P1252428413DizVe

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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

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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
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Longitudinal PBMC Analysis
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Kinetics of the Antigen Specific CD8 T cell
Response
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Kinetics of the Dbgp33-specific Response
Gated on Live CD8 T Cells
Day 8
Day 12
Day 15
Day 22
Day 34
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
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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
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
-Pep
gp33
gp276
gp118
gp365
86
93
83
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
37
20
13
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
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Recall Proliferative Capacity - Spleen Memory
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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
Recall Proliferative Capacity - Liver Memory
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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?

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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
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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
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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

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Why does Ad5 induce qualitatively sub-optimal
memory CD8 T cells?
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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
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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
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Acknowledgements
Rafi Ahmed
Surojit Sarkar Vandana Kalia Wendy Tan Bogna
Konieczny
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(No Transcript)
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PI Julie McElrath (FHCRC)

• How different adjuvants and vectors shape T-cell
  effector and memory responses?
• Quantity
• Function
• Location
• Duration

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Longitudinal PBMC Analysis
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Antigen Specific T-cell Kinetics
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Ag Specific Response
Gated on CD8 Positive cells
Day 22
Day 70
Day 47
Day 33
Day 15
Day 12
Day 8
LCMV
Ad5 1010
Db-gp33
Ad5 109
No detectable response
Ad5 108
CD8
35
CD127
CD127 Expression on GP33 Cells
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Bcl-2
Bcl-2 Expression in gp33 cells
37
CD62L
CD62L Expression on gp33 Cells
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Memory T Cell Responses in Tissues
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Absolute Number of Memory CD8 T Cells
40
Cytokine Production IFN-? Vs TNF-?
Gated on Live CD8 T Cells
-Pep
gp33
gp276
gp118
gp365
LCMV
1010 vp Ad5-gp
TNF-?
109 vp Ad5-gp
108 vp Ad5-gp
IFN-?
41
Cytokine Production IFN-? Vs IL-2
Gated on Live CD8 T Cells
-Pep
gp33
gp276
gp118
gp365
LCMV
1010 vp Ad5-gp
IL-2
109 vp Ad5-gp
108 vp Ad5-gp
IFN-?