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Ad5 Induced CD8 T Cells: Quantity and Quality

Ad5 Induced CD8 T Cells: Quantity and Quality. Surojit Sarkar. Do different vectors induce memory T-cells of different qualities?. PI: Julie McElrath (FHCRC). Ad5-GP (Gregory Spies) Ad5 backbone with E1 and E3 deletion Contains the full length LCMV glycoprotein LM-GP (Thomas Dubensky)

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Ad5 Induced CD8 T Cells: Quantity and Quality

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  1. Ad5 Induced CD8 T Cells: Quantity and Quality Surojit Sarkar

  2. Do different vectors induce memory T-cells of different qualities? PI: Julie McElrath (FHCRC)

  3. Ad5-GP (Gregory Spies) • Ad5 backbone with E1 and E3 deletion • Contains the full length LCMV glycoprotein • LM-GP (Thomas Dubensky) • LM backbone with Act A and Internalin B deletion • Contains CD8 epitope GP33-41 and CD4 epitope GP61-90 from the LCMV glycoprotein

  4. Experimental Set-up Days 8, 12, 15, 22, 30 and 45 Day 30 & 60 LM-gp SAC AND ANALYZE: -Size of Memory Response -Phenotypic Analysis -Functional Analysis -Tissue Localization -Recall Potential -Homeostatic Maintenance 1x107 cfu IV BLEED & ANALYZE: -Kinetics of the response -Phenotypic markers -Effector molecules -Survival factors Ad5-gp 1x1010 pfu IM LCMV 2x105 pfu IP

  5. Longitudinal PBMC Analysis

  6. Kinetics of the Antigen Specific CD8 T cell Response

  7. Kinetics of the Dbgp33-specific Response Gated on Live CD8+ T Cells Day 34 Day 8 Day 12 Day 15 Day 22 LM-gp Ad5-gp Db-gp33 LCMV CD8

  8. Granzyme B

  9. CD127 (IL-7 Receptor )

  10. Bcl-2

  11. Memory T Cell Responses in Tissues

  12. Red = MFI of expression Black = Percent Positive Day 32 Spleen - gp33 Gated on Dbgp33-41 Tetramer Positive Cells Gated on CD8+ Cells CD127 Bcl-2 PD-1 CD43 69 4 LM-gp 31 9 Ad5-gp Db-gp33 50 LCMV 4 CD8

  13. Red = MFI of expression Black = Percent Positive Day 32 Spleen - gp276 Gated on Dbgp276 Tetramer Positive Cells Gated on CD8+ Cells CD127 Bcl-2 PD-1 CD43 30 5 Ad5-gp Db-gp276 58 LCMV 4 CD8

  14. Day 32 - Tissue Distribution of Memory Gated on Live CD8+ T Cells PBL Spleen iLN Lung Liver LM-gp Ad5-gp Db-gp33 LCMV CD8

  15. Absolute Number of Memory CD8 T Cells

  16. Cytokine Production: IFN- Vs TNF- Gated on Live CD8+ T Cells +gp365 -Pep +gp33 +gp276 +gp118 86% 83% 93% 83% LCMV 36% 38% 60% Ad5-gp TNF- 87% LM-gp Red = % Double producers IFN-

  17. Cytokine Production: IFN- Vs IL-2 Gated on Live CD8+ T Cells +gp365 -Pep +gp33 +gp276 +gp118 25% 13% 37% 20% LCMV 6% 3% 11% Ad5-gp IL-2 19% LM-gp Red = % Double producers IFN-

  18. Recall Proliferative Capacity - Spleen Memory Day 50 Post-Infection Day 5.5 Post-Challenge LM Adoptively transfer equal numbers of gp33-specific SPLEEN cells into naïve mice (6000 Donor Cells) Analyze expansion of donor cells in Spleen and Liver Challenge with VV-33 Ad5 LCMV

  19. LM LM Ad5 Ad5 LCMV LCMV 0 50000 100000 150000 200000 0 5000 10000 15000 20000 Total Number of Donor Cells in Spleen Total Number of Donor Cells in Liver Recall Proliferative Capacity - Spleen Memory

  20. Recall Proliferative Capacity - Liver Memory Day 50 Post-Infection Day 5.5 Post-Challenge LM Adoptively transfer equal numbers of gp33-specific LIVER cells into naïve mice (6000 Donor Cells) Analyze expansion of donor cells in Spleen and Liver Challenge with VV-33 Ad5 LCMV

  21. LM LM Ad5 Ad5 LCMV LCMV 0 5000 10000 15000 20000 25000 30000 0 50000 100000 150000 200000 Total Number of Donor Cells in Liver Total Number of Donor Cells in Spleen Recall Proliferative Capacity - Liver Memory

  22. Conclusions • Both Ad5 and LCMV induce high levels of effector CD8 T cells during the expansion phase • Interestingly, there is less contraction in the Ad5 induced CD8 T cell response • Acquisition of memory properties (CD127/Bcl-2 upregulation) is impaired during an Ad5 response • Both lymphoid and non-lymphoid memory cells raised by Ad5 immunization were deficient in terms of cytokine production and recall proliferation • Used a high dose of Ad5 (1010 pfu = 5x1011 vp) • Will the Ad5 induced memory differentiation program change with lower doses of Ad5?

  23. Ad5 - Dose Response Days 8, 12, 15, 22, 30, 47, 70 Day 90 1010 vp (IM) SAC AND ANALYZE: -Size of Memory Response -Phenotypic Analysis -Functional Analysis -Tissue Localization -Recall Potential -Homeostatic Maintenance BLEED & ANALYZE: -Kinetics of the response -Phenotypic markers -Effector molecules -Survival factors 109 vp (IM) 108 vp (IM) 107 vp (IM) No detectable response

  24. Recall Proliferative Capacity Day 78 Post-Infection Day 5.5 Post-Challenge 108vp Ad5 Adoptively transfer equal numbers of gp33-specific SPLEEN cells into naïve mice (15000 Donor Cells) 109vp Ad5 Analyze expansion of donor cells in Spleen Challenge with VV-33 1010vp Ad5 LCMV

  25. Conclusions • Even at lower doses of Ad5, acquisition of memory properties appears to be impaired compared to LCMV and LM • Phenotype • Cytokine • Recall Proliferation: Lymphoid and Non-Lymphoid memory • Magnitude of Ad5 induced memory is similar compared to LCMV induced memory

  26. Why does Ad5 induce qualitatively sub-optimal memory CD8 T cells?

  27. Effector & Memory cells develop + Ag Effector cell death & Memory cell survival — Ag Expansion Contraction Relative CD8 T cell number Stabilization 0 8 15 22 30 60 Days Post-Infection CD8 T Cell Memory Differentiation Kaech et al., Cell 2002 Wherry et al., Nat. Immunol., 2003 Kaech et al., Nat. Immunol., 2003 Sarkar et al., J. Exp. Med., 2008

  28. Antigen Persistence after Ad5 Infection Days Post-Transfer Day 15 Post-Infection Day 1 Day 6 LCMV Naïve P14 Label Splenocytes With CFSE and Adoptively Transfer (CFSE Labeled Naïve Sensors) Ad5/E Ad5/GP CFSE

  29. Acknowledgements Rafi Ahmed Surojit Sarkar Vandana Kalia Wendy Tan Bogna Konieczny

  30. PI: Julie McElrath (FHCRC) How different adjuvants and vectors shape T-cell effector and memory responses? • Quantity • Function • Location • Duration

  31. Longitudinal PBMC Analysis

  32. 20 LCMV Ad5 1010 vp 15 Ad5 109 vp Ad5 108 vp 10 Percent of Db-GP33+ of CD8 5 0 0 10 20 30 40 50 60 70 Days Post-Infection Antigen Specific T-cell Kinetics

  33. Ag Specific Response Gated on CD8 Positive cells Day 8 Day 12 Day 15 Day 22 Day 33 Day 47 Day 70 LCMV Ad5 1010 Db-gp33 Ad5 109 No detectable response Ad5 108 CD8

  34. 100 90 LCMV 80 Ad5 1010 vp 70 Ad5 109 vp Ad5 108 vp 60 50 Percent CD127 Positive 40 30 20 10 0 0 10 20 30 40 50 60 70 Days Post-Infection CD127 CD127 Expression on GP33+ Cells

  35. 80 LCMV 70 Ad5 1010 vp 60 Ad5 109 vp 50 Ad5 108 vp 40 Bcl-2 MFI 30 20 10 0 0 10 20 30 40 50 60 70 Days Post-Infection Bcl-2 Bcl-2 Expression in gp33+ cells

  36. 120 LCMV 100 Ad5 1010 vp Ad5 109 vp 80 Ad5 108 vp 60 Percent CD62L Positive 40 20 0 0 10 20 30 40 50 60 70 Days Post-Infection CD62L CD62L Expression on gp33+ Cells

  37. Memory T Cell Responses in Tissues

  38. Absolute Number of Memory CD8 T Cells

  39. Cytokine Production: IFN- Vs TNF- Gated on Live CD8+ T Cells +gp365 -Pep +gp33 +gp276 +gp118 LCMV 1010 vp Ad5-gp TNF- 109 vp Ad5-gp 108 vp Ad5-gp IFN-

  40. Cytokine Production: IFN- Vs IL-2 Gated on Live CD8+ T Cells +gp365 -Pep +gp33 +gp276 +gp118 LCMV 1010 vp Ad5-gp IL-2 109 vp Ad5-gp 108 vp Ad5-gp IFN-

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