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Lee, Jenifer Widger, Tibor Keler, Lélia Delamarre and Ira MellmanBithi Chatterjee, Anna Smed-Sörensen, Lillian Cohn, Cécile Chalouni, Richard Vandlen, Byoung-Chul regulate the efficiency of cross presentation by human dendritic cellsInternalization and endosomal degradation of receptor-bound antigens

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1

Internalization and endosomal degradation of receptor-bound antigens

regulate the efficiency of cross presentation by human dendritic cells

Bithi Chatterjee1,2, Anna Smed-Sörensen2,3, Lillian Cohn2, Cécile Chalouni2, Richard

Vandlen2, Byoung-Chul Lee2, Jenifer Widger4, Tibor Keler4, Lélia Delamarre2, and Ira

Mellman2*

1Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510;

2Genentech, South San Francisco, CA 94080; 3Department of Microbiology, Tumor and

Cell Biology, Karolinska Institutet, Stockholm, Sweden; 4Celldex Therapeutics,

Phillipsburg, NJ 08865

*Correspondence should be addressed to:

Ira Mellman, Genentech, 1 DNA Way, Mail Stop 212, South San Francisco, CA 94080

email: [email protected]

Short title: Degradation controls cross presentation efficiency

Keywords: cross presentation, endocytosis, degradation, MHC class I, dendritic cells

Blood First Edition Paper, prepublished online July 12, 2012; DOI 10.1182/blood-2012-01-402370

Copyright © 2012 American Society of Hematology




2

Abstract

Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides

on to MHC class I molecules, a process termed cross presentation. The mechanisms of

cross presentation remain incompletely understood, particularly in primary human DCs.

One unknown is the extent to which antigen delivery to distinct endocytic compartments

determines cross presentation efficiency, possibly by influencing antigen egress to the

cytosol. We addressed the problem directly and quantitatively by comparing the cross

presentation of identical antigens conjugated to antibodies against different DC receptors

that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+

and monocyte-derived DCs, CD40 and mannose receptor (MR) targeted antibody

conjugates to early endosomes while DEC205 targeted antigen primarily to late

compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the

most efficient at cross presentation. This did not reflect DC activation by CD40, but rather

its relatively poor uptake or intra-endosomal degradation as compared to MR or DEC205.

Thus, while both early and late endosomes appear to support cross presentation in

human DCs, internalization efficiency, especially to late compartments, may be a negative

predictor of activity when selecting receptors for vaccine development.




3

Introduction

Dendritic cells (DCs) are professional antigen presenting cells that process proteins

and load resultant peptides on to major histocompatability complex (MHC) molecules.

Upon activation, DCs migrate to secondary lymphoid organs and present antigen to their

cognate T cells, for the induction of adaptive immune responses 1. Since DCs are

essential for the initiation of adaptive immune responses, they are attractive targets for

enhancing prophylactic and therapeutic vaccination strategies 2-4 and have been shown to

activate anti-tumor immune responses in murine cancer models 5,6.

In human cancer, clinical evidence is now accumulating to suggest that the

induction or activation of CD8+ T cell immunity can contribute to the arrest of tumor

growth and patient survival 7. In principle, targeting tumor antigens to DCs may enhance

protective CD8+ T cell responses due to the ability of DCs to cross present exogenous

antigens 8. In cross presentation, exogenous proteins are endocytosed, processed, and

loaded on to MHC class I molecules for presentation to CD8+ T cells. The efficiency of

cross presentation can be improved >100-fold when receptors found on the surface of

DCs are targeted specifically 6.

Multiple DC populations exhibit some capacity for cross presentation in vitro, but

certain subpopulations (CD8+ DCs in the mouse, BDCA3+ DCs in human) are thought to

be particularly adept in vivo 8. DCs exhibit a variety of surface receptors that can

internalize antigen for cross presentation, with some being subpopulation-specific.

Pioneering studies from Steinman and co-workers have shown cross presentation using

the C-type lectin receptor DEC205, which is expressed by CD8+ mouse DCs as well as

by multiple human DC subsets 9,10. Another lectin receptor, CLEC9A/DNGR-1, is




4

expressed by mouse CD8+ DCs and human BDCA3+ DCs, and also mediates cross

presentation 5. Indeed, a variety of receptors expressed by human or mouse DCs (DCIR,

Langerin, mannose receptor (MR), DC-SIGN, CLEC12A) enable cross presentation 10-15.

Which of these receptors is most efficient and why remains poorly understood, particularly

in human DCs.

A few recent studies have begun to address the relative roles of early endosomes

and late endosomes/lysosomes in cross presentation. Burgdorf et al. suggested for

mouse DCs that cross presentation of soluble ovalbumin internalized by MR, presumably

targeted to early endosomes, is more efficient than cross presentation following fluid

phase uptake, which also delivers an unspecified portion of soluble ovalbumin to late

endosomes 16-18. Early endosomes also may host the cross presentation of liposome-

encapsulated hen egg lysozyme, but in a fashion independent of the proteasomal

pathway 19,20. Antigen coupled to DC-SIGN antibodies also suggested that early

endosomal targeting may facilitate cross presentation by human DC-SIGN transgenic

mouse DCs 13. In all of these studies, however, there was limited quantitation or

characterization of antigen accumulation and fate, and direct and effective comparisons

were not made between the various uptake routes. Where the effects of targeting different

receptors was compared, endosomal targeting was not well studied and the conjugates

were poorly characterized for the possibility of aggregates 21,22, an important consideration

if any of these platforms are to be transferred to the clinic for use in humans. Aggregates

also tend to be transferred to lysosomes and may have altered degradation properties

23,24. Finally, suggestions that early endosomal targeting is optimal appear inconsistent

with more extensive investigations of DEC205, which is targeted primarily to late




5

endocytic compartments 25,26, yet appears to mediate the efficient cross presentation of

proteins coupled to or fused to anti-DEC205 antibodies 6,9,12.

To establish directly and quantitatively the endosomal requirements for efficient

cross presentation using a clinically relevant and scalable platform, we used elongated

peptide antigens conjugated to monoclonal antibodies against three receptors that traffic

to distinct cellular compartments: CD40, MR, and DEC205. Interestingly, we found an

inverse relationship between internalization or antigen degradation and cross

presentation: antigens destined for more degradative late endosomes were poorly cross

presented relative to the same antigens targeted to early endosomes, an effect that was

independent of the amount of antigen internalized.




6

Materials and methods

Cell isolation and culture

This study was approved by the Genentech Institutional Review Board. Our procedures

for the isolation of DCs and T cells from blood have been described previously 27. Healthy

donors were leukapheresed and enriched populations of lymphocytes and monocytes

were obtained by counterflow elutriation. BDCA1+ DCs were isolated from elutriated

monocytes using the CD1c DC isolation kit and AutoMACS technology (Miltenyi). Cells

were cultured overnight in R10 (RPMI 1640 + Glutamax with 10% FCS, 100 units/mL

penicillin + 100 μg/mL streptomycin, 10 mM Hepes) (Gibco/Invitrogen), and 2 ng/mL GM-

CSF (Peprotech). Monocyte-derived DCs (MoDCs) were derived in 5-7 d from CD14+

monocytes cultured in R10 with 100 ng/mL GM-CSF (Peprotech) and 6.5 ng/mL IL-4

(R&D Systems). To mature DCs, DCs were exposed to 0.2 μg/mL lipopolysaccharide

(LPS, Sigma) overnight. For autologous CD8+ T cell assays with MoDCs, elutriated HLA-

A*0201+ monocytes were frozen in anticipation of donor recall. CD8+ T cells were

isolated from HLA-A*0201+ elutriated lymphocytes using the CD8+ T cell isolation kit

(Miltenyi). For CD4 T cell assays using an NY-ESO peptide specific human CD4 T cell

clone (CCNE-1 415; obtained from Cassian Yee), HLA-DPB1*0401 DCs were obtained as

above.

Antibodies

Anti-DEC205 (3G9; Kd, 0.3 nM) and anti-MR (B11; Kd, 0.7 nM) used in our studies were

obtained in collaboration with Celldex Therapeutics, and the anti-CD40 (S2C6; Kd, 0.12




7

nM) was obtained in collaboration with Seattle Genetics. Binding affinities were

determined by flow cytometry.

Antibodies used for flow cytometry/immunofluorescence were as follows: anti-CD3 (SK7),

CD4 (Leu-3a/SK3), CD8 (SK1), CD8 (RPA-T8), CD11c (B-ly6), CD14 (MΦP9), CD19

(Leu-12), CD40 (5C3), CD71 (M-A712), CD83 (HB15e), CD86 (FUN-1), CD206 (19.2),

DEC205 (MG38), HLA-DR (TU36), IFNγ (B27), IL-2 (MQ1-17H12), Lamp1 (H4A3) and

TNFα (6401.1111) (BD Biosciences); anti-HLA-DR (L243) (Biolegend); anti-HLA-ABC

(W6/32) (eBioscience); anti-CD1a (NA1/34) (DAKO); anti-EEA1 (Cell Signaling);

secondary reagents (steptavidin Alexa 555/647, anti-rabbit Alexa 546, anti-FITC Alexa

488) (Invitrogen/Molecular Probes).

Synthesis of antibody-peptide conjugates

Antibodies in PBS buffer with 50mM potassium phosphate (pH 7.5) were reacted with a

10-15 fold molar excess of N-succinimidyl S-acetylthioacetate (Pierce) for 4-6 h at 20°C.

Excess reagents were removed by dialysis in PBS resulting in antibodies with 3-6 blocked

thiol groups. Blocking groups were then removed with 20 mM hydroxylamine (Sigma) and

peptides containing an N-terminal maleimide (Elim Biopharmaceuticals) were added to

approximately 5-6 fold molar excess over antibody and allowed to react with the new thiol

groups for 2-4 h at 20°C. Excess peptide and aggregated antibodies were removed by gel

filtration in PBS. Electrospray mass spectrometry was used to estimate the average

number of peptides per antibody. The peptides conjugated to antibodies were

(immunodominant epitopes underlined and sequence position indicated):




8

LTKGILGFVFTLTVPSER (influenza M1 58-66), QAGILARNLVPMVATVQGQNL

(cytomegalovirus (CMV) pp65 495-503), or QQLSLLMWITQAFLPVFLAQPPSGQRR (NY-

ESO 157-170).

Immunofluorescence

Following culture or accumulation with 1 μg/mL anti-CD40 (S2C6), anti-MR (B11), or anti-

DEC205 (3G9) antibodies covalently conjugated to Alexa 488 (Alexa 488 monoclonal

antibody kit, Invitrogen), BDCA1+ DCs or MoDCs were washed and spotted on Alcian

blue coated coverslips for 10-15 min at room temperature. Cells were fixed in 4%

paraformaldehyde (PFA, Electron Microscopy Source) for 15 min, permeabilized in 0.05%

saponin (Sigma), and counterstained. When biotinylated antibodies were used,

endogenous biotin was blocked first by using an excess of unlabeled streptavidin and

biotin (Endogenous biotin blocking kit, Invitrogen). Following labeling, coverslips were

mounted onto glass slides using Prolong Gold with DAPI (Invitrogen/Molecular Probes).

Images were acquired on a Leica SP5 confocal microscope, using a 100x oil objective

(NA: 1.47) with zoom 7, and Leica’s LAS imaging software (Leica Microsystems).

Antibody accumulation experiments

Cells were continuously incubated with 1 μg/mL anti-CD40 (S2C6), anti-MR (B11), or anti-

DEC205 (3G9) Alexa 488 antibodies for indicated times, in the presence or absence of

the serine/cysteine protease inhibitor leupeptin (5 mM) (Roche) and the acidophilic weak

base ammonium chloride (NH4Cl) (10 mM). Following incubations, cells were stained for

CD14 and maturation markers, fixed, acquired using a BD FACSCanto II, and analyzed




9

for Alexa 488 signal using FlowJo. For direct receptor comparisons, MFI was normalized

for surface antibody signal (4°C) and the number of fluors per antibody.

Antigen presentation assays

MHC class I:

HLA-A*0201 DCs were incubated for 4-6 h in the presence of antibody-peptide conjugates

(0.1-10 μg/mL) or other reagents of interest (peptide control 2.5-250 ng/mL, Proimmune;

epoxomicin, 0.1 μM, Calbiochem; DMSO, 0.1 μM, Sigma). Following antigen uptake, DCs

were washed extensively to remove free antibody, peptide, or inhibitor, and cocultured

with carboxyfluorescein succinmidyl ester (CFSE) (Invitrogen) labeled CD8+ T cells for 8-

10 d at a DC:T cell ratio of 1:30, in the presence of 20 units/mL IL-2 (Roche) and 200

ng/mL LPS (Sigma). After coculture, influenza M1 or CMV pp65 peptide specific CD8+ T

cells were labeled with a corresponding PE-pentamer (ProImmune), acquired using a BD

FACSCanto II, and gated on CD3+CD8+ (CD14/CD4/CD19-) cells. CFSElo/pentamerhi

cells were analyzed using FlowJo.

MHC class II:

HLA-DPB1*0401 DCs were incubated for 1.5 h in the presence of antibody conjugates

(0.1-10 μg/mL) or other reagents of interest (peptide control 2.5-25 μg/mL, Elim

Biopharmaceuticals; SEB 1μg/mL, Sigma). Following antigen uptake, DCs were washed

and cocultured with an NY-ESO peptide specific human CD4 T cell clone for 7-9 h at

DC:T ratios of 1:1, 1:3, and 1:9. Two hours into the coculture, Brefeldin A (BFA;

eBioscience) was added to prevent cytokine secretion. Following coculture, cells were




10

stained for surface markers, fixed, permeabilized, and labeled for intracellular IFNγ, IL-2,

and TNFα. Cells were acquired with a BD FACSCanto II, and gated on CD3+CD4+CD1c-

cells, followed by Flowjo analysis.

Pulse-chase experiments

A488 conjugated antibodies of interest were pulsed with cells at 37°C or 4°C for 1 h at 4

μg/mL, in the presence or absence of 5 mM leupeptin and 10 mM NH4Cl. Cells were

washed and chased for the indicated times, in the presence or absence of leupeptin and

NH4Cl. Cells were counterstained with CD14 and maturation markers, washed, and fixed.

Cells were acquired using a BD FACSCanto II, and analyzed for Alexa 488 signal using

FlowJo.




11

Results Antibodies are targeted to distinct cellular compartments

To compare quantitatively the ability of different DC endosomal compartments to

facilitate cross presentation, we selected three receptors suspected to have distinct

intracellular destinations or rates of internalization: mannose receptor (MR), CD40, and

DEC205. We first confirmed their surface expression on primary human BDCA1+ DCs

and monocyte-derived DCs (MoDCs) (Fig. 1A-C). With the exception of MR on BDCA1+

DCs, both CD40 and DEC205 were expressed at variably high levels by both immature

and mature BDCA1+ DCs and MoDCs. MR expression was low but detectable on

immature BDCA1+ DCs, but this amount further decreased upon maturation. As expected,

maturation enhanced CD40 and DEC205 expression in both DC types. When comparing

relative receptor expression levels, both DC types expressed significant amounts of each

receptor before and after maturation by LPS, except for MR whose expression was low in

immature BDCA1+ DCs (Fig. 1D). All antibodies exhibited sub-nanomolar affinity (anti-

CD40: 0.12 nM, anti-DEC205: 0.3 nM, anti-MR: 0.7 nM), and were used at saturation.

We next characterized the ability of each receptor to be internalized and its

subsequent subcellular localization in both BDCA1+ DCs and MoDCs. As expected from

previous studies, anti-DEC205 antibodies accumulated largely in late endosomes. Very

little was found on the surface of BDCA1+ DCs and MoDCs (Fig. 2A and 2D, first panel),

and a small amount appeared to be associated with early endosomes as marked by EEA1

(Fig. 2A and 2D, second panel). The bulk of anti-DEC205 antibody overlapped with the

late endosomal marker Lamp1 (Fig. 2A and 2D, third panel), and the signal was enhanced

in cells treated with the protease inhibitor leupeptin together with the acidophilic weak




12

base ammonium chloride (inh), indicating that antibody degradation was occurring in this

compartment (Lamp1; Fig. 2A and 2D, fourth panel). Therefore, in untreated cells the

amount of anti-DEC205 delivery to late compartments was underestimated.

When MR antibody was incubated continuously with BDCA1+ DCs and MoDCs, it

was mainly found intracellularly with relatively little on the cell surface (Fig. 2B and 2E,

first panel). Anti-MR was found in early endosomes as indicated by the partial overlap with

EEA1 (Fig. 2B and 2E, second panel) and minimal overlap with Lamp1 (Fig. 2B and 2E

third panel). This pattern was maintained even in the presence of protease and

acidification inhibitors (Fig. 2B and 2E fourth panel), suggesting that anti-MR antibodies

did not reach late compartments as significantly as anti-DEC205 and did not escape

detection due to their degradation.

Following continuous incubation in BDCA1+ DCs and MoDCs, anti-CD40 antibody

was largely found on the surface (Fig. 2C and 2F, first panel), in early endosomes (EEA1;

Fig. 2C and 2F, second panel), and minimally in late endosomes (Lamp1; Fig. 2C and 2F,

third panel). This pattern was similar in the presence of protease and acidification

inhibitors (Fig. 2C and 2F, fourth panel). Thus, like MR, anti-CD40 internalized to early

endosomes, but appeared to do so much less efficiently. Similar results were obtained for

all antibodies at earlier and later time points of internalization (data not shown), and the

extent of overlap was quantified (Fig. S1).

The patterns observed by antibody internalization were confirmed by indirect

immunofluorescence detection of the receptors themselves: CD40 was mainly found on

the surface, while MR and DEC205 were mainly found intracellularly, in early and in late

endosomes, respectively (data not shown). In sum, we have characterized the targeting




13

and localization of antibodies against three receptors in two different human DC types:

DEC205 in late endosomes, MR in early endosomes, and CD40 on the surface and in

early endosomes.

CD40 accumulates the least efficiently of the three receptors

To quantify the ability of DEC205, MR, and CD40 to accumulate antigen, we

determined the time course of continuous anti-receptor antibody uptake by flow cytometry.

As shown in Figure 3 for BDCA1+ DCs (A) and MoDCs (B), MR antibody was more

effectively accumulated than either CD40 or DEC205 antibodies (top panels). Inhibiting

proteolysis by the addition of the protease and acidification inhibitors (inh) did not

markedly affect the accumulation of either MR or CD40 antibodies, but did significantly

enhance the amount of cell-associated DEC205 antibody, particularly in MoDCs (Fig. 3B,

bottom panel; p <0.05). This finding is consistent with the observations that MoDCs exhibit

higher levels of lysosomal enzymes than BDCA1+ DCs 28,29 and that DEC205 antibodies

are delivered predominantly to protease-containing late endosomes. In all cases, isotype

controls were minimally internalized (muIso corresponds to anti-CD40; huIso corresponds

to anti-MR and anti-DEC205). Taken together, these data indicate that MR and DEC205

antibodies are internalized by both DC types to a greater extent than CD40 antibodies.

Targeting CD40 and MR is more efficient for cross presentation than targeting

DEC205

Having characterized the internalization and intracellular destinations of antibodies

to CD40, DEC205, and MR, we next compared their relative abilities to mediate cross




14

presentation. For this purpose, each high affinity antibody was chemically coupled to

either of two immunodominant HLA-A*0201 epitopes, influenza M1 (58-66) and

cytomegalovirus (CMV) pp65 (495-503), using the strategy outlined in Figure 4A. Both

epitopes (red residues, Fig. 4A) were extended by several residues of naturally occurring

peptide sequence on the amino- and carboxy-terminal ends (black residues, Fig. 4A). We

determined that the antibodies were conjugated to similar numbers of peptides per

molecule by mass spectrometry and remained >95% monomeric by gel filtration

chromatography (data not shown).

Using these antibody conjugates, we performed recall cross presentation assays

using human HLA-A*0201+ DCs and autologous CD8+ T cells from donors with

detectable memory responses against influenza or CMV (Fig. S2A). The percent of total

CD8+ T cells that were dividing and M1 or pp65 specific was assessed by CFSE dilution

and pentamer staining. In immature BDCA1+ DCs and MoDCs fed with M1 peptide

coupled antibodies, targeting antibodies to the early endosomal receptors CD40 and MR

was generally superior for cross presentation as compared to DEC205 (Fig. 4B and Fig.

S2B), which surprisingly, was the most inefficient for cross presentation. Interestingly, the

receptor that was least efficient in accumulating antigen, CD40, was the most efficient for

cross presentation, indicating that intracellular antigen accumulation was not the rate-

limiting step in determining cross presentation efficiency.

We also assessed the ability of mature DCs to cross present receptor targeted

antigen, as matured mouse DCs are able to present antigens captured via receptor-

mediated endocytosis 26. We found that mature BDCA1+ DCs and MoDCs were able to

induce efficient cross presentation via CD40, and in mature MoDCs, targeting MR also led




15

to efficient cross presentation (Figure 4C). Targeting MR in mature BDCA1+ DCs resulted

in little to no cross presentation, likely due to the absence of MR on the surface upon

maturation (Fig. 1A). In both immature and mature DCs, targeting DEC205 resulted in

similarly low levels of cross presentation (Fig. S3), and was unable to induce cross

presentation as efficiently as targeting MR or CD40, except at the highest antibody

concentrations used in immature MoDCs (Fig. 4B). Figure 4D shows donor matched pre-

processed HLA-A*0201+ peptide controls, with mature DCs presenting surface loaded

influenza M1 peptide more efficiently than immature DCs, in line with their higher surface

expression of MHC class I. CD40 antibodies coupled to the CMV pp65 peptide also

resulted in superior cross presentation as compared to DEC205-pp65 antibodies (Fig. 4E).

Thus, receptor-mediated delivery to early endosomes is a relevant determinant of cross

presentation efficiency for multiple epitopes. We determined whether a cytosolic

intermediate was involved for antibody-targeted cross presentation to both early and late

endosomes by inhibiting proteasomal activity. Cross presentation via all three receptors

required proteasomal degradation for the generation of peptides, while surface peptide

loading and presentation remained intact in the presence of epoxomicin (Fig. 4F).

Having shown that MHC class I cross presentation was superior when targeting

CD40 or MR over DEC205, we next asked if the same held true for processing and

presentation on MHC class II. We chemically coupled anti-CD40 and anti-DEC205 to an

extended MHC class II peptide from the NY-ESO protein that had a cysteine to alanine

mutation to facilitate conjugation (Fig. 5A, mutated residue bolded). The mutation did not

appear to reduce the ability of the peptide to be recognized by NY-ESO specific T cells

(Fig. 5B). Therefore, we next incubated BDCA1+ DCs with the antibody conjugates, and




16

cocultured DCs together with the NY-ESO peptide specific CD4 T cell clone; activation

was measured by monitoring the production of three cytokines (IFNγ, IL-2, and TNFα). As

found for cross presentation of other antigens on MHC class I, anti-CD40 was far more

efficient at mediating the presentation of a NY-ESO MHC class II epitope than was anti-

DEC205 over a 100-fold range of antibody concentrations (Fig. 5C, 5D, and 5E) and at

different DC: T cell ratios (Fig. 5F). Thus, antibody targeting to CD40 is not only superior

for cross presentation, but also for MHC class II presentation.

Taken together, these data indicate that antigen accumulation is not necessarily a

good predictor of cross presentation efficiency. Instead, receptors that target to early

compartments are quantitatively more adept at delivering antigen into the cross

presentation pathway than late endosomes, a finding that also seems to hold true for

MHC class II presentation. Interestingly, both endosomal destinations required

proteasomal degradation. Additionally, a receptor (CD40) that internalizes slowly but

targets to early endosomes appeared superior for antigen cross presentation.

Anti-CD40 does not enhance cross presentation by triggering DC maturation

We next asked if the anti-CD40 antibody’s superiority for cross presentation might

reflect its ability to generate an activation signal in DCs, especially since CD40 signaling is

known to enhance cross presentation in general 6,30. MoDCs were incubated overnight

with unlabeled CD40, MR, or DEC205 antibodies and assayed for maturation. Anti-CD40

slightly activated MoDCs, as gauged by CD86 and CD83 upregulation on a small cell

population. HLA-ABC (MHC class I) surface levels remained unaffected by the addition of

anti-CD40, indicating that the upregulation of HLA-ABC by anti-CD40 signaling was not




17

responsible for enhanced cross presentation (Fig. 6A). Anti-DEC205 and anti-MR

antibodies did not cause upregulation of any surface markers (Fig. 6A). Analysis of

supernatants collected from MoDCs treated overnight with these antibodies showed non-

detectable levels of IL-12p70 and minimal amounts of TNFα in response to anti-CD40 (Fig.

6B). These experiments indicate that the addition of the anti-CD40 antibody used here

(S2C6) induced a very low but detectable level of DC activation, consistent with this

antibody’s previous characterization as a very weak agonist in vitro.

To determine if the low level of CD40-mediated DC activation might itself enhance

cross presentation, we asked if the addition of anti-CD40 antibodies would enhance cross

presentation by the anti-DEC205-M1 and anti-MR-M1 antibody conjugates. DCs were

incubated with either anti-DEC205-M1 or anti-MR-M1 together with 1 μg/mL unconjugated

anti-CD40 or isotype control during the antigen uptake phase. In immature and mature

MoDCs (Fig. 6C-D) and in immature BDCA1+ DCs (Fig. 6E), the addition of CD40

antibodies did not enhance DEC205 cross presentation. In co-uptake experiments, we

observed that both MR and CD40 likely trafficked through the same compartments (data

not shown), and it is thought that antigen presentation is enhanced when both antigen and

agonist originate from the same compartment 31. However, when we co-administered MR-

M1 and anti-CD40, this did not enhance cross presentation via MR (Fig. 6C-E), indicating

that the addition of this anti-CD40 antibody did not enhance cross presentation by

triggering DC activation. Indeed, adding anti-CD40 antibodies also did not improve MHC

class II presentation by DEC205 (Fig. 6F), indicating that any anti-CD40 signal imparted

was negligible for both antigen presentation pathways tested.




18

DEC205 antibodies are degraded over time, whereas CD40 and MR antibodies

remain stable

Having ruled out a role for CD40 signaling in enhancing cross presentation, and

having established that the extent of antigen uptake is not a key factor in cross

presentation efficiency, we wished to understand if differences in antibody degradation

were responsible, as suggested by our earlier accumulation experiments. To more directly

monitor antibody degradation, we pulsed MoDCs with Alexa 488 conjugated antibodies for

1 h at 4°C or 37°C, followed by washing and chasing for the indicated times. After 24 h,

there was a significant loss of anti-DEC205 signal (Fig. 7A) while both anti-CD40 and anti-

MR signal remained relatively constant (Fig. 7B, C), indicating that anti-DEC205 was

exposed to greater degradative forces. Parallel experiments were performed in cells

treated with leupeptin and ammonium chloride (inh) to inhibit endosomal-lysosomal

proteolysis. While we did not see an appreciable change in anti-MR and anti-CD40

fluorescence levels upon inhibitor addition (dashed lines), the inhibitors did rescue the

anti-DEC205 antibody signal, consistent with this antibody having been delivered to late

endosomes and subjected to degradation.

Inhibiting degradation rescues DEC205 targeted cross presentation

Conceivably, delivery to degradative compartments reduces cross presentation by

causing intra-lysosomal degradation of the internalized antibody-peptide conjugates prior

to release into the cytosol for proteasomal processing. Indeed, this difference might be

accentuated in our system, which relied on the use of relatively small peptide conjugates

that could be more easily digested than intact protein antigens. Resistance to degradation




19

is well known to be important for MHC class II presentation 1,32-34, with antigens more

resistant to degradation being more efficiently processed and presented. Our MHC class

II data may also reflect the differences in degradative capacity between the endosomal

compartments, as targeting CD40 elicited superior CD4+ T cell responses as compared to

DEC205 (Fig. 5). The same may be true in the case of cross presentation, given studies

showing enhancement when antigen presenting cells were treated with chloroquine 19,35.

To examine this possibility, we added protease and acidification inhibitors during the

antigen capture phase of our CD8+ T cell assay. As shown in Figure 7, the addition of

protease and acidification inhibitors enhanced cross presentation via DEC205 in both

immature (panel D) and mature DCs (panel E). This was true for influenza M1 and CMV

pp65 peptide conjugates (Fig. 7D, G). Interestingly, addition of increasing concentrations

of protease inhibitors reduced cross presentation via CD40 (Fig. 7F), indicating that some

level of proteolysis was required for escape to the cytosol and subsequent cross

presentation. Presentation of unconjugated peptide was largely unaffected by the inhibitor

treatments, except at the very highest concentrations (Fig. 7H). Thus, cross presentation

of antigens delivered to highly degradative compartments could be enhanced by inhibiting

the degradative capacity of these compartments. In sum, by targeting three receptors with

distinct endosomal localization patterns, we show that targeting early endosomal

receptors with antibody-peptide conjugates results in more efficient cross presentation,

due to the lowered degradative capacity of this compartment. Additionally, we find that

under conditions where proteases are inhibited, both late and early endosomal

compartments are able to support cross presentation, arguing against the existence of

specializations unique to a single endosome population.




20

Discussion DCs harbor a variety of specializations that together enable their capacity for

antigen presentation and the control of T cell stimulation 1. Although a number of these

specializations affect the endosomal and phagosomal apparatus 36,37, the formation of

MHC class II-peptide complexes does not appear to be linked to any one intracellular site.

The situation is less clear in the case of cross presentation on MHC class I molecules.

Although antigen can be cross presented regardless of internalization mode 38-40, whether

it must access a specific endosomal compartment was uncertain. Previous reports have

implicated early endosomes as essential for cross presentation, based on a qualitative

comparison of receptor-mediated versus fluid phase ovalbumin uptake and the possible

presence of the TAP2 translocator by immunofluorescence microscopy 16,18, while other

studies with little emphasis on intracellular trafficking have determined that many

receptors are able to cross present equivalently with great efficiency 10,12,21,22.

We avoided making any assumptions concerning which endosomal compartment

was essential or what specializations it might possess for cross presentation by using high

affinity antibodies that targeted antigen to distinct compartments. This approach allowed a

direct, quantitative assessment of the relative importance of antigen internalization,

localization, and degradation in determining the efficiency of cross presentation in two

different human DC populations. Our results suggest that both early and late endosomal

compartments are capable of serving as antigen portals for cytosolic entry and cross

presentation. Late compartments appear to be less efficient for some antigens, however,

given their higher concentration of lysosomal enzymes, which degrade antigens before

they can be released into the cytosol. Accordingly, we find that the rapidity of degradation




21

can explain at least some of the compartment-specific differences in cross presentation.

The fact that inhibiting proteolysis enhances the ability of late endosomes and lysosomes

to allow cross presentation of accumulated antigens also means that it is unlikely that

early endosomes alone contain the specializations required for antigen egress or for re-

importation for loading onto endosomal MHC class I molecules, as proposed previously 18.

Our ability to detect a relationship between degradation and presentation from late

compartments may reflect the nature of the antigen delivery platform we have developed,

i.e., the use of extended peptides chemically coupled to anti-receptor antibodies. Previous

work has relied mostly on the use of heavy chain fusions of full-length antigens 9,10, which

may be inherently more resistant to degradation. In our hands, such fusions also have a

significant tendency to aggregate and to be proteolysed during production, which would

complicate potential scale up for application in human patients. Our peptide platform

offers the advantage of scalability and combinatorial flexibility (allowing for delivery of

different peptides), although the potential decrease in peptide antigen stability may require

the targeting of such constructs to early endosomes. Indeed, protein stability may be a

reason why targeting DEC205 is inferior for cross presentation; peptide antigens are likely

more susceptible to degradation, which would decrease their ability to survive long

enough to escape into the cytosol. The use of more susceptible peptide antigens may

also explain why anti-CD40 was superior to anti-DEC205 for targeting MHC class II

presentation. Given that an inverse relationship between antigen stability and formation of

peptide-MHC class II complexes is known to exist 33, it is perhaps not too surprising that a

similar situation might exist for cross presented antigens.




22

We were, however, surprised by the observation that targeting CD40 mediated

more efficient cross presentation than MR. Both receptors targeted internalized peptide-

antibody conjugates to early endosomes, but CD40 was substantially less efficient at

antibody uptake. The enhanced cross presentation was not due to maturation effects of

anti-CD40, despite the fact that CD40 stimulation can be important for activating cross

presentation by mouse DCs 6,30. The one obvious difference was in the localization of

antibodies even after extended time points. Whereas the bulk of anti-MR was internalized,

most anti-CD40 remained at the plasma membrane from which it was presumably

continuously but more slowly internalized, or to which it was more rapidly recycled.

Conceivably, maximum antigen stability may be achieved when antibodies are

internalized slowly to early and recycling endosomes. This limits the time that the

antibody-peptide conjugate spends intracellularly, where early endosomal proteases

(albeit fewer) gain access to antigens 41,42, and provides a continuous “time-release” pool

of antigen that might be used over extended periods for the continuous formation of

peptide-MHC class I complexes. Indeed, preliminary cross presentation data from another

slowly internalizing receptor, CD11c, appears to support this hypothesis (B.C. and I.M.,

unpublished data, February 1, 2012). Whatever the underlying mechanism, this

observation was unexpected and indicates that rapid endocytosis and antigen

accumulation may not always be the best criteria for choosing receptors to target vaccines

to DCs.

Our data may additionally point to some cell type specific differences in cross

presentation efficiency. Both DC types examined exhibited similar patterns of receptor

expression, localization, antibody accumulation, and cross presentation. However, while




23

MoDCs were more endocytic overall, they also appeared to be more degradative,

especially when we assayed for DEC205 targeting. We generally see that immature

MoDCs exhibit lower cross presentation efficiency than BDCA1+ DCs, and one reason for

this may be due to higher protease levels in MoDCs as compared to BDCA1+ DCs 28,29.

The existence of distinct DC subsets that have variable capacities for cross presentation

may mean that any such specializations could also be cell type specific. Indeed, recent in

vivo work in the mouse has demonstrated that as long as a given antibody (DEC205,

Langerin, CLEC9A/DNGR-1) can target CD8α+ cross presenting DCs, cross presentation

will occur 10. Recent studies on DC subtypes have implicated BDCA3+ DCs as the human

equivalent of the mouse CD8α+ DCs 43-46, a DC type that is key for efficient cross

presentation in vivo in mice 47-49. Initial data from these groups indicates that BDCA3+

DCs are superior for cross presentation, compared to BDCA1+ DCs. Our data indicate

that degradation of antigen affects the efficiency of cross presentation by human DCs,

including BDCA1+ DCs, and that slower antigen internalization to early endosomes may

be superior. Therefore, it may be possible that differential antigen handling by BDCA3+

DCs may be the reason for these differences, potentially through altered antigen

accumulation or degradation, or perhaps through increased access to the cytosol.

Further studies to elucidate the requirements for efficient cross presentation will be

key for efficient DC targeting. Since we see that multiple DC types are able to cross

present, it will be advantageous to target more than one type of DC simultaneously to

elicit the most efficient CD8+ T cell responses. Based on our results, this will best be

accomplished not only by taking into account surface receptor expression, but also by

determining the intracellular localization and degradation of the trafficking antibodies of




24

interest.




25

Authorship

Contributions: B.C. and I.M. designed the experiments with help from A.S.-S.; B.C.

performed the experiments; B.C. and I.M. analyzed the data; A.S.-S., L.D., and I.M.

supervised the project; L.C. assisted with primary DC isolation; C.C. assisted with imaging

analysis; R.V. and B-C. L. generated the antibody-peptide conjugates; J.W. and T.K.

provided the B11 and 3G9 antibodies; B.C. and I.M. wrote the paper; and all authors

approved this manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests. B.C.,

L.C., C.C., R.V., B-C. L, L.D., and I.M. are employees of Genentech. J.W. and T.K. are

employees of Celldex Therapeutics.

Acknowledgements We would like to thank the Genentech Research Blood Program and Blood Centers of the

Pacific for donor coordination and leukapheresis, as well as Laurie Gilmour and Yelena

Dayter for technical assistance with elutriations. We would especially like to thank

members of the Mellman lab for advice and fruitful discussions, and Peter Ebert for the

critical reading of this manuscript. The authors dedicate this manuscript to the memory of

our mentor and dear friend, Ralph Steinman.




26

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33. Delamarre L, Couture R, Mellman I, Trombetta ES. Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis. J Exp Med. 2006;203(9):2049-2055.




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34. Ziegler HK, Unanue ER. Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci U S A. 1982;79(1):175-178.

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41. Chapman HA. Endosomal proteases in antigen presentation. Curr Opin Immunol. 2006;18(1):78-84.

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29

Figure legends Figure 1: Human BDCA1+ DCs and MoDCs express mannose receptor, CD40, and

DEC205.

Donor-matched BDCA1+ DCs, or MoDCs were washed and incubated with (A) anti-

mannose receptor (MR), (B) anti-CD40 or (C) anti-DEC205 antibodies for 25 min at 4°C.

DCs were washed, fixed, and analyzed by flow cytometry. Histograms show one

representative of ten independent donors. (D) BDCA1+ DCs (closed symbols) and

MoDCs (open symbols) were incubated with Alexa 488 conjugated anti-MR, CD40, or

DEC205 for 25 min at 4°C, washed, and analyzed by flow cytometry. MFI was normalized

for the number of fluorophores per antibody, in order to directly compare surface levels

between receptors. The graph shows data from at least three independent donors with the

mean MFI depicted.

Figure 2: Targeting to DEC205 results in late endosomal localization while CD40

and mannose receptor antibodies are largely excluded from late endosomes.

(A-C) BDCA1+ DCs or (D-E) MoDCs were continuously incubated with 1 μg/mL of

fluorescently labeled (A and D) anti-DEC205, (B and E) anti-MR or (C and F) anti-CD40

antibodies for 6 h at 37°C. In parallel, DCs were incubated with antibodies in the presence

of protease and acidification inhibitors leupeptin and ammonium chloride (inh). DCs were

seeded on coverslips and fixed, followed by staining for the cell surface (HLA-DR) and

permeabilization and labeling of early endosomes (EEA1) or late endosomes (Lamp1).

Arrows indicate areas of overlap. Images were acquired on a Leica SP5 confocal




30

microscope, 100x oil objective (N.A: 1.47), zoom 7. All immunofluorescence experiments

were repeated in at least five independent donors. Scale bar is 5 µm.

Figure 3: Antibodies to DEC205 and mannose receptor are accumulated more

efficiently by DCs than anti-CD40.

(A) BDCA1+ DCs or (B) MoDCs were continuously incubated with 1 μg/mL of

fluorescently labeled anti-MR, anti-CD40, or anti-DEC205 for the indicated times, washed,

counterstained, and fixed (top panels). Middle and bottom panels: data from the top panel,

together with matched samples treated with leupeptin and ammonium chloride (inh)

during the continuous incubation. MuIso is the isotype for anti-CD40, while huIso is the

isotype for anti-MR and anti-DEC205. Graphs depict mean normalized MFI from at least

three independent donors ± SEM. Normalized MFI indicates amount of accumulation; MFI

was calculated by removing the contribution of surface fluorescence (4°C control) and

normalizing the MFI for the number of fluorophores per antibody. Differences in antibody

accumulation in the absence or presence of inhibitors were assessed using the paired t

test and statistically significant differences depicted (*), p<0.05.

Figure 4: Targeting CD40 and mannose receptor leads to superior cross

presentation compared to targeting DEC205.

(A) Schematic for the generation of antibody-peptide conjugates. On average, our peptide

conjugates had 1 peptide per antibody. (B) Matched immature or (C) mature HLA-

A*0201+ DCs were incubated with various concentrations of anti-CD40-M1, anti-MR-M1,

and anti-DEC205-M1 for 4-6 h, washed, and cocultured with autologous CFSE labeled




31

CD8+ T cells for 8-10 d in the presence of LPS and IL-2. “Immature” or “mature” refers to

the activation state of the DC prior to antigen uptake and coculture. MuIso-M1 is the

isotype for anti-CD40-M1, while huIso-M1 is the isotype for anti-MR-M1 and anti-DEC205-

M1. Graphs depict frequencies of total CFSElo and Influenza M1 (58-66) specific CD8+ T

cells. One representative experiment of six to eight independent donors (immature

samples) or three to four independent donors (mature samples) is shown. Targeting via

anti-CD40 and MR was judged superior to targeting via anti-DEC205 across six to eight

independent donors using a paired t test at 1 μg/mL for immature DCs (CD40 vs.

DEC205: MoDC p=0.0270, BDCA1+ DC p=0.0337; MR vs. DEC205: MoDC p=0.0264;

BDCA1+ DC was not statistically significant possibly due to variable surface MR

expression). (D) Influenza M1 (58-66) non-extended peptide control for antigen

presentation assay. (E) (Top panel) as in (B), DCs were incubated with anti-CD40-pp65 or

anti-DEC205-pp65 conjugates. (Bottom panel) CMV pp65 (495-503) non-extended

peptide control. (F) DCs were incubated with 1 μg/mL antibody-M1 conjugates or 25

ng/mL M1 peptide ± 0.1 μM epoxomicin or DMSO for 4-6 h, followed by washing and

coculture with autologous CFSE labeled CD8+ T cells. “No ag” shows the background

when DCs and CD8+ T cells are cocultured in the absence of antigen. Graphs depict

frequencies of total CFSElo and influenza M1 (58-66) specific CD8+ T cells. One

representative experiment from at least three independent donors is shown.

Figure 5: Targeting CD40 results in superior MHC class II presentation than

targeting DEC205.




32

(A) NY-ESO extended peptide sequence. The amino acid change from cysteine �

alanine is indicated in bold. (B) DPB1*0401+ BDCA1+ DCs were incubated with indicated

doses of irrelevant HIV reverse transcriptase peptide (HIV RTp), WT NY-ESO peptide

(WT NYESOp), or C�A mutated NY-ESO peptide (mut NYESOp) for 1.5 h, followed by

washing and coculture with an NY-ESO peptide specific CD4+ T cell clone. Percentage of

CD4+ T cells with intracellular IFNγ results are shown as one representative of three

independent DC donors. (C-E) 0.1-10 μg/mL antibody-NY-ESO peptide conjugates were

fed to BDCA1+ DCs for 1.5 h, followed by washing and coculture with an NY-ESO specific

CD4+ T cell clone at a DC: T cell ratio of 1:1, and subsequent staining for intracellular

cytokines. The percentage of CD4+ T cells that are positive for (C) IFNγ, (D) IL-2, and (E)

TNFα are depicted. One representative of three independent DC donors is shown. (F) As

in (C-E), using 1 μg/mL antibody-NY-ESO conjugates with varying DC:T cell ratios. The

percentage of CD4+ T cells that are positive for IFNγ is depicted. One representative of

three independent DC donors is shown.

Figure 6: Anti-CD40 does not enhance cross presentation of mannose receptor or

DEC205.

(A) 1 μg/mL of anti-CD40 (S2C6), anti-MR (B11), anti-DEC205 (3G9), isotype (iso), or 200

ng/mL LPS were added to MoDCs overnight. Following overnight culture, supernatants

were harvested and DCs were labeled for surface markers that are normally upregulated

following maturation, as indicated. One representative of three independent donors is

shown. (B) Supernatants from (A) were analyzed for cytokine production using Luminex

technology. IL-12p70 levels were insignificant, except for LPS controls. (C-D) MoDCs or




33

(E) BDCA1+ DCs were incubated with 1 μg/mL antibody-M1 peptide conjugates alone or

with antibody-M1 conjugates + 1 μg/mL unconjugated CD40 (S2C6) antibody or 1 μg/mL

isotype (muIso) together for 4-6 h, followed by washing and coculture with autologous

CFSE labeled CD8+ T cells. Graphs depict frequencies of total CFSElo and Influenza M1

(58-66) specific CD8 T cells. One representative of three independent donors is shown.

(F) BDCA1+ DCs were incubated with 1 μg/mL DEC205-NY-ESO peptide conjugates

alone or with DEC205-NY-ESO peptide conjugates + 1 μg/mL unconjugated CD40

(S2C6) antibody or 1 μg/mL isotype (muIso) together for 1.5 h, followed by washing and

coculture with an NY-ESO specific CD4+ T cell clone. Graphs depict percentage of IFNγ+

CD4+ T cells. One representative of two independent DC donors is shown.

Figure 7: DEC205 targeted cross presentation is rescued by the addition of

protease and acidification inhibitors.

4 μg/mL Alexa 488 covalently conjugated (A) anti-DEC205 (3G9), (B) anti-CD40 (S2C6),

or (C) anti-MR (B11) antibodies were internalized by MoDCs for 1 h at 37°C. Cells were

washed extensively and chased for indicated times ± leupeptin and ammonium chloride

(inh), followed by fixation and analysis by flow cytometry. Graphs depict the mean MFI of

three independent donors ± SEM. MFI differences in the absence or presence of inhibitors

were assessed using the paired t test and statistically significant differences depicted (*),

p<0.05. (D) Immature or (E) mature BDCA1+ DCs were incubated with 1 μg/mL DEC205-

M1 conjugates ± indicated inhibitors for 4-6 h, followed by washing and coculture with

autologous CFSE labeled CD8+ T cells. Graphs depict frequencies of CFSElo, influenza

M1 (58-66) specific CD8+ T cells. One representative of three independent donors is




34

shown for (D) and one representative of two independent donors is shown for (E). (F)

Immature BDCA1+ DCs were incubated with 1 μg/mL CD40-M1 conjugates ± indicated

inhibitors for 4-6 h, followed by washing and coculture with autologous CFSE labeled

CD8+ T cells. Graphs depict frequencies of total CFSElo and Influenza M1 (58-66) specific

CD8+ T cells. One representative of three independent donors is shown. (G) BDCA1+

DCs were incubated with 1 μg/mL DEC205-pp65 conjugates ± indicated inhibitors for 4-6

h, followed by washing and coculture with autologous CFSE labeled CD8+ T cells. Graphs

depict frequencies of CFSElo, CMV pp65 (495-503) specific CD8+ T cells. One

representative of two independent donors is shown. (H) CMV pp65 (495-503) non-

extended peptide control (25 ng/mL).




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