Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact.
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS, MACSQuant, and PepTivator are registered trademarks or trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
Excerpt from MACS&more Vol 15 – 1/2013
Efficient and rapid in vitro generation of fully functional multi-virus-specific CD4+ and CD8+ T cellsAnna Foerster-Marniok, Verena Traska, Olaf Brauns, Sven Kramer, Jurgen Schmitz, Mario Assenmacher, and Anne RichterMiltenyi Biotec GmbH, Bergisch Gladbach, Germany
efficient and rapid in vitro generation of fully functional multi-virus-specific CD4+ and CD8+ T cells
Anna Foerster-Marniok, Verena Traska, Olaf Brauns, Sven Kramer, Jurgen Schmitz, Mario Assenmacher, and Anne RichterMiltenyi Biotec GmbH, Bergisch Gladbach, Germany
IntroductionAntigen-specific T cells play a critical role in the regulation of immune responses and the elimination of virus-infected or malignant cells in the human body. T cells are essential, e.g., for the control of the outgrowth of Epstein-Barr virus (EBV)-infected B cells. CD4+ and CD8+ T cells specific for the EBV antigens BZLF1¹-⁴ and EBNA-1⁵-¹¹ have been found in EBV-infected individuals. Virus-specific T cells also hold great potential for clinical use. The adoptive transfer of clinical-grade CD4+ and CD8+ T cells specific for adenovirus (AdV) hexon, cytomegalovirus (CMV) pp65, and EBV antigens is a valuable tool for the treatment of AdV¹², CMV¹³-¹⁶, and EBV¹⁷,¹⁸ infections after hematopoietic stem cell transplantation. To further enhance research on virus-specific T cells, we have established a protocol for the efficient and rapid generation of EBV-, AdV-, and CMV-specific (tri-virus-specific) T cells. The virus-specific CD4+ and CD8+ T cells were stimulated in vitro using peptide pools, and subsequently magnetically enriched according to their IFN-γ secretion. The tri-virus-specific T cell population could be easily expanded without major alterations in the proportions of the respective specificities.
Materials and methodsGeneration of multi-virus-specific T cells For the generation of multi-virus-specific T cells we stimulated 10⁹ PBMCs from leukapheresis products of several healthy
donors with a combination of four PepTivator® Peptide Pools (Miltenyi Biotec) covering CMV pp65 or IE-1, AdV hexon, or EBV EBNA-1 or BZLF-1 for four hours. The simultaneous addition of four peptide pools to a single
Figure 1 Experimental procedure for the generation of multi-virus-specific T cells. For details see the materials and methods section.
Generation of multi-virus-specific T cells
Separate antigen loading
Mixed antigen loading
Analysis for specificities
Intracellular cytokine staining after in vitro restimulation with a mixture and individual peptide pools
PBMCs PBMCs
1/4Loading
antigen 1
1/4Loading
antigen 2
1/4Loading
antigen 3
1/4Loading
antigen 4
Enrichment of IFN-γ–secreting T cells
Stimulation
T cell expansion
Stimulation with a mix of antigens
1+2+3+4
PBMCs
Enrichment of IFN-γ– secreting T cells
T cell expansion
Vol 15 • 1/2013 MACS & more24
RePoRT
www.miltenyibiotec.com
sample might decrease the activation efficacy for each peptide pool due to competition of peptides for MHC binding. As a control, we divided the PBMC samples into four aliquots, incubated each aliquot with a single peptide pool for two hours, and recombined the four aliquots for T cell stimulation for another four hours. Afterwards, we used the Large Scale IFN-γ Secretion Assay – Enrichment Kit, human, from Miltenyi Biotec to magnetically
Figure 2 Enrichment of multi-virus-specific T cells after stimulation with individual peptide pools or a combination thereof. PBMCs from four different donors were stimulated in two different ways as described in the materials and methods section: i) Cells were stimulated with a combination of pp65, IE-1, hexon, and BZLF1 peptide pools in a single batch (mixed antigen loading), or ii) cells were loaded with the individual peptide pools in four aliquots, and aliquots were subsequently combined for further stimulation (separate antigen loading). Subsequently, virus-specific T cells were enriched according to IFN-γ secretion. Before and after enrichment IFN-γ–secreting CD4+ and CD8+ T cells were analyzed by flow cytometry. (A) Results from one representative donor are shown. Numbers indicate frequencies among CD4+ or CD8+ cells. (B) Numbers of IFN-γ–positive CD4+ and CD8+ T cells enriched from PBMCs of four different donors.
Figure 3 Restimulation of enriched and expanded multi-virus-specific T cells. Enriched IFN-γ–secreting cells were expanded for 9 to 13 days. Subsequently, cells were restimulated with a mixture of peptide pools or were left unstimulated. Cells were analyzed for IFN-γ production by intracellular staining and flow cytometry. (A) Results from one representative donor are shown. Numbers indicate frequencies among CD4+ and CD8+ T cells, respectively. (B) Frequencies of IFN-γ–positive CD4+ and CD8+ T cells. Results from four different donors are shown.
CD4+ T cells
CD8+ T cells
Num
ber
of e
nric
hed
IFN
-γ+ T
cel
ls
per
10⁹
lym
ph
ocy
tes
1.4×10⁶
1.2×10⁶
1.0×10⁶
8.0×10⁵
6.0×10⁵
4.0×10⁵
2.0×10⁵
1.0×10³
Num
ber o
f IFN
-γ+
T c
ells
out
of 1
0⁹ ly
mph
ocyt
es
LP 3/1 mix
LP 3/1 separate
LP 19/2 mix
LP 19/2 separate
LP 6/2 mix
LP 6/2 separate
LP 11/2 mix
LP 11/2 separate
CD4+ T cells CD8+ T cells
LP 3/1 mixLP 3/1 separateLP 19/2 mixLP 19/2 separateLP 6/2 mixLP 6/2 separateLP 11/2 mixLP 11/2 separate
1×10⁶
1×10⁷
1×10⁵
1×10⁴
* among CD4+/CD8+ T cells
CD
4
0.42 %* 98.6 %*
Before enrichment
After enrichment
secreted IFN-γ
CD
8
1.45 %* 99.8 %*
Mixed antigen loading
CD
4
0.46 %* 98.4 %*
Before enrichment
After enrichment
secreted IFN-γ
CD
8
1.80 %* 99.5 %*
Separate antigen loading
0
10
20
30
40
50
60
70
80
90
100
% IFN-
_+ T cells
LP 3/1 mix
LP 3/1 separate
LP 19/2 mix
LP 19/2 separate
LP 6/2 mix
LP 6/2 separate
LP 11/2 mix
LP 11/2 separate
* among CD4+/CD8+ T cells
CD
4
Unstimulated sample
Stimulated sample
CD
8
CD
4
0.04%* 51.40%*
Unstimulated sample
Stimulated sample
intracellular IFN-γ
CD
8
0.08%* 61.74%* % IF
N-γ
+ T
cel
ls
CD4+ T cells
CD8+ T cells
0.11%* 31.39%*
0.03%* 33.99%*
Mixed antigen loading Separate antigen loading
select antigen-activated IFN-γ–secreting CD4+ and CD8+ T cells. Purities of enriched cells were determined by flow cytometry using the MACSQuant® Analyzer (Miltenyi Biotec). Enriched multi-virus-specific CD4+ and CD8+ T cells were expanded in the presence of IL-2 and feeder cells for 9–13 days. A flowchart illustrating the experimental procedure is shown in figure 1.
Analysis of PBMCs and multi-virus-specific T cells for antigen specificityFor the analysis of antigen specificity and functionality of the enriched, expanded multi-virus-specific T cells, we examined the IFN-γ response after short-term in vitro restimulation with individual peptide pools or a mixture thereof. For comparison, we also restimulated PBMCs. The IFN-γ response was determined by intracellular cytokine staining using an
A
A
B
B
Vol 15 • 1/2013 MACS & more 25
RePoRT
www.miltenyibiotec.com
Anti-IFN-γ-PE antibody (Miltenyi Biotec). CD4+ and CD8+ cells were then analyzed by flow cytometry using the MACSQuant Analyzer. Based on total cell numbers and the frequencies of IFN-γ–positive T cells among PBMCs and multi-virus-specific T cells upon restimulation with individual peptide pools, we calculated the relative frequencies and absolute cell numbers of T cells with a single antigen specificity.
Results and discussionMagnetic enrichment of multi-virus-specific CD4+ and CD8+ T cellsUsing the Large Scale IFN-γ Secretion Assay – Enrichment Kit, we were able to consistently enrich virus-specific CD4+ and CD8+ T cells to purities higher than 90%. Flow cytometric analyses of IFN-γ–secreting CD4+ and CD8+ T cells in PBMCs before and after magnetic enrichment are shown in figure 2A for one
representative donor. Prior to enrichment, cells were stimulated in two different ways: Cells were either stimulated with a combination of pp65, IE-1, hexon, and BZLF1 peptide pools in a single batch, or cells were loaded with the individual peptide pools in four aliquots, and aliquots were subsequently combined for further stimulation. Both stimulation procedures resulted in comparable frequencies of IFN-γ–secreting T cells before enrichment
Figure 4 Analysis of individual virus-specific T cell populations. Multi-virus-specific T cells were enriched as indicated in figure 2 and expanded. PBMCs and multi-virus-specific T cell lines were restimulated with individual peptide pools or a mixture thereof. The relative frequencies and absolute cell numbers of T cells with a single antigen specificity were calculated based on total cell numbers and the frequencies of IFN-γ–positive T cells among PBMCs and multi-virus-specific T cells upon restimulation with individual peptide pools. Data from different donors are shown as indicated.
LP 6/2 mix LP 6/2 separate
LP 3/1mix LP 3/1 separate
LP 19/2 mix LP 19/2 separate
LP 11/2 mix LP 11/2 separate
PBM
C
PBM
C
PBM
C
PBM
C
mix
mix
mix
mix
sep
arat
e
sep
arat
e
sep
arat
e
sep
arat
e
Rela
tive
freq
uenc
y of
sp
ecifi
c C
D4+
T c
ells
(%)
donor 3/1 donor 19/2 donor 11/2PB
MC
PBM
C
PBM
C
mix
mix
mix
sep
arat
e
sep
arat
e
sep
arat
ePBMC mix separate PBMC mix separate PBMC mix separate
Rela
tive
freq
uen
cy o
f sp
ecifi
c C
D4+
T c
ells
(%)
100
donor 3/1 donor 19/2 donor 11/2
BZLF1
EBNA-1
AdV hexon
IE-1
pp65
90
80
80
60
50
40
30
20
10
0
Rela
tive
freq
uenc
y of
sp
ecifi
c C
D8+
T c
ells
(%) donor 3/1 donor 19/2 donor 6/2 donor 11/2
PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate
Rela
tive
freq
uen
cy o
f sp
ecifi
c C
D8+
T c
ells
(%)
100
donor 3/1 donor 19/2 donor 6/2 donor 11/2
BZLF1
EBNA-1
AdV hexon
IE-1
pp65
90
80
80
60
50
40
30
20
10
0
BZLF1
EBNA-1
AdV hexon
IE-1
pp65
PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate
Rela
tive
freq
uen
cy o
f sp
ecifi
c C
D4+
T c
ells
(%)
100
donor 3/1 donor 19/2 donor 6/2 donor 11/2
BZLF1
EBNA-1
AdV hexon
IE-1
pp65
90
80
80
60
50
40
30
20
10
0
CMV pp65
Num
ber
of
IFN
γ+C
D4+
T c
ells
1×10⁴
1×10⁵
1×10⁶
1×10⁷
1×10⁸
PBMC afterexpansion
1×10⁴
1×10⁵
1×10⁶
1×10⁷
1×10⁸
CMV pp65
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
1×10⁷
CMV IE-1
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
EBV BZLF1
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
1×10⁷
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
1×10⁷
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
1×10⁷
PBMC afterexpansion
1×10⁴
1×10⁵
1×10⁶
1×10⁷
1×10⁸
AdV hexon
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
EBV EBNA-1
PBMC afterexpansion
1×10³
1×10⁴
1×10⁵
1×10⁶
1×10⁷
PBMC afterexpansion
CMV IE-1 AdV hexon EBV EBNA-1 EBV BZLF1
Num
ber
of
IFN
γ+C
D8+
T c
ells
A
B
Vol 15 • 1/2013 MACS & more26
RePoRT
www.miltenyibiotec.com
MACS Product* Order no.
PepTivator CMV pp65, humanPepTivator CMV IE-1, humanPepTivator AdV5 Hexon, humanPepTivator EBV EBNA-1, humanPepTivator EBV BZLF1, human
For further specificities visit www.peptivator.com
Large Scale IFN-γ Secretion Assay – Enrichment Kit, human
130-091-329
IFN-γ Secretion Assay – Cell Enrichment and Detection Kit (PE), human
130-054-201
MACSQuant Analyzer 130-092-197
Anti-IFN-γ, CD4, and CD8 antibodies Visit www.macsantibodies.com
Human IL-2 and other cytokines Visit www.macscytokines.com
* Products are for research use only.
(fig. 2A). Likewise, purities of enriched cells (fig. 2A and data not shown) and numbers of enriched cells (fig. 2B) were similar in the respective samples from all tested donors, regardless of whether the samples underwent mixed or separate antigen loading.
Specificity and functionality of the enriched and expanded multi-virus-specific T cellsWithin 9 to 13 days cell populations that were generated by either mixed or separate antigen loading expanded between 25- and 496-fold (data not shown). Both cell lines were restimulated with a mixture of peptide pools and analyzed for intracellular IFN-γ production. The percentage of IFN-γ–re-expressing CD4+ T cells derived from mixed antigen loading and separate antigen loading amounted to 23.8–90.5% and 32.5–84.8%, respectively. Similarly, the percentage of CD8+ T cells amounted to 31.8–88.0% and 34.0–79.2%, respectively (fig. 3A,B). These results confirm the high specificity and functionality of the T cell lines. T cell expansion rates and frequencies of T cells re-expressing IFN-γ were similar, regardless of whether the cell lines were originally generated by mixed or separate antigen loading (fig. 3B).
Individual virus-specific T cell populations show similar enrichment and expansion ratesThe strategy for the enrichment of virus-specific T cells from PBMCs is based on IFN-γ expression induced by stimulation with the peptide pools. Therefore, differences between stimulation efficacies of individual peptide pools would result in differences in IFN-γ responses of the respective virus-specific T cells, and ultimately in a biased proportion of specificities within the enriched multi-virus-specific T cell population. To ensure that the proportions of the different specificities are similar before and after enrichment, we compared the relative frequencies (fig. 4A) and absolute numbers (fig. 4B) of T cells specific for each single antigen in PBMCs and in the T cell lines. The relative frequencies of T cells specific for the individual CMV, EBV, and AdV antigens were about the same in PBMCs and in both T cell lines. This demonstrates that all individual virus-specific T cell populations within PBMCs were effectively stimulated and enriched. Moreover, regardless of whether the
original PBMCs were loaded with a mixture of peptide pools or separately with single peptide pools, the obtained T cell lines were comparable with respect to the expansion rate of the individual specificities.
Conclusion• AcombinationofseveralPepTivatorPeptide
Pools enables the simultaneous, effective activation of CMV-, EBV-, and AdV-specific (tri-virus-specific) CD4+ and CD8+ T cells.
• Activated tri-virus-specific T cells can beco-enriched using the Large Scale IFN-γ Secretion Assay – Enrichment Kit.
• Enriched tri-virus-specific T cells can beexpanded without significantly altering the proportion of the individual specificities.
• Enrichedtri-virus-specificTcellsarefullyfunctional and re-express IFN-γ upon restimulation.
References1. Bogedain, C. et al. (1995) J. Virol. 69: 4872–4879. 2. Saulquin, X. et al. (2000) Eur. J. Immunol. 30:
2531–2539. 3. Precopio, M.L. et al. (2003) J. Immunol. 170:
2590–2598. 4. Tynan, F.E. et al. (2005) J. Exp. Med. 202: 1249–
1260.5. Blake, N. et al. (2000) J. Immunol. 165: 7078–7087. 6. Khanna, R. et al. (1995) Virology 214: 633–637. 7. Paludan, C. et al. (2002) J. Immunol. 169: 1593–
1603. 8. Leen, A. et al. (2001) J. Virol. 75: 8649–8659. 9. Voo, K.S. et al. (2004) J. Exp. Med. 199: 459–470. 10. Voo, K.S. et al. (2002) Cancer Res. 62: 7195–7199. 11. Long, H.M. et al. (2005) J. Virol. 79: 4896–4907.12. Feuchtinger, T. et al. (2006) Br. J. Haematol. 134:
64–76.13. Feuchtinger, T. et al. (2010) Blood 116: 4360–4367.14. Einsele, H. et al. (2002) Blood 99: 3916–3922. 15. Mackinnon, S. et al. (2008) Blood Cells Mol. Dis.
40: 63–67. 16. Peggs, K.S. et al. (2011) Clin. Infect. Dis. 52: 49–
57. 17. Moosmann, A. et al. (2010) Blood 115: 2960–2970.18. Icheva, V. et al. (2013) J. Clin. Oncol. 31: 39–48.
Vol 15 • 1/2013 MACS & more 27
RePoRT
www.miltenyibiotec.com