8μg/mL for 13-QY9-6) for 30
minutes at 37°C in the absence of serum and used as targets in a 4-hour 51Cr release assay
with HLA-matched, epitope-specific CTL clones at a 4:1 effector-to-target ratio. The lysis %
were compared with the lysis of B cells pulsed with undigested long peptides and optimal
epitope titrations (104, 106).
3.5.3. Identification and semi-quantification of degradation products by MS
The identity of the peptides generated during in vitro degradation was determined by tandem
MS. Equal amounts of peptide degradation samples generated at different time points were
injected into a Nano-HPLC (Eksigent) coupled to an Orbitrap MS (LTQ Orbitrap Discovery,
Thermo) at a 400nL/min flow rate. A Nano cHiPLC trap column (200µm x 0.5mm ChromXP
c18-CL 5µm 120Å; Eksigent, USA) was used to remove salts in the sample buffer. Peptides
were separated in a Nano cHiPLC column (75µm x 15cm ChromXP c18-CL 5µm 300Å; Eksigent,
USA) with 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B).
The conditions for the HPLC separation prior to MS analysis can be found in table 3. Mass
spectra were recorded in the range of 370 to 2000Daltons. In tandem MS mode, the eight
most intense ions were selected with a 1Da window for fragmentation in a collision cell using
Helium with 35V as collision voltage. MS fragments were searched against source peptide
databases with Protein Discoverer 1.4 (Thermo Scientific, USA). For semi-quantification the
integrated area under a peak generated by a given peptide is proportional to the abundance
of that peptide. Each degradation time point was run on the MS at least twice.
Materials & Methods 31
Table 3. HPLC gradient used to fractionate degradation products prior to MS analysis.
Time (min) % buffer A % buffer B
0.0 98.0 2.0
0.1 98.0 2.0
5.0 98.0 2.0
100.0 40.0 60.0
100.5 2.0 98.0
107.5 2.0 98.0
108.0 98.0 2.0
125.0 98.0 2.0
125.1 98.0 2.0
3.5.4. Intracellular peptide stability assay
To analyze the intracellular stability of optimal MHC I epitopes, highly purified peptides (>98%
pure) were degraded with 15μg of whole cell extracts at 37°C in 50μL of degradation buffer
(50mM Tris-HCl, 137mM potassium acetate, 1mM magnesium chloride, 1mM ATP; pH7.4 or
pH4.0) (56, 105). Aliquots were taken at 0, 10, 30, and 60 minutes, and the reaction was
stopped with 5% trifluoroacetic acid to inactivate proteases. Peptides incubated in
degradation buffer without cell extracts were used as controls.
3.5.5. Quantification of epitope degradation by reversed-phase HPLC analysis
To quantify the amount of remaining peptide in the digestion mix at each time point, we used
reversed-phase high-performance liquid chromatography (RP-HPLC; Waters, USA) analyses.
RP-HPLC involves the separation of molecules on the basis of their hydrophobicity. The
separation depends on the hydrophobic interaction of molecules from the mobile phase to
the immobilized hydrophobic ligands attached to the stationary phase. The digestion mix is
initially applied to the stationary phase in the presence of aqueous buffers. To elute the
peptides in order of increasing hydrophobicity, increasing amounts of organic solvents over
time are applied.
Peptides were fractionated on a Biosuite 4.6mmx50mm C18 column or a xBridge
4.6mmx150mm C18 column (Waters, USA) in water/0.01% formic acid and
acetonitrile/0.005% formic acid (gradient 1) or water/0.05% trifluoroacetic acid and 50%
acetonitrile/0.04% trifluoroacetic acid (gradient 2). The degradation of the original peptide
resulted in the reduction of its peak and the appearance of additional peaks corresponding to
truncated peptides (Figure 7A). The amount of peptide at each time point was calculated by
the integration of the area under each peptide peak. The identity of the peak corresponding to
the original peptide was determined by comparing its elution time to that of pure peptide in
degradation buffer without cell extracts. The relative amount of each peptide at each time
Materials & Methods 32
point was plotted over time, whereby 100% represents the amount of peptide detected at
time 0 minutes. The time at which 50% of the peptide was degraded defines its half-life
(Figure 7B, left panel). A stability rate of each peptide was calculated by a nonlinear regression
(one-phase exponential decay) of the degradation profile obtained over the 60-minute
incubation (Figure 7B, right panel).
Figure 7. Defining the intracellular stability of peptides by RP-HPLC quantification.
A. The peptides present in the digestion mix at each time point were quantified by RP-HPLC
analysis. Each peptide in the digestion mix corresponded to a distinct peak. The degradation of
the original peptide resulted in the reduction of its peak and the appearance of additional
peaks corresponding to truncated peptides. The amount of each peptide was calculated by the
integration of the area under the peptide peak. The identity of each peak was determined by
comparing its elution time to that of known synthetic peptides.
B. The relative amount of each peptide at each time point was plotted against the time. 100%
represents the amount of peptide detected at time 0 minutes. The time at which 50% of the
peptide was degraded defines its half-life. A stability rate of each peptide was calculated by a
nonlinear regression (one-phase exponential decay) of the degradation profile obtained over
the 60-minute incubation (blue line).
Table 4. HPLC gradient 1 to analyze the degradation products of B57-ISW9, B57-KF11, B57-
TW10, B57-FF9 and A11-ATK9.
Time (min) % buffer A % buffer B
0.00 95.0 5.0
4.00 90.0 10.0
23.00 30.0 70.0
23.50 10.0 90.0
27.00 10.0 90.0
28.00 100.0 0.0
Materials & Methods 33
Table 5. HPLC gradient 2 to analyze the degradation products of A03-RK9 and A03-KK9.
Time (min) % buffer A % buffer B
0.00 100.0 0.0
2.00 100.0 0.0
16.50 10.0 90.0
19.00 0.0 100.0
19.50 95.0 5.0
30.0 95.0 5.0
3.6. Cross-presentation of exogenous HIV proteins by DCs and Møs
3.6.1. ELISPOT cross-presentation assay
To measure HIV-specific CD8+ T cell responses to cross-presenting DCs and Møs we used the
enzyme-linked immunospot (ELISpot) assay. This assay detects antigen-induced secretion of
interferon-gamma (IFN-y) by CD8+ T cells trapped by an immobilized antibody, which is
visualized by an enzyme-coupled secondary antibody.
Immature DCs and Møs were incubated with recombinant HIV-1 p24-protein or HIV-1 p55-
protein (Protein Sciences Corporation, USA) for 1hr at 37ºC. As a control, cells were exposed
to the same concentrations of control protein (Protein Sciences Corporation, USA). Where
indicated, immature DCs or Møs were incubated with inhibitors for proteasome (10µM
MG132; Enzo Life Sciences, USA) or cysteine proteases (5µM E64; Sigma-Aldrich, USA) for 45
minutes at 37ºC before addition of recombinant proteins. Cells pulsed with equivalent molar
concentrations of the optimal epitope HLA-B57 ISW9, HLA-B57 KF11, HLA-B57 TW10, and HLA-
A03 RK9 were used as controls for the antigen presentation and CD8+ T cell clone specificity.
Following incubation with proteins, DCs and Møs were thoroughly washed with ice-cold PBS
and co-cultured overnight with epitope-specific CD8+ T cell clones at a 2:1 effector-to-target
ratio in 96-well plates (Millipore, USA) coated with anti-IFN-y mAb 1-D1K (Mabtech, USA). On
the next day, ELISPOT plates were washed and developed, and the detection of spot forming
cells (SFC) was performed as described previously (107). The number of SFC was calculated by
subtracting the number of spots in the negative-control wells from the number of spots in
each experimental well. Responses were regarded as positive if they had at least three times
the mean number of SFC in the three negative-control wells. The magnitude of the epitope-
specific response was reported as the number of SFC per million cells.
3.6.2. P24 uptake assay
To assess the amount of exogenous p24 protein taken up by immature DCs and Møs we used
an enzyme-linked immunosorbent assay (ELISA) to quantify the amount of intracellular p24
Materials & Methods 34
protein. This assay is based on the binding of an antigen to its specific antibody, which allows
the detection of very small amounts of antigen. Briefly, an immobilized antibody captures the
p24 antigen. The captured antigen is complexed with a biotinylated polyclonal antibody to the
p24 antigen, followed by a streptavidin-horseradish peroxidase (HRP) conjugate. The resulting
complex is detected by incubation with ortho-phenylenediamine-HCl (ODP), which produces a
yellow color that is directly proportional to the amount of HIV-1 p24 captured. The
absorbance of each microplate well is determined using a microplate reader and calibrated
against the absorbance of an HIV-1 p24 antigen standard curve.
Immature DCs or immature Møs (1x106cells/mL) were exposed to the following cocktail of
protease inhibitors for 45 minutes at 37ºC to reduce intracellular degradation of internalized
protein before cell lysis: 10µM MG132 (Enzo Life Sciences, USA), 5µM E64 (Sigma-Aldrich,
USA), 10µM cathepsin S inhibitor Z-FL-COCHO (Calbiochem, USA), 10µM leupeptin (Enzo Life
Sciences, USA), 120µM bestatin (Sigma-Aldrich, USA). Following incubation with inhibitors,
DCs and Møs were exposed to different concentrations of recombinant HIV-1 p24 protein and
incubated for 1hr at 37ºC or 4ºC. Afterwards, all samples were thoroughly washed with ice-
cold PBS and immediately treated with 3mg/mL pronase E (Sigma-Aldrich, USA) in AIM-V
medium without serum for 10 minutes on ice. Cells were washed once in AIM-V medium
supplemented with 10% HSA and three times in ice-cold PBS to eliminate pronase E and then
resuspended in 0.5% Triton X-100 containing lysis buffer. The amount of p24 protein was
determined using a HIV-1 p24 antigen ELISA kit according to the manufacturer’s instructions
(Perkin Elmer, USA). The absorbance of each microplate well was determined using a
microplate reader (Tecan, Switzerland).
3.7. Statistical analysis
Spearman’s rank correlation coefficient was used to examine bivariate associations. The
paired t-test, Wilcoxon signed rank test, and Kruskal-Wallis test were used to compare
measurements between groups. All p values are two-sided and p values of <0.05 were
considered significant. In figures, p-value criteria are assigned as *p<0.05, **p<0.01 and
***p<0.001. Statistical analyses were conducted using GraphPad Prism (GraphPad Prism
Software, La Jolla, CA).
Results 35
4. Results
4.1. Hydrolytic activities of cytosolic proteases involved in antigen
processing in DCs and Møs
We first analyzed the hydrolytic activities of several cytosolic proteases involved in the direct
antigen-processing pathway in immature and mature monocyte-derived dendritic cells (iDCs,
mDCs) and macrophages (iMøs, mMøs). Maturation of cells was achieved by addition of LPS
(TLR4 agonist), CL097 or R848 (TLR7/8 agonists) for two days. Proteasomal catalytic subunits
(caspase-like, tryptic, and chymotryptic) and post-proteasomal activities (aminopeptidases,
TPP II, and TOP) in live, intact DCs and Møs were measured using protease-specific fluorogenic
peptidic substrates, and fluorescence emission upon peptide hydrolysis was monitored over
time as previously done (106, 108, 109).
4.1.1. Proteasomal hydrolytic activities in DCs and Møs change differently upon
maturation with TLR4 or TLR7/8 ligands
In immature DCs and Møs we detected comparable proteasome caspase-like, tryptic and
chymotryptic activities. Maturation of cells for 2 days with LPS or R848, resulted in significant
differences in proteasomal hydrolytic activities between mMøs and mDCs (1.8-, 2.1-, and 1.5-
fold higher in LPS-matured Møs than DCs, and 1.5-, 1.8-, and 1.3-fold higher in R848-matured
Møs, respectively) (Figure 8A). Similarly, in CL097-matured Møs significant higher proteasomal
hydrolytic activities were observed compared to CL097-matured DCs (1.2-, 2.0-, and 2.1-fold
higher in CL097-matured Møs than DCs) (Figure 8B).
To compare how each TLR agonist altered cytosolic antigen processing activities in DCs or Møs,
we calculated a ratio of activities in mature cells over their immature counterpart (data not
shown). Both TLR4 and TLR7/8 stimulations increased proteasomal caspase-like, tryptic, and
chymotryptic hydrolytic activities in Møs by 20% to 30%. In contrast, matured DCs showed
decreased proteasomal activities upon maturation with a 30% lower caspase-like activity, and
a 40% to 50% lower tryptic activity in LPS- and R848-matured DCs. We then plotted the
hydrolytic activity of each subunit in immature and mature DCs against the % of CD86+ CD83+
DCs. The expression of both markers is upregulated upon maturation of DCs with various TLR
ligands. Caspase-like, tryptic, and chymotryptic activities inversely correlated with the
expression of the maturation markers CD83+ and CD86+ after LPS-, R848- or CL097-induced
maturation (rs=-0.4191, p=0.042; rs=-0.4200, p=0.029; and rs=-0.6018, p=0.002, respectively),
Results 36
which demonstrated decreased proteasomal activities upon maturation of DCs (Figure 8C).
Together, these results indicated that TLR-induced maturation triggered significant changes in
all three proteasomal hydrolytic activities of DCs and Møs in divergent ways.
Figure 8. Proteasomal activities in monocyte-derived DCs and Møs change differently upon
maturation with TLR4 or TLR7/8 ligands.
A. Proteasomal caspase-like, tryptic, and chymotryptic activities were measured with
peptidase-specific fluorogenic substrates in live, intact iDC (), iMø (), mDC LPS (), mMø
LPS (), mDC R848 () and mMø R848 (). Results are from n>10 healthy donors.
B. The same activities as in (A) were measured in iDC (), iMø (), mDC LPS (), mMø LPS (),
mDC CL097 (), and mMø CL097 (). n>10 healthy donors.
C. Proteasomal activities in iDC and mDCs were plotted versus the percentage of CD86+ CD83+
DCs for each experiment. Surface expression was analyzed by flow cytometry. Comparison by
Spearman test is indicated. n>23 measurements.
4.1.2. Hydrolytic activities of cytosolic aminopeptidases and TPP II are comparable
in DCs and Møs, whereas TOP activities are significantly higher in Møs
Proteasomal degradation products can be further trimmed by cytosolic peptidases before
translocation into the ER for loading onto MHC I or they can be degraded into fragments that
Results 37
are too short to enter the ER (33). To assess the hydrolytic activity of cytosolic
aminopeptidases, thimet oligopeptidase (TOP), and tripeptidyl peptidase II (TPP II) we used
protease-specific fluorogenic peptide substrates (Figure 9). We detected comparable
aminopeptidases hydrolytic activities between immature and mature DCs and Møs, whereas
TOP activities were approximately 2-fold higher in immature, LPS- and R848-matured Møs
compared to their DCs counterparts (Figure 9A). Similar findings were observed in CL097-
matured Møs (Figure 9B). Hydrolytic activities of TPP II were comparable between immature,
LPS- and CL097-matured DCs and Møs (Figure 9B). In contrast to proteasomal activities, we
did not detect any changes in amínopeptidases, TOP, and TPP II activities between the
immature and mature state of each cell subset.
These results indicated that TLR-induced maturation did not result in differences in post-
proteasomal hydrolytic activities between DCs and Møs.
Figure 9. DCs and Møs have comparable aminopeptidases and TPP II activities, whereas TOP
activities are higher in Møs.
A. Post-proteasomal activities for aminopeptidases and TOP were measured in iDC (), iMø
(), mDC LPS (), mMø LPS (), mDC R848 () and mMø R848 ().
B. Aminopeptidases, TOP and TPP II activities were measured in iDC (), iMø (), mDC LPS (),
mMø LPS (), mDC CL097 (), and mMø CL097 (). For (A-B) results are from n>7 healthy
donors.
Results 38
4.1.3. TLR-mediated maturation of DCs and Møs results in early changes of
proteasomal activities and reflect changes detected upon HIV infection
To assess how TLR stimulation changes protease activities in maturing DCs, we measured
proteasome and aminopeptidases activities at 5h, 24h, and 48h post stimulation with LPS or
CL097. A ratio of activities in maturing cells over their immature counterpart at each time
point was calculated (Figure 10). In line with our previous results, LPS- and CL097-maturing
DCs showed 30% decreased proteasomal caspase-like and tryptic activities as early as 24h
post stimulation (Figure 10A). Further maturation with both TLR ligands decreased caspase-
like activities by 40% and tryptic activities up to 50% compared to immature cells.
Chymotryptic activities were increased as early as 5h upon maturation with LPS but were not
affected by CL097 stimulation at any time point (Figure 10A).
We next analyzed whether HIV-1 infection of DCs and Møs affected the antigen processing
activities. Single-round infection with VSVg-pseudotyped lentivirus expressing HIV-1 NL4.3
Env resulted in infection rates of 14.4% and 23.7% of DCs and Møs 6 days post infection, as
measured by the expression of GFP (Supplemental Figure 2). Addition of SIV viral protein X
(Vpx) increased the infection rate of Møs (34.7% vs. 23.7% infected Møs in the presence or
absence of Vpx), whereas no effect was observed in DCs (Supplemental Figure 2). Previous
studies demonstrated increased infection rates of human monocyte-derived DCs after
incubation with Vpx, which targets the restriction factor SAMHD1 for degradation by
proteasomes (110). However, differences in the concentration of Vpx used in this study and
the incubation time of Vpx with cells prior to the addition of the virus may explain the
different infection rates observed.
In line with results from TLR-matured cells, we detected 20% lower caspase-like activity, and
30% lower tryptic activities in infected DCs (Figure 10B). Chymotryptic activities in DCs did not
change during the first three days of infection and showed a 30% increase after 6 days post
infection. In Møs, we detected 10-20% increased proteasomal caspase-like, tryptic, and
chymotryptic hydrolytic activities upon infection (data not shown). Aminopeptidases activities
in DCs and Møs did not change upon maturation with LPS or CL097 or HIV-1 infection (Figure
10C and data not shown).
These results indicated that TLR-induced maturation affected proteasomal activities within
24h post stimulation and resulted in significant differences between DCs and Møs, which
reflected changes seen upon HIV-1 infection.
Results 39
Figure 10. Changes in antigen processing activities in maturing monocyte-derived DCs reflect
changes observed upon HIV infection.
A. Ratios of proteasomal activities in TLR-maturing DCs over immature cells of the same donor
were calculated and represented as mean ± SD at the indicated time points post stimulation.
B. Immature DCs were infected with VSVg-ΔEnv NL4.3 in the presence or absence of Vpx for the
indicated time points. At each time point proteasomal caspase-like, tryptic and chymotryptic
activities were measured using protease-specific fluorogenic substrates. Ratios of activities in
infected cells over non-infected cells from the same donor were calculated and represented as
mean ± SD.
C. Ratios of aminopeptidases activities in TLR-maturing DCs (left panel) or infected DCs (right
panel) over immature or non-infected cells of the same donor were calculated and represented
as mean ± SD at the indicated time points post stimulation or infection. For (A-C) results are
from n=2 healthy donors.
Results 40
4.2. Expression of cytosolic and ER-resident proteases involved in
antigen processing in DCs and Møs
To examine whether the elevated hydrolytic activities observed in mature Møs compared to
mature DCs were due to different expression levels of peptidases, we analyzed cytosolic
extracts by dual infrared fluorophore Western blotting for the expression of structural and
catalytically active proteasomal subunits, cytosolic post-proteasomal peptidases, the
proteasome activator, the ER-resident aminopeptidases ERAP1 and ERAP2 and the chaperone
Hsp90. This technique allows for multiplex detection of signals over a wider quantifiable linear
range than chemiluminescence (111) (Figure 11).
Figure 11. Higher expression levels of proteins involved in antigen processing in Møs
compared to DCs.
Cell extracts from immature, LPS-matured, or CL097-matured DCs and Møs were probed in
Western blots for the expression of chaperone Hsp90, constitutive proteasome catalytic
subunits β1, β2, and β5, proteasomal lid S1, ER-resident aminopeptidase ERAP1, proteasome
regulator PA28α, and proteasomal core α7 using actin as a loading control.
4.2.1. Higher expression levels of the catalytical and structural subunits of the
constitutive and immunoproteasome in Møs compared with DCs
The expression levels of the constitutive proteasomal subunits responsible for caspase-like
(β1), tryptic (β2), and chymotryptic activities (β5) were 2.4-fold higher in immature Møs
compared to immature DCs (Figure 12A). Similar differences were detected between LPS-,
CL097-, and R848-matured Møs and DCs (2.3-, 2.8-, and 2.4-fold in LPS-matured Møs, and 1.8-,
2.5-, and 2.4-fold in CL097-matured Møs, and 2.4-, 3.6-, and 2.0-fold in R848-matured Møs,
respectively). In immature and LPS-matured Møs, approximately 2-fold and 1.6-fold higher
expression levels of immunoproteasome catalytic subunits β1i and β5i were detected (Figure
12B), whereas the expression levels of the proteasome activator subunit PA28α were
comparable between immature and mature DCs and Møs. The lack of a sensitive anti-β2i
Results 41
antibody prevented us from analyzing the tryptic immunoproteasome subunit. Similar to the
constitutive proteasomal subunits, the expression levels of proteasomal core α7 were
significantly higher in immature and mature Møs compared to DCs, whereas the expression of
the lid S1 tended to be higher in Møs compared to DCs, but these differences were not always
significant (Figure 12C).
These results demonstrated that, regardless of cell maturation states and variability among
donors, the higher expression of the catalytic subunits of proteasomes in Møs likely
contributed to the elevated hydrolytic activities observed in Møs compared to DCs.
Figure 12. Higher expression levels of catalytically active and structural proteasome subunits
in Møs compared to DCs.
Densitometric quantification normalized to actin was performed for the expression of (A)
constitutive proteasomal catalytic subunits β1, β2, and β5; (B) the proteasomal activator
PA28α, and for immunoproteasome catalytic subunit β1i and β5i; (C) proteasomal lid S1 and
proteasomal core α7. Paired t-tests were performed between matched iDC (), iMø (), mDC
LPS (), mMø LPS (), mDC CL097 (), mMø CL097 (), mDC R848 () and mMø R848 ().
Maturation was induced for 48h with the indicated TLR ligands. Mean ± SD is shown.
Results 42
4.2.2. Møs have higher expression levels of cytosolic TOP and ER-resident ERAP1
and ERAP2 compared to DCs
Analysis of the expression levels of post-proteasomal TPP II and leucine aminopeptidases
(LAP) showed comparable expression levels between immature and mature DCs and Møs.
Higher TOP expression levels were detected in immature and mature Møs compared to DCs,
without reaching statistical significance (1.9-fold in immature Møs, 2.0-fold in LPS-matured
Møs, and 1.7-fold in R848-matured Møs) (Figure 13A). The expression levels of the chaperone
Hsp90 were similar between DCs and Møs in the immature and mature state. The expression
of the ER-resident aminopeptidases ERAP1 and ERAP2 was higher in Møs compared to DCs,
specifically for immature and LPS-matured cells (1.3- and 1.5-fold in immature Møs; 1.6- and
2.2-fold in LPS-matured mMøs for ERAP1 and ERAP2, respectively) (Figure 13B).
These results indicated that higher TOP expression levels detected in immature and mature
Møs might contribute to the higher TOP activities measured in Møs compared to DCs.
Figure 13. Comparable expression levels of cytosolic post-proteasomal activities in DCs and
Møs.
Densitometric quantification normalized to actin was performed for the expression of (A) post-
proteasomal cytosolic proteases TOP, TPP II, and LAP and (B) cytosolic chaperone Hsp90, and
ER-resident aminopeptidases, ERAP1 and ERAP2. Paired t-tests were performed between
matched iDC (), iMø (), mDC LPS (), mMø LPS (), mDC CL097 (), mMø CL097 (), mDC
R848 () and mMø R848 (). Maturation was induced for 48h with the indicated TLR ligands.
Mean ± SD is shown.
Results 43
4.3. HIV-1 epitope processing by cytosolic peptidases in DCs and Møs
We next aimed to determine whether higher expression levels and proteolytic activities of the
antigen processing machinery in Møs compared to DCs led to differences in the processing of
HIV-derived epitopes. We previously developed an in vitro degradation assay to analyze
epitope processing in cytosolic extracts (79, 106). This assay uses cell extracts as a source of
cytosolic proteases to degrade synthetic HIV-1 peptides. Peptides generated during the
degradation were identified by MS and their antigenicity was analyzed using a 51Cr release
assay. This assay allowed us to compare the degradation of peptides by cytosolic proteases
from DCs and Møs in order to analyze the production of multiple epitopes located within a
peptide independent of the HLA of the donor.
4.3.1. Proteasomal activities in cell extracts of DCs and Møs reflect those measured
in intact, live cells
A prerequisite to using this assay was to confirm that the differences in peptidase activities
measured in live, intact DCs and Møs were replicable in cytosolic extracts used for the epitope
processing assay. To measure proteasomal caspase-like, tryptic, and chymotryptic activities in
cell extracts from DCs and Møs we used equal amounts of extracts normalized to actin levels
(Figure 14). In line with our previous results, we observed in cytosolic cell extracts from Møs
matured with TLR4 and TLR7/8 ligands significant higher proteasomal activities compared to
cell extracts from mature DCs. In cell extracts from LPS-matured Møs we detected 5.4-fold,
6.5-fold, and 2.8-fold higher caspase-like, tryptic, and chymotryptic activities, respectively,
compared to cell extracts from LPS-matured DCs. Similar trends were observed in cytosolic
extracts from R848- and CL097-matured DCs and Møs (Figure 14A-14B). Differences in values
of peptidase activities between intact, live cells and cell extracts may be due to variations in
uptake of substrates between DCs and Møs, differences in ratios between peptidase and
substrate between extracts and intracellular volumes of live cells, and the fact that
proteasome activities measured in live cells include both cytosolic and nuclear proteasomes.
Despite differences in values, all three proteasomal activities measured in cells correlated
significantly with their activities measured in cytosolic cell extracts (rs=0.32, p=0.002, caspase-
like; rs=0.30, p=0.010, tryptic; rs=0.23, p=0.025, chymotryptic; Figure 14C).
Together these results demonstrated that alterations of peptidase activities induced by TLR
ligands and infection occurred similarly in live cells and cell extracts and therefore validated
the use of cytosolic extracts to compare epitope processing in DCs and Møs using our in vitro
degradation assay.
Results 44
Figure 14. Changes in proteasomal activities in cell extracts reflect those observed in live,
intact DCs and Møs.
A. Proteasomal caspase-like, tryptic, and chymotryptic activities were measured in cytosolic
extracts from iDC (), iMø (), mDC LPS (), mMø LPS (), mDC R848 () and mMø R848 ()
using protease-specific fluorogenic substrates. Results are from n>16 healthy donors.
B. Proteasomal caspase-like, tryptic, and chymotryptic activities were measured as described in
(A) including cytosolic extracts from mDC CL097 (), and mMø CL097 (). Results are from
n>13 healthy donors and show mean ± SD. Paired t-tests and Wilcoxon signed rank test were
performed (*p<0.05, **p<0.01, ***p<0.001).
C. The proteasomal hydrolytic activities measured in live, intact immature or mature DCs ()
and Møs () were plotted against their activities in corresponding cell extracts. A partial
correlation on Spearman ranked data was performed to control for cell type-dependent effects.
Results are from n>73 measurements.
4.3.2. The kinetics and amounts of optimal HIV-1 epitopes produced in cytosolic
extracts of DCs and Møs vary among epitopes
We first used cytosolic extracts from DCs and Møs to degrade a synthetic 16-mer peptide
representing HIV-1 reverse transcriptase (RT), 5-ATK9-2 (WKGSPAIFQSSMTKIL, aa 153-168),
which contains the subdominant HLA-A03/A11-restricted ATK9 epitope (AIFQSSMTK, aa 158-
166) (112). At different time points during the degradation, generated peptides were
Results 45
identified by MS (79, 106, 109). The degradation of 5-ATK9-2 led to the production of various
fragments representing peptides containing ATK9 with extensions at the N- and C-terminus,
peptides with N-terminal extensions that could be further trimmed into optimal ATK9, the
optimal ATK9 epitope, and also fragments lacking intact ATK9, called antitopes (Figure 15). Mø
cell extracts produced a large variety of fragments containing ATK9 with N-terminal extensions
at each degradation time point and optimal epitope ATK9 was detectable within 3 minutes,
whereas it appeared after 10 minutes in DC cell extracts. Similar results were observed with
cell extracts of immature and mature DCs and Møs (Supplemental Figure 3).
Figure 15. Mø cell extracts produce HLA-A11-ATK9 epitope more efficiently than DCs.
Four nmol of 5-ATK9-2 (aa 153-168 in HIV-1 RT) were degraded with 30µg of cytosolic extracts
from CL097-matured DCs (A) or Møs (B) for 3, 10, 30, 60 and 120 minutes. Peptides containing
epitope HLA-A11-restricted ATK9 (black bars), or no epitope, called antitopes (white bars) were
identified by MS. Optimal epitope ATK9 () is indicated. The data are representative of three
independent experiments from different donors.
Results 46
To assess and compare the production of peptides in both cell subsets over time, we
calculated for each time point the contribution of each category of peptides (epitope,
antitopes, extended epitopes, original fragment 5-ATK9-2) to the total degradation products
(Figure 16). Each peptide is defined by a peak at a specific retention time and an area under
the peak, which we previously showed to be proportional to the amount of the corresponding
peptide (94, 113). In cell extracts of Møs matured with CL097, optimal ATK9 and fragments
containing N-extended ATK9 represented the major products detected as early as 3 minutes
and throughout the degradation reaction, whereas in cell extracts of DCs similarly matured,
longer peptides were produced in accordance with lower hydrolytic activities. Moreover,
regardless of the maturation state of the cells, Mø cell extracts always produced more N-
extended ATK9-containing fragments and optimal ATK9 than DC cell extracts.
Figure 16. Mø cell extracts produce more A11-ATK9 epitope and epitope precursors
compared to DC cell extracts.
All degradation products of 5-ATK9-2 generated in cell extracts from CL097-matured DC and
Mø were identified by MS at each time point. The peak area of each identified peptide was
determined using Proteome Discoverer 1.4. Peptides were grouped into antitopes that are
lacking intact ATK9 (blue), optimal ATK9 epitope (black), fragments containing N-terminal
(white), C-terminal (red) or N- and C-terminal extensions (green) of ATK9, and the original
fragment (gray). The contribution of each category of peptides to the total intensity of all
degradation products is shown at each time point.
The area of the peak corresponding to ATK9 detected by MS increased over the course of 210
minutes of degradation in Mø cell extracts to up to 11.4-fold higher than during the
degradation in DC cell extracts (Figure 17A). To independently confirm the difference in
production of ATK9 epitope between DCs and Møs, we measured the antigenicity of the
degradation peptides produced over 210 minutes as previously done (79, 106, 109).
Degradation peptides were purified and pulsed onto an HLA-A11 B cell line used as target cells
in a killing assay with ATK9-specific CTLs. Degradation peptides generated in Møs became
increasingly antigenic over the course of the experiment, with lysis % plateauing around 120
Results 47
minutes at 38% lysis for CL097-matured Mø digests, whereas it reached only 3% lysis for
CL097-matured DC digests – a 12.6-fold difference in antigenicity of the degradation products
(Figure 17B). The antigenicity of the degradation products measured by CTL killing assay
correlated strongly with the production of optimal ATK9 epitope detected by MS (rS=0.6068,
p<0.0001), providing additional validation of the semi-quantification of epitope production by
MS (Figure 17C).
These results showed that the processing of ATK9 epitope from HIV-1 RT-derived precursor
peptide, 5-ATK9-2, was faster and yielded more antigenic peptides in Mø than in DC cytosolic
extracts independent of the maturation state.
Figure 17. Higher amounts of HLA-A11 ATK9 produced by Mø cell extracts resulted in higher
lysis of target cells by ATK9-specific CTLs.
A. Kinetics of optimal ATK9 production by iDC (), iMø (), mDC LPS (), mMø LPS (), mDC
CL097 (), and mMø CL097 () detected by MS.
B. 5-ATK9-2 degradation products were purified and pulsed onto HLA-A03/A11 B cell targets.
ATK9-specific CTL responses were assessed by 51Cr release assay. For (A-B), data are
representative of three independent experiments with extracts from different healthy donors.
C. ATK9 peak areas in DCs () or Møs () detected by MS in three independent experiments
were plotted against the target cell lysis values generated by the corresponding degradation
peptides. Spearman correlation test was performed; n=106 data points. For (A-C) the 30
minutes degradation time points for iMø were lost.
Results 48
To analyze whether differences in the processing of HLA-A11 ATK9 in cytosolic extracts of DCs
and Møs reflected differences seen upon HIV-1 infection of both cell subsets, we infected
immature DCs and Møs with VSVg-ΔEnv NL4.3 alone or in combination with Vpx and analyzed
the activation of HLA-A11 ATK9-specific CTLs by IFN-gamma ELISPOT assay (data not shown).
Due to low infectivity of DCs and Møs as shown before and insufficient amount of cells to sort
infected cells we were not able to directly compare the endogenous processing and
presentation of HLA-A11 ATK9 by DCs and Møs to A11-ATK9-specific CTL clones.
We next analyzed the processing of a 28-mer Nef-derived precursor peptide 13-QY9-6
(AQEEEEVGFPVTPQVPLRPMTYKAAVDL, aa 60-87 in Nef) which contains HLA-B35-restricted
QY9 and 16 other epitopes (114). Cell extracts from Møs and DCs similarly degraded 13-QY9-6
at a slow rate with 85% of the total peak intensity contributed by peptides longer than 26
amino acids after 10 minutes and approximately 40% after 210 minutes (Figure 18A). In
contrast to A11-ATK9, HLA-B35-restricted QY9 epitope (QVPLRPMTY, aa 73-81) was efficiently
generated by both cell subsets (Figure 18B). There was little difference in the antigenicity of
peptides generated in cell extracts from DCs and Møs (26% and 22% lysis for CL097-matured
DCs and Møs, respectively) (Figure 18C), and no significant differences were observed among
TLR4, 7 and 8 stimulated cells (data not shown). The antigenicity of the degradation products
correlated strongly with the detection of optimal QY9 epitope by MS, again validating the use
of MS to semi-quantify epitope production (rS=0.8329, p<0.0001; results from two
independent experiments) (Figure 18D).
Moreover, we analyzed the production of the 16 well described HIV-1 CD8+ T cell epitopes and
epitope precursors (defined as peptides with the correct C-terminus and extended by up to
three residues at the N-terminus that could be further trimmed in the ER) located in this 28-
mer. Three epitopes (19%) were produced in DC and Mø cell extract at each time point during
degradation, whereas three epitopes (19%) were only produced at later time points during
degradation. Furthermore, seven epitopes (44%) were not produced in either DC or Mø cell
extracts at any time point, while three epitopes (19%) were only produced in cell extracts
from Møs (data not shown).
Thus due to heterogeneity in peptidase activities among DCs and Møs, peptides presented by
HIV-infectable subsets may differ in their timing of presentation, amount and length (optimal
or extended epitopes), all of which could affect recognition by HIV-specific CD8+ T cells.
Results 49
Figure 18. Extracts from DCs and Møs generate similar amounts of Nef-derived antigenic
peptides at similar rates.
A. Two nmol of 13-QY9-6 (aa 60-87 in Nef) were degraded with 45µg of cytosolic extracts from
mDC CL097 and mMø CL097 for 10, 120, and 210 minutes. Degradation products identified by
MS were grouped according to their lengths of fragments: longer than 26 aa (blue), 19-25 aa
(orange), 13-18 aa (gray), 8-12 aa (red) and fragments equal or shorter than 7 aa (light gray).
The contribution of each category of peptides to the total intensity of all degradation products
is shown at each time point.
B. QY9 epitope production in extracts from mDC CL097 () and mMø CL097 () detected by
MS at the different time points.
C. The 13-QY9-6 degradation products were purified and pulsed onto HLA-B35 B cell targets.
QY9-specific CTL responses were assessed by a 51Cr release assay. For (A-C), data are
representative of three independent experiments with different healthy donors.
D. MS peak areas of peptide QY9 produced in extracts from mDC CL097 () and mMø CL097
() from two independent experiments were plotted against their respective target cell lysis
values from the 51Cr release assay. Spearman correlation test was performed; n=27 data points.
Results 50
4.4. Processing of HIV-1 peptides by cytosolic and endo-lysosomal
peptidases in DCs and Møs
To assess how degradation of HIV-1 peptides along the cross-presentation pathway of
immature and mature DCs and Møs affected the degradation patterns, we used the previously
described degradation assay recapitulating degradation in cell compartments involved in
cross-presentation (105). This assay allowed the simultaneous analysis of degradation
products by cytosolic, endosomal and lysosomal peptidases from the same cells using MS.
4.4.1. Lysosomal activities in live intact DCs and Møs correlate with activities
measured in cell extracts
To confirm that hydrolytic activities of endo-lysosomal proteases in cell extracts used for
degradation of HIV-1 peptides reflected activities in live, intact DCs and Møs, we first analyzed
the hydrolytic activities of cathepsin S and omni cathepsins, which measures the combined
activities of cathepsins B, L, and S in live, intact DCs and Møs. In line with previous studies we
detected lower omni cathepsins and cathepsin S activities in live, intact DCs compared with
Møs (49). Similar to proteasomal activities in mature DCs, we demonstrated that hydrolytic
activities of cathepsin S and omni cathepsins decreased in DCs upon maturation as shown by
an inverse correlation between both activities and the % of CD86+ and CD83+ DCs (Figure 19A).
We then analyzed the same activities in cell extracts from DCs and Møs at pH4.0 to specifically
activate cathepsins. In line with our previous data using cytosolic extracts, we found that
cathepsin S and omni cathepsins activities in live, intact cells significantly correlated to their
counterparts in corresponding cell extracts, which validated the use of cell extracts to analyze
the degradation of HIV-1 peptides by endo-lysosomal proteases in vitro (Figure 19B).
Maturation of phagosomes to endosomes and lysosomes correlates with a drop in the pH and
the sequential activation of different cathepsins (53). To confirm that different cathepsins
activities can be activated in vitro at different pH values, we measured the proteolytic activities
of omni cathepsins, cathepsin S, cathepsin B, and cathepsin D in cell extracts from immature
DCs at pH4.0, pH5.5, and pH7.4, respectively (Figure 19C). All analyzed cathepsins showed
higher hydrolytic activities at acidic pH values. However, whereas cathepsin D was more active
at pH4.0, cathepsin B showed higher activity at pH5.5, demonstrating different activation of
cathepsins during maturation of mild acidic early endosomes to strong acidic lysosomes.
Similar results were observed using cell extracts from Møs (data not shown).
Together these results demonstrated that different pH values activated cathepsins from
endosomes and lysosomes, which allowed us to analyze the processing of HIV-1 peptides by
endo-lysosomal proteases in vitro.
Results 51
Figure 19. Lysosomal activities in DC and Mø cell extracts reflect activities in live, intact cells
and can be activated at different pH values.
A. Omni cathepsins and cathepsin S activities in immature and TLR-matured DCs were plotted
against the % of CD86+ CD83+ DCs for each experiment. Surface expression was analyzed by
flow cytometry. Comparison by Spearman test is indicated. n≥26 measurements.
B. Omni cathepsins and cathepsin S hydrolytic activities measured in live, intact immature or
mature DCs () or Møs () were plotted against their activities in corresponding cell extracts
at pH4.0. A partial correlation on Spearman ranked data was performed to control for cell type-
dependent effects. Results are from n≥30 measurements.
C. Omni cathepsins activities (combined cathepsin B, L, S activities), cathepsin S, cathepsin B,
and cathepsin D were measured with specific fluorogenic substrates in whole cell extracts of
immature DCs at pH4.0, pH5.5, and pH7.4, respectively. n≥5 independent donors.
Results 52
4.4.2. Preferential production of immunodominant epitopes KF11 and TW10 in
cytosolic and endo-lysosomal cell extracts
We first analyzed the degradation of a synthetic 35-mer peptide containing the HLA-B57-
restricted subdominant epitope ISW9 and the immunodominant epitope KF11
(MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa 10-44 in Gag p24) (104-106) by cytosolic
(pH7.4) and endosomal and lysosomal (pH5.5 and pH4.0) proteases. Degradation of this
fragment resulted in the production of peptides of variable lengths at the different pH values
over time indicating the degradation by different proteases with different cleavage
preferences (Supplemental Figure 4). To assess and compare the production of peptides in
each cell subset and cell compartment, we used the peak area under each of the MS identified
peptides. Peptides were grouped according to their lengths and the contribution of each
category of peptides to the total degradation products was calculated for each time point.
Degradation at pH4.0 in cell extracts from immature DCs yielded shorter fragments compared
with degradation at pH7.4, with majority of fragments being 8-12 and 13-18 aa long and
contributing to 45% and 51% of total peptide intensity at 120 minutes, respectively. At pH7.4,
fragments of 8-12 and 13-18 aa length contributed to only 26% and 37% of the total peptide
intensity, whereas fragments of 19-25 aa length represented 31% of total peptide intensity at
120 minutes (Figure 20A, upper left panel). Then we grouped all degradation products
according to their epitope content and showed that the degradation of fragments containing
both epitopes (ISW9+/KF11+) resulted in the preferential production of B57-KF11 epitope-
containing fragments (ISW9-/KF11+), and only small amounts of B57-ISW9 epitope-containing
fragments (ISW9+/KF11-) in extracts of immature DCs at all pH values tested (Figure 20B, upper
left panel). KF11- and ISW9-containing fragments were produced more efficiently at pH7.4
than at pH5.5 and pH4.0, suggesting that both epitopes may most likely be presented in the
direct presentation pathway or if epitope precursors may escape from endo-lysosomes into
the cytosol quickly after internalization. Similar results were observed for immature Møs
(Figure 20A/B, lower left panel), in line with comparable endocytic hydrolytic activities in cell
extracts from immature monocyte-derived DCs and Møs (115). We then analyzed the
degradation of the 35-mer in extracts from LPS-matured DCs and Møs and detected similar
degradation patterns with fragments of comparable lengths and higher amounts of fragments
containing immunodominant epitope B57-KF11 (Figure 20A/B, right panel).
To analyze the cleavage patterns by proteases active in different cell compartments involved
in cross-presentation, we grouped degradation products identified by MS according to their N-
terminus or C-terminus and determined the contribution of each group of peptides to the
total intensity of all degradation products (Figure 21, upper or lower graph of each panel).
Results 53
Figure 20. The immunodominant epitope B57-KF11 is more efficiently produced in cross-
presentation-competent compartments than subdominant epitope B57-ISW9.
A. Two nmol of p24-35mer (MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa 10-44 in Gag
p24) were degraded in 15µg of whole cell extracts from immature and mature DCs and Møs for
10, 30, 60, and 120 minutes in degradation buffer at pH7.4, pH5.5 or pH4.0. Degradation
products identified by MS were grouped according to their lengths of fragments: equal or
longer than 26 aa (blue), 19-25 aa (orange), 13-18 aa (gray), 8-12 aa (red), and fragments
equal or shorter than 7 aa (light gray). The peak area of each identified peptide was
determined using Proteome Discoverer 1.4 and the contribution of each category of peptides to
the total intensity of all degradation products is shown at each time point.
B. All degradation products of p24-35mer were identified as described in (A). Peptides were
grouped into fragments containing B57-ISW9 and B57-KF11 epitopes (black), containing only
B57-KF11 epitope (red), containing only B57-ISW9 epitope (blue), or neither epitope (gray). For
(A-B) data are representative of three independent experiments with three different donors.
After 30 minutes of degradation at pH7.4 several minor cleavage sites produced ISW9- and
KF11-containing fragments, whereas at pH5.5 and pH4.0 major cleavage sites within ISW9 and
KF11 destroyed the epitopes. Further N- and C-terminal trimming resulted in the appearance
of new cleavage sites, which still preserved KF11-containing peptides at pH7.4, whereas at
pH5.5 and pH4.0 both epitopes were further destroyed.
Together these data indicated that this p24 35-mer was sensitive to degradation by proteases
active in all three cell compartments involved in cross-presentation in DCs and Møs and
favored the production of dominant epitope B57-KF11 over that of B57-ISW9.
Results 54
Figure 21. Major cleavage sites within p24-35mer destroy the B57-ISW9 epitope.
Cleavage patterns of p24-35mer incubated with whole cell extracts from immature DCs for 30
minutes (upper panel) or 120 minutes (lower panel) at pH7.4, pH5.5, and pH4.0 are shown as
the contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a
specific amino acid, to the total intensity of all degradation products. Data are representative
of three independent experiments with three different donors.
Moreover, we analyzed the production of 16 well described HIV-1 CD8+ and CD4+ T cell
epitopes and epitope precursors located in this 35-mer (116). Epitope precursors, defined as
peptides with the correct C-terminus and extended by up to three residues at the N- terminus
that could be further trimmed in the ER (Supplemental Figure 5). Peptides were produced in
extracts of both cell subsets at all pH values tested (B57-KF11, B15-HL9), preferentially
produced at pH7.4 (B57-ISW9, A02-TV9), or at pH5.5 and pH4.0 (B45-VI11, B15-VF9, B44-EV9)
or not produced at any time (B07-SV9). These results highlighted a variable production or
degradation of epitopes at different rates in different cell compartments which might affect
their capacity to activate CD8+ or CD4+ T cells during infection or cross-presentation.
We next extended the analysis to another HIV-1 Gag p24-derived peptide containing the HLA-
B57-restricted epitope TW10 (GSDIAGTTSTLQEQIGWMTNNPPIPVGGEIY, aa 101-131 in Gag
p24) (Figure 22). In contrast to p24-35-mer, the majority of degradation products identified
after 10 to 120 minutes in extracts from immature DCs and Møs at pH7.4, pH5.5 and pH4.0
represented the original fragment or long fragments of mostly >26aa (Figure 22A), containing
the TW10 epitope with N- and C-terminal extensions (Figure 22B). Similarly, degradation in
Results 55
extracts from mature DCs and Møs showed comparable kinetics of degradation and resulted
in the production of fragments with similar lengths.
Figure 22. Slow degradation of TW10-containing peptides results in high amounts of B57-
TW10 available for direct or cross-presentation.
A. Two nmol of p24-31mer (GSDIAGTTSTLQEQIGWMTNNPPIPVGGEIY, aa 101-131 in Gag p24)
were degraded in 15µg of whole cell extracts from immature and mature DCs and Møs for 10,
30, 60, and 120 minutes in degradation buffer at pH7.4, pH5.5 or pH4.0. Degradation products
identified by MS were grouped according to their lengths of fragments as described before.
B. All degradation products of p24-31mer were grouped into fragments containing B57-TW10
(black), B57-TW10 epitope with N-terminal extensions (white), B57-TW10 epitope with C-
terminal extensions (red), B57-TW10 epitope with N- and C-terminal extensions (green),
antitopes defined as fragments lacking B57-TW10 (blue), or the original peptide (gray),
respectively. For (A-B) the contribution of each category of peptides to the total intensity of all
degradation products is shown at each time point.
In line with the slow degradation of this fragment, the few cleavage sites identified after 30 or
120 minutes were mostly located outside B57-TW10 at all three pH values (Supplemental
Figure 6). The relative resistance of the B57-TW10-containing fragment to intracellular
degradation contrasted with the rapid degradation of the p24 fragment containing B57-ISW9
and B57-KF11 which may contribute to a higher amount of TW10-containing peptide available
for presentation by direct or cross-presentation, thus contributing to the dominance of TW10-
specific CTL responses during acute HIV-1 infection.
Results 56
4.5. Cross-presentation of HIV-1 proteins by DCs and Møs
We next analyzed whether differences in the processing of HIV-1 peptides observed in cell
extracts from DCs and Møs at pH4.0, pH5.5 and pH7.4 reflected differences in the actual cross-
presentation of the epitopes by live, intact DCs and Møs. We therefore incubated cells with
exogenous recombinant HIV-1 proteins and quantified epitope-specific CTLs responses to
cross-presenting DCs and Møs by IFN-gamma ELISPOT assay.
4.5.1. Cross-presentation of the immunodominant Gag p24 B57-TW10 and B57-KF11
epitopes is more efficient than that of subdominant B57-ISW9 epitope
We first analyzed the cross-presentation of the three optimally defined HLA-B57 restricted HIV
epitopes originating from HIV-1 p24 protein by immature monocyte-derived DCs and Møs:
subdominant HLA-B57-restricted ISW9 (ISPRTLNAW, aa 15-23 in Gag p24), dominant HLA-B57-
restricted KF11 (KAFSPEVIPMF, aa 30-40 in Gag p24), and dominant HLA-B57-restricted TW10
(TSTLQEQIGW, aa 108-117 in Gag p24) (116, 117).
We detected 28-fold and 94-fold lower B57-ISW9-specific CTL responses to cross-presenting
DCs compared with B57-KF11 and B57-TW10-specific responses, respectively. In addition, B57-
KF11 specific CD8+ T cell responses to cross-presenting DCs were 3-fold lower compared with
B57-TW10 without reaching significance (Figure 23A, left panel). Similar results were observed
with cross-presenting Møs, with 47-fold lower B57-ISW9-specific CTL responses compared
with B57-KF11 and B57-TW10-specific CTL responses (Figure 23A, right panel). We then
analyzed the capacity of DCs and Møs pulsed with increasing amounts of the synthetic optimal
B57-ISW9 and B57-TW10 epitopes to activate epitope-specific CTLs. Similar activation of B57-
ISW9 and B57-TW10-specific CTLs were detected at each epitope concentration (Figure 23B).
Previous studies showed comparable affinities of ISW9, KF11 and TW10 for HLA-B57 (118),
suggesting that differences in CTL responses to cross-presenting DCs and Møs were not due to
differences in peptide avidity among the clones, but due to differences in the intracellular
processing of epitope-containing fragments as shown by our in vitro degradation assay.
These findings were in line with our previous results in cell extracts from DCs and Møs
demonstrating higher production of TW10- and KF11-containing peptides compared to ISW9
in all cell compartments involved in cross-presentation. Moreover, these results indicated that
processing of peptides in the cross-presentation pathway might contribute to
immunodominance patterns.
Results 57
Figure 23. The immunodominant HLA-B57-restricted TW10 and KF11 epitopes are more
efficiently cross-presented than subdominant ISW9 epitope.
A. Immature DCs (left panel) and Møs (right panel) were incubated with recombinant HIV-1 p24
protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with
HLA-B57 ISW9-specific (), KF11-specific (), and TW10-specific () CTL clones as effector
cells. CTL responses in form of SFCs are shown for n≥10 donors.
B. CTL responses to immature DCs (left panel) and Møs (right panel) pulsed with different
concentrations of optimal epitope B57-ISW9 () or B57-TW10 () were measured. Data are
representative of two independent experiments with different donors.
To ensure that epitopes cross-presented by DCs and Møs were endogenously processed we
measured the intracellular concentrations of HIV-1 p24 protein after uptake at 37ºC or 4ºC. In
both cell subsets the intracellular concentration of HIV-1 p24 increased with the amount of
p24 used for uptake at 37ºC whereas the uptake at 4ºC was minimal (Figure 24A). The
intracellular p24 concentrations in immature Møs were at least 5-fold lower compared with
DCs, which may indicate a faster degradation of internalized protein by Møs (49). Moreover,
B57-KF11-specific CTL responses increased with the amount of exogenous p24 protein added
to cells, in accordance with higher amount of intracellular p24 leading to higher amount of
peptide presentation (Figure 24B). Together, these data showed that the higher CTL responses
against dominant TW10 and KF11 epitopes after uptake of HIV-1 p24 by DCs and Møs were
due to cross-presentation of higher amounts of both peptides compared with subdominant
ISW9.
Results 58
Figure 24. The amount of exogenous p24 protein internalized by DCs and Møs affects the
amount of epitope cross-presented to CTLs.
A. Immature DCs (red bars) and immature Møs (blue bars) were pre-treated with a cocktail of
protease inhibitors to reduce intracellular degradation before addition of different
concentrations of recombinant HIV-1 p24 protein at 37ºC or 4ºC. Internalized p24 protein was
determined by a standard p24 ELISA assay using whole cell extracts from lysed DCs and Møs.
Results are from three independent experiments with different donors. Mean ± SD is shown.
B. Immature DCs (red bars) or immature Møs (blue bars) were incubated with different
concentrations of recombinant HIV-1 p24 protein and used as antigen-presenting cells to B57-
KF11-specific CTLs. Responses in form of SFC are shown. Data are representative of two
independent experiments with different donors.
4.5.2. Protease activities in cross-presentation competent cell compartments
differently affect processing of HIV-1 epitopes in DCs and Møs
We next aimed to identify factors contributing to the production or destruction of the three
epitopes in live, intact DCs and Møs along the cross-presentation pathway. Incubation of
immature DCs with proteasome inhibitor MG132 resulted in a 43-fold and 4-fold increased
presentation of B57-ISW9 and B57-KF11 epitopes, respectively, suggesting that proteasomal
degradation of epitope-containing peptides limits the amount of ISW9 and KF11 available for
presentation (Figure 25A). Incubation of DCs with epoxomicin, which selectively inhibits the
proteasome, showed a 11-fold increase in B57-KF11-specific CTL responses (data not shown).
Results 59
In contrast, inhibition of cysteine proteases by E64 had no effect on the cross-presentation of
both epitopes, indicating that fragments escape early into the cytosol before trafficking to
compartments with high cysteine protease activity. B57-TW10-specific CTL responses to cross-
presenting immature DCs decreased approximately 3-fold upon inhibition of proteasomes,
suggesting that proteasomal processing is required for efficient presentation of TW10. In
contrast to DCs, the cross-presentation of B57-ISW9 by immature Møs was not affected upon
inhibition of proteasomes, suggesting that the cross-presentation of ISW9 in Møs is
proteasome-independent (Figure 25B). B57-KF11 and B57-TW10 CTL responses decreased 2-
and 3-fold respectively upon proteasome inhibition with MG132 or epoxomycin in Møs,
suggesting that the processing of both epitopes requires proteasome processing in Møs.
Inhibition of cysteine proteases in Møs did not affect the cross-presentation of ISW9, KF11
and TW10. Together, these results indicated that cross-presentation of HIV-1 p24 involved
distinct proteases in DCs and Møs, which might be essential or detrimental for the processing
of epitopes.
Figure 25. Inhibition of proteasomes increases B57-KF11 and B57-ISW9 epitope cross-
presentation by DCs but not Møs.
Immature DCs (A) and immature Møs (B) were pretreated with proteasome inhibitor (MG132)
or a broad inhibitor of cysteine proteases (E64) before addition of recombinant HIV-1 p24
protein. CTL responses specific to HLA-B57-ISW9 (), HLA-B57-KF11 (), and HLA-B57-TW10
() were measured as described in Figure 23. Results are from n≥5 different donors.
Results 60
4.5.3. Low degradation of RK9-containing fragments results in high amounts of HLA-
A03 RK9 epitope cross-presented by DCs and Møs
We next analyzed the processing of another immunodominant epitope located in a different
HIV-1 protein and restricted by a different HLA allele in the cross-presentation pathway. The
A03-RK9 epitope (RLRPGGKKK, aa 20-28 in Gag p17) has been shown to be efficiently
produced by cytosolic peptidases in the endogenous processing pathway and presented to
A03-RK9-specific CTLs (37, 79).
We first analyzed the degradation of a HIV-1 p17-derived peptide (RWEKIRLRPGGKKKYKL, aa
15-31 in p17) containing the epitope A03-RK9 by proteases active at pH7.4, pH5.5, and pH4.0.
The degradation patterns at all pH values demonstrated minimal degradation of the epitope
(Figure 26). These data indicated that the low degradation of this peptide in all compartments
involved in cross-presentation may result in more fragments available for loading onto MHC I
and therefore better cross-presentation compared with B57-ISW9 and B57-KF11 epitopes.
Figure 26. The RK9-containing fragment shows minor cleavage sites by cytosolic or endo-
lysosomal proteases.
Cleavage patterns of p17-17mer incubated with whole cell extracts from immature DCs for 120
minutes at pH7.4, pH5.5, and pH4.0 are shown as the contribution of each cleavage site,
presented as cleavage N-terminal or C-terminal to a specific amino acid, to the total intensity of
all degradation products. Data are representative of two independent experiments with
different donors.
To test the cross-presentation of the A03-RK9 epitope by immature DCs and Møs, we
incubated cells with recombinant HIV-1 p55 protein and measured the activation of A03-RK9-
specific CTLs to cross-presenting DCs and Møs. Indeed, A03-RK9 CTL responses to cross-
presenting cells were as strong as cells exogenously pulsed with 1.2 ug/ml RK9 (Figure 27A-B).
Moreover, pre-incubation of DCs and Møs with inhibitors of the proteasome or cysteine
Results 61
proteases, showed no effect on A03-RK9 cross-presentation, in line with the limited sensitivity
of proteasome and cathepsin-mediated degradation resulting in high amounts of peptide
available for maximum A03-RK9-specific CTL stimulation (Figure 27A). Similar to our previous
findings with HIV-1 p24 protein, the incubation of DCs or Møs with different concentrations of
HIV-1 p55 protein resulted in concentration-dependent A03-RK9-specific CTL responses
(Figure 27C).
Together, these findings indicated that the high stability of A03-RK9-containing peptides
against degradation by proteases from cross-presentation competent cell compartments
resulted in high amounts of epitope available for cross-presentation.
Figure 27. Limited degradation of RK9-containing fragments results in cross-presentation of
high amounts of HLA-A03 RK9 epitope.
A. Immature DCs (left panel) or Møs (right panel) were incubated with recombinant HIV-1 p55
protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with
HLA-A03 RK9-specific CTLs as effector cells. CTL responses as SFC are shown for n≥5 donors.
B. CTL responses to immature DCs and immature Møs pulsed with different concentrations of
optimal epitope A03-RK9 were measured as described before.
C. Immature DCs (left panel) or Møs (right panel) were incubated with different concentrations
of recombinant HIV-1 p55 protein and used as antigen presenting cells in an overnight IFN-
gamma ELISPOT assay with HLA-A03 RK9-specific CTLs as effector cells. CTL responses in form
of SFC are shown.
Results 62
4.6. Intracellular stability of HIV-1 epitopes in cytosolic and lysosomal
cell extracts
The variable stability of epitopes in cytosolic extracts of PBMCs contributes to the amount of
peptide available for presentation to T cells (37, 38, 113). We hypothesized that peptide
stability in cytosol and endo-lysosomes of DCs and Møs may contribute to the relative
efficiency of cross-presentation of immunodominant epitopes.
4.6.1. Intracellular epitope stability of HIV-1 epitopes in cytosolic and lysosomal cell
extracts follows CTL responses hierarchy
We first measured the stability of A03-RK9, a dominant epitope in the acute phase, and B57-
KF11, a dominant epitope in the chronic phase, in whole cell extracts of immature DCs and
Møs at pH7.4 and pH4.0. In cell extracts of immature DCs at pH7.4, we detected a more rapid
degradation of B57-KF11 epitope compared to the A03-RK9 epitope (approximate half-lives of
20 minutes versus 61 minutes). In cell extracts of immature DCs at pH4.0, we detected an 11-
fold faster degradation of B57-KF11 epitope (half-life: 1.9 minutes), whereas the A03-RK9
epitope showed a 20-fold higher half-life (half-life: 1223 minutes) (Figure 28, left panel).
Similar results were observed in cell extracts from immature Møs (Figure 28, right panel).
Figure 28. Variable stability of B57-KF11 and A03-RK9 in cytosolic and lysosomal cell extracts
of immature DCs and Møs.
One nmol of highly purified HLA-B57-restricted KF11 () and HLA-A03-restricted RK9 () were
degraded in 15µg of immature DC extracts (left panel) or Mø extracts (right panel) in
degradation buffer at pH7.4 (solid line) or pH4.0 (dashed line). Degradation products were
analyzed by RP-HPLC after 10, 30, and 60 minutes. 100% represents the amount of peptide
detected at time 0, calculated as the surface area under the peptide peak. Data are
representative of three independent experiments using extracts from different healthy donors.
We next compared the intracellular stability of seven well-defined dominant and subdominant
MHC I epitopes located in HIV Gag p24, Gag p17 and RT, and examined whether their stability
Results 63
corresponded to immunodominance patterns observed in HIV-1 infection. To rank epitopes
we calculated for each epitope a stability rate as done before (37, 113). In cell extracts of
immature DCs at pH7.4 the dominant epitopes A03-RK9 and B57-TW10 showed approximately
5-fold and 4-fold higher stability rates compared with the subdominant epitopes B57-ISW9
and A11-ATK9, respectively (Figure 29, upper left panel). At pH4.0 we detected dramatically
reduced stability rates for B57-ISW9, B57-KF11, B57-FF9, and A11-ATK9, indicating a more
rapid proteolysis by endo-lysosomal proteases. However, the observed stability rates of all
epitopes formed the same hierarchy as seen at pH7.4. Similar results were observed in cell
extracts of immature Møs (Figure 29, lower left panel) and in cell extracts from LPS-matured
DCs and Møs (Figure 29, upper and lower right panel).
Figure 29. The intracellular stability of HIV-1 epitopes in the cytosol and lysosomes follows
CTL responses hierarchy.
The stability rate of optimal epitopes ISW9, KF11, FF9, TW10, ATK9, RK9, and KK9 at pH7.4 and
pH4.0 was calculated by a nonlinear regression (one-phase exponential decay) of the
degradation profile obtained over a 60-minute incubation in extracts of immature and mature
DCs (upper panel) and Møs (lower panel). N=3 and mean ± SD is shown.
Together, these results showed that the intracellular stability of optimal HIV epitopes was
highly variable in DCs and Møs and in different cell compartments, but followed similar
hierarchies and might contribute to differences seen in cross-presentation and
immunodominance patterns observed in HIV-1 infection.
Results 64
4.7. The impact of HIV-1 escape mutations on epitope production and
peptide stability in the cross-presentation pathway
Immune pressure exerted by T cell immune responses leads to predictable mutations within
and outside epitopes altering viral fitness or epitope presentation. Many mutations altering
proteasomal or aminopeptidase peptide processing lead to immune escape (37). However the
impact of HLA-restricted mutations on the processing of epitopes in the cross-presentation
pathway and immune escape is not known despite being a compartment involved in virus
uptake.
4.7.1. Frequent escape mutations in immunodominant epitopes reduce epitope
production and peptide stability in the cross-presentation pathway
In HLA-B57+ patients TW10 rapidly mutates at residue 3 and 9 during acute infection (119,
120) and the dominant TW10 CTL response wanes and KF11-specific CTL response becomes
dominant (64, 121). To assess the impact of escape mutations on degradation patterns in the
cross-presentation pathway, peptides containing the TW10 epitope or its naturally occurring
variants TW10 T3N or TW10 T3N/G9A were degraded in whole cell extracts from immature
DCs and Møs at pH7.4 and pH4.0. Degradation at pH7.4 showed comparable kinetics with 67-
87% of original peptide and N- and C-extended TW10 fragments left after 60 minutes (Figure
30A). However, both mutants produced less N- and C-extended TW10-containing peptides
than the WT at pH7.4, with peptides contributing to 23% of the total peptide intensity in
TW10 T3N variant, 19% in the TW10 T3N/G9A variant and 45% in the TW10 WT at 60 minutes,
respectively. Degradation at pH4.0 demonstrated faster kinetics and generated a majority of
fragments lacking part of the epitope, called antitopes. After 10 minutes of degradation
antitopes contributed to 39% of the total peptide intensity in TW10 WT, whereas in T3N and
T3N/G9A mutants antitopes contributed to 65% and 99% of total peptide intensity.
The analysis of the N- and C-terminal cleavage sites showed that TW10 WT and TW10 T3N
sequences were spared from degradation at pH7.4 after 10 minutes, whereas a cleavage site
between tryptophane and alanine destroyed the TW10 T3N/G9A variant within 10 minutes of
degradation (Figure 30B, upper panel). At pH4.0, in line with the faster degradation of the
peptides into antitopes, two major cleavage sites produced fragments with an isoleucine at
the N-terminus or a glutamine at the C-terminus thereby destroying TW10 and its variants.
These cleavage sites were 2.8-fold and 2.6-fold higher in the TW10 T3N/G9A variant compared
with the TW10 WT and the TW10 T3N variant (Figure 30B, lower panel). Similar results were
observed with cell extracts from immature Møs (data not shown).
Results 65
Figure 30. Escape mutations in immunodominant epitopes reduce epitope production in the
cross-presentation-competent cell compartments.
A. Two nmol of 5-TW10-3 (DIAGTTSTLQEQIGWMTN, aa 103-120 in Gag p24), 5-TW10-3 T3N
(DIAGTTSNLQEQIGWMTN), and 5-TW10-3 T3N/G9A (DIAGTTSNLQEQIAWMTN) were degraded
in 15µg of whole cell extracts from immature DCs and Møs for 10, 30 or 60 minutes in
degradation buffer at pH7.4 and pH4.0. (Continued on next page)
Results 66
Degradation products identified by MS were grouped into fragments containing the optimal
epitope TW10 or its mutant (black), the epitope/mutant with N-terminal extensions (white), the
epitope/mutant with C-terminal extensions (red), the epitope/mutant with N- and C-terminal
extensions (green), antitopes defined as fragments lacking part of the epitope/mutant (blue),
or the original peptide (gray), respectively. The contribution of each category of peptides to the
total intensity of all degradation products is shown at each time point.
B. Cleavage patterns of 5-TW10-3, 5-TW10-3 T3N, and 5-TW10-3 T3N/G9A incubated with
whole cell extracts from immature DCs for 10 minutes at pH7.4 (upper panel) or at pH4.0
(lower panel) are shown as the contribution of each cleavage site, presented as cleavage N-
terminal or C-terminal to a specific amino acid, to the total intensity of all degradation
products. For (A-B) data are representative of two independent experiments with different
donors.
We then compared the cytosolic and lysosomal stability of TW10 epitope and its variants in
DCs and Møs (Figure 31). Similar intracellular stabilities were observed for B57-TW10 and
TW10 T3N in each compartment whereas the stability of TW10 T3N/G9A was reduced by 7- to
8-fold in both compartments, in line with the faster degradation of the fragment containing
the TW10 T3N/G9A mutant.
Figure 31. Reduced intracellular stability of TW10 escape mutants in cytosolic and lysosomal
cell extracts.
One nmol of highly purified HLA-B57-restricted TW10, TW10-T3N or TW10-T3N/G9A mutants
were degraded in 15µg of immature DCs or Møs extracts (right and left panel) in degradation
buffer at pH7.4 or pH4.0. A stability rate was calculated as described before. Bars represent the
mean ± SD of three independent experiments with extracts from different healthy donors.
These results demonstrated that the low stability of TW10 T3N/G9A variant might reduce the
epitope presentation in both direct and cross-presentation pathways in line with our previous
findings in cells infected with TW10 variants (37). This represents the first demonstration of an
escape mutation affecting the cross-presentation of an HIV-1 epitope.
Discussion 67
5. Discussion
In this study we comprehensively analyzed and compared the processing of HIV-derived
epitopes by monocyte-derived DCs and Møs in the two antigen processing pathways: the
direct/endogenous pathway and the cross-presentation pathway.
We found significant differences in the hydrolytic activities and expression levels of the
cytosolic antigen processing machinery in monocyte-derived DCs and Møs upon maturation
with TLR4 and TLR7/8 ligands or HIV-1 infection, with mature Møs having highest activities.
These differences led to variations in the kinetics and the amount of HIV-1 epitopes produced
and affected the recognition by HIV-specific CTLs. DCs and Møs have the ability to internalize
exogenous antigens, such as virions, antibody-coated particles or viral vectors, into endosomes
and lysosomes for processing and presentation by MHC II to CD4+ T cells or cross-presentation
by MHC I to CD8+ T cells. Here, we showed that degradation patterns in the cross-presentation
pathway of APCs favored the production of HIV-1 immunodominant MHC I-restricted epitopes,
which led to highest CTL responses to these epitopes cross-presented by DCs and Møs. During
disease progression, the appearance of escape mutations can impair epitope production and
presentation, which results in shifts in immunodominance patterns and less efficient clearance
of infected cells. In this study we provided the first demonstration of antigen processing
mutations affecting the cross-presentation of HIV-1 epitopes by DCs and Møs to CTLs.
Together, in the context of HIV infection the processing of an antigen will most likely be
affected by (i) the processing machinery in different HIV-infectable cell subsets, (ii) the mode
of viral entry and the pathway used by the antigen to traffic through subcellular
compartments, (iii) the recognition of viruses by pathogen-recognition receptors and the
subsequent activation of cells, (iv) the hydrolytic activities of proteases located in different cell
compartments harboring specific cleavage site preferences, and (v) the cleavability of HIV-1
sequences by these proteases. All these aspects will affect the presentation of peptides by
MHC I or MHC II for recognition and activation of HIV-specific CD8+ or CD4+ T cells. A better
understanding of each of these factors will be crucial for HIV-1 vaccine design.
5.1. Antigen processing in HIV-infectable cell subsets
HIV-1 infects several CD4-expressing cell subsets, including CD4+ T cells, monocytes, Møs and
DCs, which should be targeted by HIV-specific CD8+ T cells for clearance of infection. Whether
all HIV-infectable cell subsets process and present equivalent amounts of epitopes and are
equally recognized by HIV-specific CTLs is poorly defined. If all HIV-infectable cell subsets
display sufficient amount of peptides upon infection, they may be recognized and cleared by
Discussion 68
HIV-specific CD8+ T cells, and this broader recognition of infected targets may contribute to the
superior antiviral function of these CD8+ T cells - independently of their poly-functionality,
avidity, and TCR (122-124). However, if peptide presentation by one subset is slower or below
threshold of detection for a specific TCR, these cells may produce and propagate virus before
immune recognition occurs. The processing and presentation of MHC I epitopes is a multistep
pathway including several proteases in different cell compartments (125, 126), which can
produce or destroy epitope-containing peptides. Previously, it has been shown that significant
higher proteasome and aminopeptidases activities in monocytes compared to CD4+ T cells
from the same donor affected the processing of several HIV-1 epitopes and resulted in clear
differences in the kinetics and the amount of antigenic peptides produced (106). In line with
these findings, other studies demonstrated differences in the processing of LCMV proteins by
fibroblasts and DCs in mice (127) or during degradation of HIV-1 epitopes by purified
proteasomes from DCs or CD4+ T cells (128). In this study we show that differences in the
expression levels and proteolytic activities of proteases involved in the processing of antigens
in immature and mature DCs and Møs affect the production of several HIV-1 epitopes and lead
to uneven recognition by epitope-specific CTLs (104). Elevated levels of immunoproteasomes
can lead to faster kinetics and higher amounts of epitope-containing fragments available for
presentation (129-131). As we detect more ATK9-containing fragments in Møs compared to
DCs it is tempting to link the production of ATK9 to higher proteolytic activities of the
constitutive proteasome or immunoproteasome. This would be in line with previous findings in
monocytes, which demonstrated higher proteasomal activities compared to CD4+ T cells and
produced more ATK9 epitope (106). However, processing of peptides by proteasomes may also
lead to higher degradation of peptides, as we show for the B57-ISW9 and B57-KF11 epitopes
during cross-presentation by DCs, thus reducing the amount of epitope available for
presentation (28, 32) and the processing of some epitopes has been shown to be less efficient
by immunoproteasomes (132). Moreover, differences in other cytosolic or ER-resident
peptidases, such as TOP or ERAP1 or 2 (which are higher in Møs compared to DCs) may lead to
different epitope processing. Due to low infectivity of DCs and Møs, we were not able to
directly compare the endogenous processing and presentation of the HLA-A11-restricted ATK9
epitope by both cell subsets to A11-ATK9-specific CTL clones. However, the similar changes in
antigen-processing activities observed in DCs and Møs upon TLR-stimulation or HIV infection
suggest that our findings using the in vitro degradation assay are relevant to epitope
production and presentation in the context of HIV-1 infection.
Human DCs represent a very heterogeneous population that consists of different DC subsets,
each with distinct functional characteristics. In the blood at least three subsets can be
Discussion 69
distinguished (BDCA1+ DCs, BDCA3+ DCs and pDCs) and in the skin at least two main subsets
(Langerhans cells and CD14 DCs). Similarly, multiple Møs subsets (133), monocyte subsets
(134), and CD4+ T cell subsets (4) have been identified, which may have different capacities to
process HIV-1 epitopes. In addition, cell-specific peptidases such as a serine protease uniquely
expressed in monocytes (135), may lead to cell-specific processing of epitopes and may
contribute to the differential epitope presentation and priming of immune responses by
different DC subsets shown previously (136, 137). Further transcriptomics (138, 139),
proteomics and functional analyses are needed to identify additional cell subset-specific
peptidases that may shape epitope presentation by various APC subsets.
Besides differences in the proteolytic activities between different cell subsets, the presence of
natural peptides, usually five to six amino acids long, has been shown to modulate the
proteasome activity and may contribute to differences in peptide processing (140). Further
studies are required to evaluate the relevance of these peptides in different cell subsets during
HIV-1 infection.
Together, differences in the antigen processing activities between HIV-infectable cell subsets
may result in differences in the timing and the efficiency of epitope production, which may
lead to unequal recognition and killing by HIV-specific CTLs.
5.2. Viral entry and intracellular trafficking of antigens
Viral fusion may occur either at the cell membrane or from within the endosomes, which may
be dependent on the cell subset that is targeted by HIV-1. The relative contribution of either
route to infection of target cells may be affected by the activity of endocytic proteases (higher
endocytic activities will lead to higher degradation of incoming virions), the pH within endo-
and lysosomes (more acidic pH will lead to the activation of multiple endo-lysosomal
proteases) and the fusogenicity of the virus. Thus, the same virus may fuse primarily at the
plasma membrane if endocytosis rates are low or from within the endosomes when
endocytosis activities are high.
The route of virus entry determines the intracellular trafficking of antigens and thus the
proteases that the antigen will encounter. The presence of different proteases in various cell
compartments in HIV-infectable cell subsets may affect the degradation patterns of antigens.
Previous studies showed different degradation patterns by proteases located in endo-
lysosomes, the cytosol or the ER (78, 105, 141). Internalization of virions into endocytic
compartments will lead to degradation by compartment-specific peptidases (different
cathepsins, IRAP, asparaginyl endopeptidase, gamma-interferon-inducible lysosomal thiol
reductase). Processed antigens can then enter the cytosol for further processing by the
Discussion 70
proteasome and eventually cross-presentation by MHC I. In contrast, viral fusion at the cell
membrane may lead to the transport of the same viral antigens into the cytoplasm where the
proteasome will degrade the antigen and thus may result in different cleavage patterns (141).
Degradation of HIV-1 peptides in cytosolic and endo-lysosomal cell extracts shown in this study
demonstrate that some MHC I epitopes are similarly processed in both cell compartments
while others are better processed in one or the other compartment as shown before (105).
The trafficking of antigens in DCs and Møs may also depend on the receptor used to internalize
the antigen. Different receptors can transport their cargo into different endocytic
compartments, such as early or late endosomes or lysosomes, where peptides can be loaded
onto MHC II or escape into the cytosol for cross-presentation by MHC I.
Whether differences in the intracellular trafficking of antigens (direct presentation vs. cross-
presentation) affect the kinetics of epitope presentation remains open, despite the importance
of early immune recognition and killing of infected cells before viral spreading occurs. Our in
vitro degradation data show different kinetics and amounts of MHC I and MHC II peptides,
which may lead to differences in the kinetics of epitope presentation to CD8+ or CD4+ T cells.
To address this question a previously developed real-time cytotoxicity assay can be used (142).
This assay measures the presentation of epitopes by live, intact APCs to epitope-specific CTLs
or CD4+ T cells and subsequent killing or activation in real-time.
Together, the route of virus entry into the cell, either by membrane fusion and release of viral
RNA into the cytoplasm or by endocytosis of viral particles into endosomes, will affect (i) the
efficiency of infection of target cells (143), (ii) the route of antigen trafficking through various
subcellular compartments and (iii) may affect the receptors involved in pathogen sensing and
therefore the signaling cascade utilized to activate cells.
5.3. Viral recognition and cell activation upon HIV-1 infection
DCs and Møs express a variety of TLRs for recognition of pathogen-associated molecular
patterns (PAMPs), such as glycoproteins, lipopolysaccharides, and nucleic acid motifs. From
the first contact of HIV-1 with its receptors at the plasma membrane to its replication in the
cytoplasm, viral proteins and nucleic acids can be detected and trigger activation of immune
cells (144). Different DC subsets express distinct TLRs, which determine their responses to
products derived from pathogens (145, 146) and lead to changes in the capacity to internalize
antigens (147), modify the intracellular antigen processing machinery (104) and shape the
adaptive immune response by producing different cytokines (148, 149). All DC subsets express
TLR7 and/or TLR8, which are expressed in endosomes and can sense single-stranded RNA
oligonucleotides from HIV-1. Upon viral recognition a downstream signaling cascade activates
Discussion 71
the expression of genes required for inflammation and adaptive immune activation (150). We
show that TLR4 and TLR7/8 activation of DCs and Møs leads to significant changes in the
hydrolytic activities of various cytosolic and endo-lysosomal proteases between both cell
subsets (104), which modifies the kinetics and the amount of epitope production and leads to
differences in CTL activation. The presence of multiple DCs and Møs subsets in vivo with
different TLR ligands and different susceptibilities to HIV-1 infection, suggest that there are
most likely multiple maturation phenotypes and/or infection states of cell subsets (immature
cells, mature cells, and cells undergoing maturation), which will affect their capacity to
produce and present epitopes. Thus, we cannot rule out that the heterogeneous populations
of both cell subsets in vivo would result in more complex patterns of epitope production and
presentation.
The activation of TLR7/8 requires the endocytosis of extracellular viral particles or the cellular
process of autophagy, which transports cytosolic viral RNA into lysosomes (151). In order to
recognize viral RNA in the cytoplasm many cells express RIG-I-like receptors (RLRs), which
utilize the same signaling cascade as TLR7 and TLR8 (152). Whether sensing through RLRs
results in similar kinetics of cell activation and changes in antigen processing activities as
shown in this study using TLR ligands needs to be evaluated in future studies.
During HIV-1 infection a massive production of pro-inflammatory cytokines and type I and II
interferons can be detected (25, 26). Interferon-gamma, LPS and TNF-alpha can induce the
expression of catalytically active immunoproteasome subunits and several other cytosolic and
ER-resident peptidases in cells (153-155), which can affect the processing of HIV-1 epitopes
and thus modifies CTL recognition. Recent studies reported of intermediate proteasomes, in
which only one or two catalytic subunits were replaced and which displayed additional
cleavage specificity, even enlarging the repertoire of antigens presented on MHC I (156).
Moreover, changes in the protease composition in different cell subsets may not only affect
the processing of HIV-1 peptides, but also of peptides from other pathogens or co-infections
thereby affecting the hierarchy of immune responses against these pathogens. Previous
studies have shown that even the self-peptidome displayed by HIV-1 infected cells can change
during HIV-1 infection (157). In addition, activation of CD4+ T cells by TCR stimulation can affect
protease activities and modify the processing of HIV-1 peptides, which may affect the capacity
of HIV-specific CD8+ T cells to recognize and kill these cells.
Moreover, treatment of HIV-1 infected patients with antiretroviral drugs can also affect
antigen processing. HIV protease inhibitors, which target HIV-1 protease to prevent virus
maturation, have been shown to alter proteasome and aminopeptidases activities (113, 158,
Discussion 72
159). These changes modified the degradation patterns of HIV-1 peptides and the production
and presentation of epitopes to HIV-specific CTLs.
Thus, besides differences in the expression levels and hydrolytic activities of proteases
involved in antigen processing between different HIV-infectable cell subsets, the recognition of
PAMPs and the subsequent production of pro-inflammatory cytokines as well as extrinsic
factors such as drugs used to treat HIV-infected individuals can affect the processing of
epitopes and therefore the activation of CD8+ and CD4+ T cells.
5.4. Epitope processing in different subcellular compartments
DCs and Møs express a variety of antigen capture and sensing receptors, which will most likely
affect their capacity to acquire and process internalized antigens (160). Previous studies
showed that targeting antigens to specific receptors will result in the transport of its cargo to
different cell compartments and results in presentation by MHC I or MHC II (51, 161, 162).
Various endocytic proteases, including cysteine cathepsins, serine proteases, and aspartic
proteases are located in endocytic vesicles, which are tightly regulated by the pH. DCs have
been shown to maintain a more alkaline pH within endosomes and phagosomes compared to
Møs (55), which results in lower hydrolytic activities of various cathepsins. In line with these
findings, we show that different cathepsins can be activated at different pH values and
cathepsins activities in intact, live DCs are lower than in Møs. However, using whole cell
extracts as a source of proteases from endosomes and lysosomes activated at pH5.5 and pH4.0
may simplify the actual processing of antigens in different cell compartments of DCs and Møs.
Nevertheless, as antigen processing involves multiple proteases, the use of cell extracts for
protein degradation allows us to consider all these peptidases present in a given compartment,
even though we cannot exclude that by lysing cells and disrupting the cell structure, proteases
from different compartments will affect the degradation patterns. Despite these limitations,
we show that the degradation patterns observed in our in vitro degradation assay correspond
to the cross-presentation patterns of the epitopes by intact, live DCs and Møs, validating the
use of our in vitro degradation assay to analyze processing of peptides in the cross-
presentation pathway. Moreover, other studies showed similar correlations between
degradation of peptides in cytosolic extracts and epitope presentation and killing of infected
cells by CTLs (37, 79, 113). Ideally, different cell compartments, such as early and late
endosomes or lysosomes, would be separated at different time points during cross-
presentation and analyzed for the presence of degradation products. This approach is limited
by the large number of cells required to separate sufficient amounts of different cell
compartments and the fast sample preparation to avoid further peptide degradation. Thus,
Discussion 73
our method represents a simple and fast approach to analyze the degradation of antigens by
proteases from different cell compartments at different time points.
Since antigens intended for cross-presentation traffic through the endocytic system and the
cytosol, the stability of an antigen contributes to the amount of peptide available for
presentation. Indeed, we observed poor cross-presentation of the B57-ISW9 epitope in DCs
and Møs compared to the A03-RK9 epitope, due to a low intracellular stability of the optimal
epitope B57-ISW9 and multiple cleavage sites within ISW9-containing peptides during
degradation of long HIV-1 peptides. This suggests that cross-presentation requires high
stability against degradation in phagosomes to preserve potential MHC I binding epitopes.
However, some degree of degradation is required for cross-presentation, probably because
small molecules are exported to the cytosol with a higher efficiency than large ones (163).
The export of antigens from endosomes and lysosomes into the cytosplasm may be an
additional step determining the efficiency of cross-presentation and whether antigens are
presented by the vacuolar pathway or the cytosolic cross-presentation pathway. Previous
studies showed that induction of macropinocytosis in Møs promotes antigen export to the
cytoplasm, whereas DCs that macropinocytose constitutively also showed constitutive export
of antigens into the cytoplasm (163-165). In line with these findings, we observed an increased
presentation of B57-ISW9 and B57-KF11 epitopes by DCs upon inhibition of the proteasome,
indicating the export of epitope-containing peptides into the cytoplasm whereas in Møs the
processing of both epitopes seemed to be proteasome-independent. However, even though
cross-presentation of some epitopes requires proteasomal processing and TAP transport,
there is no clear evidence for peptide import and loading in the ER during cross-presentation.
The detection of MHC I peptide complexes in phagosomes and endosomes (166, 167), the
localization of TAP to ER membranes (168) and the identification of an endosomal N-terminal
peptidase (IRAP) (44) indicate that after antigen export to the cytoplasm and processing by the
proteasome, peptides may translocate back into endosomes and phagosomes by TAP for
loading onto MHC I. Therefore, we cannot exclude that loading of B57-ISW9 and B57-KF11
onto HLA-molecules during cross-presentation in DCs may occur in endocytic compartments.
To confirm the transport of B57-ISW9- and B57-KF11-containing peptides into the ER during
cross-presentation by DCs, either TAP or ERAP need to be inhibited. Moreover, as different
receptors transport exogenous antigens into different subcellular compartments, it is most
likely that even the same epitope can be loaded onto MHC I in endosomes or the ER
depending on the trafficking of the antigen after internalization. However, it is still very
difficult to evaluate the relevance and the relative contribution of these two pathways to
cross-priming in vivo.
Discussion 74
5.5. Cross-presentation contributes to immunodominance patterns
During acute and chronic HIV infection a predictable hierarchy of CTL responses can be
identified, demonstrating consistent recognition of specific epitopes, whereas others are not
recognized. In line with previous data using cytosolic cell extracts from PBMCs (79) or purified
proteasomes (78), we observe a preferential production of peptides containing
immunodominant epitopes in all cell compartments involved in cross-presentation, which
leads to highest CTL responses to immunodominant epitopes cross-presented by DCs and Møs.
Previous studies showed that the number of peptide/MHC-complexes needed to target a cell
for lysis by epitope-specific CTLs may be fewer than 10 complexes per cell (169, 170), whereas
the amount of peptide/MHC-complexes required to stimulate proliferation of CD8+ T cells is
much greater (171, 172). The higher production and cross-presentation of B57-TW10
compared to B57-ISW9 may lead to better priming of naïve CD8+ T cells and thus to
immunodominance of B57-TW10 observed during acute HIV-1 infection. Besides differences in
the amount of epitope produced, we find highly variable intracellular stabilities of multiple
optimal MHC I epitopes in cytosolic and lysosomal cell extracts with immunodominant
epitopes having highest stabilities. Higher intracellular stability may result in more epitope
available for presentation and thus higher amounts of peptide/MHC-complexes available for
priming of naïve CD8+ T cells. These data demonstrate that the production of peptides during
cross-presentation as well as the intracellular stability may contribute to immunodominance
patterns. However, as immunodominance patterns also depend on the binding affinity of the
epitope to MHC I, the binding of the TCR to the peptide/MHC I-complex as well as the
frequency of T cell precursors, the establishment of immunodominant CD8+ T cell responses
may be determined by the contribution of each of these factors.
Moreover, differences in endo-lysosomal activities in DCs and Møs in vivo (115) as well as
different capacities to cross-present exogenous antigens by different DCs subsets (173, 174)
may affect the ability of each subset to prime CD8+ T cells in vivo. Whether the source of
antigen, such as viral particles, cell-associated antigens from apoptotic infected cells or
antibody-conjugated antigens, will result in similar cross-presentation patterns as shown in
this study requires further analysis.
Priming of naïve CD8+ T cells depends on CD4+ T cell help. Therefore it is important to better
understand the processing and presentation of MHC II epitopes during the generation of anti-
viral immune responses. Previous studies showed that degradation of HIV-1 peptides in
cytosolic extracts affected the kinetics of epitope production (79, 104, 113), and its efficiency
was defined by intraepitopic and flanking motifs (78, 79, 94, 175). Current studies analyze
whether similar motifs can be identified for the processing of peptides in endo-lysosomes,
Discussion 75
which will be important for the induction of CD4+ T cell responses during priming of naïve CD8+
T cells. Together, these data highlight the importance of degradation patterns and intracellular
epitope stability in the cross-presentation pathway to immunodominance patterns.
5.6. The impact of viral escape mutants on cross-presentation
HLA-restricted escape mutations can be frequently detected in the acute and chronic phase of
HIV-1 infection (83, 91, 176). Constant immune pressure on selected epitopes results in
variants that impair epitope processing, reduce binding to MHC I or to the TCR and thus avoid
CTL recognition (90, 95).
This study provides the first demonstration that a frequently detected HLA-restricted mutation
during acute HIV-1 infection affects the cross- and direct presentation of an epitope in DCs and
Møs. While the wild-type epitope B57-TW10, dominant in the acute phase, is efficiently
processed and highly stable in endo-lysosomes and the cytosol of DCs and Møs its mutant
TW10 T242N/G248A is faster degraded. The lower amount of epitope available for
presentation may lead to less epitope presented to T cells and therefore results in immune
escape. Whether all known escape mutants impair epitope processing in the cross-
presentation pathway is currently studied. If an escape mutant impairs the processing of an
epitope in the cross-presentation pathway and in the direct presentation pathway it will not be
presented by any HIV-infectable cell subset. However, if an escape mutant impairs the
processing by cytosolic proteases, but is produced in endo-lysosomes, it may still be presented
by DCs and Møs in the vacuolar pathway. This may suggest that DCs can prime new CD8+ T cell
responses against these mutants – assuming the mutant can bind to MHC I and is recognized
by a TCR. In line with this hypothesis, previous studies showed that some mutants did not
affect epitope presentation and primed novel CTL responses (91, 177). Whether these novel
CTL responses can recognize only cell subsets able to internalize and process escape mutants
in the vacuolar pathway or also recognize cells that transport mutants from the cytosol into
the vacuolar pathway by macroautophagy requires further studies.
Some mutants occurring outside of defined CTL epitopes may correspond to compensatory
mutations enabling the virus to achieve improved fitness after a deleterious mutation arising
elsewhere (98). Previous studies showed that escape mutants in flanking sequences affect
MHC I epitope production in the cytosol or the ER (92-94). Whether these mutants affect the
processing of MHC I epitopes in endo-lysosomes and therefore the cross-presentation of these
mutants is currently investigated.
Due to their loose interaction with the MHC II binding groove, MHC II epitopes containing
mutants may still bind to MHC II molecules and get presented. However, if MHC I-restricted
Discussion 76
escape or compensatory mutants insert major cleavage sites into overlapping MHC II peptides
this would result in impaired MHC II peptide production and represent a new mechanism for
MHC II-restricted escape. Thus, mutants in MHC I epitopes may not only affect their own
processing but also the production of overlapping MHC II epitopes.
Previous studies showed that transmission of the TW10 T242N/G248A variant to HLA-B57
negative individuals resulted in reversion of the T242N mutation to the wild-type, whereas the
G248A mutant persisted and became another’s person wild-type virus indicating different
impacts on the viral fitness (96). The fast degradation of peptides containing the G248A
mutation, as shown in this study, may affect the processing and stability of other MHC I-
restricted epitopes overlapping this mutant. If an immunodominant epitope in the newly
infected individual overlaps with the G248A mutant this may lead to impaired epitope
production of that epitope and hence affect the CTL recognition and immunodominance
patterns in the newly infected individual. Therefore it is important to detect the accumulation
of mutations that do not impact viral fitness cost and are transmitted during HIV-1 infection, as
these mutations can potentially impact the processing and presentation of other MHC I or
MHC II epitopes.
5.7. Impact of antigen processing and cross-presentation on vaccine
design
Vaccines are classified into two main types: preventative and therapeutic vaccines.
Preventative vaccines typically elicit the generation of humoral and adaptive immune
responses, which block the spread of pathogens. Therapeutic vaccines are designed as a
treatment to eradicate the cause of disease by activating or inducing CTLs to eliminate virally
infected cells.
The critical role of DCs to prime CD8+ T cells makes them an attractive target for vaccination
against HIV-1 and other viruses for which cellular immunity seems to be a crucial part of the
immune response. One approach for therapeutic vaccination is the administration of
autologous monocyte-derived DCs loaded ex vivo with a variety of antigens (178, 179).
However, this approach is laborious and expensive and therefore probably not useful in the
context of the HIV-1 epidemic. Alternatively, the selective targeting of antigens to DC-specific
endocytic receptors by monoclonal antibodies coupled to the desired antigen presents a very
efficient alternative and offers a way for antigen presentation on both MHC I and MHC II to
CD8+ and CD4+ T cells (180). Depending on the receptor targeted, immunogens may be taken
up into different subcellular compartments in which different proteases result in different
degradation patterns of peptides. To evaluate the capacity of proteins/immunogens to
Discussion 77
generate peptides compatible with loading onto a variety of MHC I or MHC II molecules
independent of a given HLA it is necessary to better understand how HIV-1 proteins are
degraded into peptides within cells. Our in vitro degradation data demonstrate that MHC I and
MHC II epitopes are variably produced in different subcellular compartments with different
kinetics and different amounts, which will affect the presentation of epitopes to CD8+ and CD4+
T cells. To prime an effective HIV-specific CD8+ T cell response, DCs also have to present MHC II
peptides to generate CD4+ T cell help required for priming. This suggests that vaccines should
target different receptors on DCs to transport antigens either to the MHC I presentation
pathway or into the MHC II pathway. This strategy would also allow to balance the
presentation of MHC I and MHC II epitopes and to minimize the activation of CD4+ T cells to
avoid HIV-1 spreading.
HIV-1 infection leads to a predictable hierachy of T cell responses with narrow
immunodominance of a few T cell responses for each HLA type (64, 83, 84). Since these
immune responses are not able to clear infection, a vaccine leading to the same
immunodominance patterns as natural HIV-1 infection will most likely not clear infection as
shown in previous vaccine trials (181, 182). These data suggest that breaking natural
immunodominance patterns may be an interesting alternative. To break immunodominance
patterns mutations can be inserted into epitope sequences or into regions flanking epitopes to
increase the production of protective epitopes and decrease the production of non-protective
epitopes (37, 79). Understanding the cleavage patterns in various subcellular compartments
will help to design immunogens that are efficiently processed in these compartments and lead
to sufficient amounts of epitopes for presentation. Moreover, the usage of chimeric proteins
that contain only portions of HIV-1 proteins, thus eliminating non-protective or “non-
functional” parts of the protein may be another alternative. However, the success of this
approach requires the identification of protective CD8+ and CD4+ T cell responses, which is still
limited (8, 183).
During transition from acute to chronic infection escape mutations within and outside of
immunodominant epitopes result in changes of CTL response hierachies. This may suggest that
simultaneous delivery of wild-type sequences and escape variants in form of a mosaic
immunogen may represent an effective approach to increase immune pressure on the virus,
thus pushing the virus to mutants that affect viral fitness (99, 100). Understanding the
degradation of escape mutants during cross-presentation will be critical for the design of
immunogens containing frequently detected escape mutants that will traffic through various
cell compartments where efficient processing is required for the induction of effective immune
responses. Moreover, this strategy requires further identification of all HLA-restricted escape
Discussion 78
mutations within an epitope and its flanking residues, which is difficult due to the continuous
evolving of escape mutants to evade the host’s immune response.
Another way to escape immune recognition used by HIV-1 is the expression of viral proteins
that interfere with antigen processing and presentation (184). Vaccination with complete HIV
proteins or with vectors that induce the expression of these proteins will most likely result in
interference of viral proteins with the antigen processing machinery of the APC. Cross-
presentation of exogenous HIV-1 proteins may circumvent this effect by pre-processing
proteins in endosomes and lysosomes before transport into the cytoplasm. However, as shown
in this study cross-presentation of exogenous complete HIV-1 proteins results in
immunodominance patterns similar to those detected during acute HIV-1 infection, which do
not result in clearance of HIV-1 infection.
Recent studies in rhesus macaques showed that immunization with a CMV vector expressing
complete SIV proteins led to clearance of SIV infection in 50% of the animals and led to broad
CD4+ and CD8+ T cell responses, highlighting the importance to generate MHC I and MHC II
epitopes with an immunogen. Interestingly, most SIV-specific CD8+ T cell responses recognized
epitopes presented by MHC II (185-187). Further studies need to analyze the mechanisms
leading to these unconventional CD8+ T cell responses and whether this approach is applicable
to humans. In addition, future studies need to analyze the impact of viral vectors used for
vaccination on the antigen processing machinery in the APCs targeted and whether these
vectors elicit immune responses, which may affect the overall hierarchy of immune responses.
Based on these data, it would be interesting to test whether conjugation of SIV proteins to DC-
specific antibodies would result in similar CD8+ and CD4+ T cell responses.
Previous studies showed that HIV Gag-specific CD4+ T cell responses increased after acute HIV
infection in individuals who control HIV-1 infection (6) and were significantly associated with
control of viral replication (7, 8). These findings suggest, that HIV-specific CD4+ T cell responses
are required for efficient HIV-specific CD8+ T cell responses (188) and should be included in an
immunogen. However, in the context of HIV-1 infection, the activation of CD4+ T cells can
always result in increased infection of CD4+ T cells and thus disease progression.
The type of adjuvant used for vaccination will trigger changes in the hydrolytic activities of
proteases and affect epitope production and presentation, as shown in this study, and will
affect the quality and type of immune response generated. Therefore it is important to analyze
the impact of different adjuvants on antigen processing in subcellular compartments to better
understand the production of MHC I and MHC II epitopes for presentation to CD8+ and CD4+ T
cells.
Discussion 79
Together, various factors impact the processing of immunogens, including (i) the antigen
processing machinery in the different APC subsets, (ii) the pathway used by the antigen to
traffic through subcellular compartments in which different proteases with different
proteolytic activities can cleave the antigen, (iii) the presence or absence of cleavable motifs in
the antigen, and (iv) the presence of TLR ligands used as adjuvants. Future studies need to (i)
identify protective immune responses covering various HLA types across multiple clades, (ii)
design immunogens encompassing these responses, and (iii) identify intracellular pathways
that lead to proper processing and presentation of these MHC I and MHC II epitopes in order
to induce effective immune responses able to recognize and kill all infected cell subsets.
References 80
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Supplemental Figures 100
7. Supplemental Figures
Supplemental Figure 1. Phenotypic analysis of DCs and Møs cell populations.
Immature DCs or Møs were phenotypically analyzed by flow cytometry. Cells were stained with
antibodies against human CD11c, BDCA-1 and CD14 (black lines) or isotype controls (gray lines)
to assess the purity of each cell population. The percentage of cells expressing each marker is
indicated. The level of surface expression of each marker is indicated as mean fluorescence
intensity (MFI) (upper right corner).
Supplemental Figures 101
Supplemental Figure 2. Infection of immature DCs and Møs with VSVg ΔEnv NL4.3.
Immature DCs (A) or Møs (B) were infected with VSVg ΔEnv NL4.3 in the presence of Vpx. On
day 1, 3, and 6 post infection the infection rate, measured by the expression of GFP, was
analyzed by flow cytometry.
(C) Bars represent the mean ± SD of two independent experiments from different donors.
Supplemental Figures 102
Supplemental Figure 3. Degradation patterns of a HIV-1 RT 16-mer fragment in cytosolic
extracts of immature and mature DCs and Møs.
Four nmol of 5-ATK9-2 (aa 153-168 in HIV-1 RT) were degraded with 30µg of cytosolic extracts
from iDCs, LPS-, or CL097-matured DCs (A) or iMøs, LPS-, or CL097-matured Møs (B) for 120
minutes. Peptides containing (black bars) or lacking intact ATK9 (white bars) were identified by
MS. Optimal ATK9 () is indicated. The data are representative of three independent
experiments from different donors.
Supplemental Figures 103
Supplemental Figure 4. Degradation of a HIV-1 Gag p24 35mer in DC cell extracts at pH4.0,
pH5.5 and pH7.4.
The HIV-1 peptide p24-35mer (MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa 10-44 in Gag
p24) was degraded in 15µg of whole cell extracts from immature DCs for 60 minutes in
degradation buffer at pH4.0, pH5.5 or pH7.4. Peptides containing the epitopes B57-ISW9 and
B57-KF11 (black bars), B57-ISW9 epitope (blue bars), B57-KF11 epitope (red bars) or lacking
both epitopes (gray bars) were identified by MS. Optimal B57-ISW9 (blue star) and B57-KF11
(red star) are indicated. Data represent one of three independent experiments from different
donors.
Supplemental Figures 104
Supplemental Figure 5. Variable production of 16 HIV-1 epitopes in cytosolic and endo-
lysosomal cell extracts of DCs and Møs.
A. The map shows the location of 12 MHC I epitopes (black arrows) and 4 MHC II epitopes (gray
arrows) within the sequence of Gag p24-35mer (aa 10-44).
B. Summary of the relative amount of optimal epitopes and corresponding N-terminal
extensions detected by MS after 10, 30, 60, and 120 minutes degradation in extracts of
immature DCs, LPS-matured DCs, immature Møs, LPS-matured Møs at pH7.4, pH5.5 and pH4.0.
Epitope precursors, defined as peptides with the correct C-terminus and extended by up to
three residues at the N-terminus, could be further trimmed in the ER. Numbers represent the
contribution of optimals and N-extended optimals to the total intensity of all degradation
products at each time point. The presence of optimal epitopes is indicated (*). Data represent
one of three MS analyses from independent experiments.
Supplemental Figures 105
Supplemental Figure 6. Minor cleavage sites within p24-31mer results in less degradation of
B57-TW10-containing peptides.
Cleavage patterns of p24-31mer incubated with whole cell extracts from immature DCs for 30
minutes (left panel) or 120 minutes (right panel) at pH7.4, pH5.5, and pH4.0 are shown as the
contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a specific
amino acid, to the total intensity of all degradation products. Data are representative of three
independent experiments with three different donors.
Abbreviations
AMC 7-amino-4-methylcoumarin
APC Antigen presenting cell
ATP Adenosine triphosphate
BDCA-1 Blood Dendritic Cell Antigen 1
CCR Chemokine receptor
CMV Cytomegalovirus
51Cr Chromium 51 isotope
CTL Cytotoxic T lymphocyte
DC Dendritic cell
DC-SIGN Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-
integrin
DRiPs Defective ribosomal products
DTT Dithiothreitol
EBV Epstein-Barr virus
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-Linked ImmunoSorbent assay
ELISPOT Enzyme-Linked ImmunoSpot
Env Envelope
ER Endoplasmic reticulum
ERAP Endoplasmic reticulum aminopeptidase
FBS Fetal bovine serum
Gag Group-specific antigen
GM-CSF Granulocyte macrophage colony-stimulating factor
HAART Highly active antiretroviral therapy
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV Human immunodeficiency virus
HLA Human leukocyte antigen
HRP Horseradish peroxidase
HSA Human serum albumin
Hsp90 Heat shock protein 90
IFN-γ Interferon-gamma
IL Interleukin
IRAP Insulin-regulated aminopeptidase
LAP Leucine aminopeptidase
LCMV Lymphocytic choriomeningitis
LPS Lipopolysaccharide
Mø Macrophage
MS Mass spectrometer
NOX2 NADPH oxidase 2
ODP o-Phenylenediamine
PA28 Proteasome activator
PAMPs Pathogen-associated molecular patterns
pDC Plasmacytoid dendritic cell
PBMCs Peripheral blood mononuclear cells
PBS Phosphate Buffered Saline
PSA Puromycin-sensitive aminopeptidase
PVDF Polyvinylidene fluoride
RLR RIG-I-like receptor
RP-HPLC Reversed-phase high-performance liquid chromatography
SAMHD1 SAM domain and HD domain-containing protein 1
SFC Spot-forming cell
TAP Transporter associated with antigen processing
TCA Trichloroacetic acid
TCR T-cell receptor
TFA Trifluoroacetic acid
TLR Toll-like receptor
TOP Thimet oligopeptidase
TPPII Tripeptidyl-peptidase II
