active stat5 promotes long lived cytotoxic cd8 t cells
TRANSCRIPT
Active STAT5 promotes long‐lived cytotoxic CD8 T cells that
induce regression of autochthonous mouse melanoma.
SITC 2011
Grégory Verdeil
No relationships to disclose
‐ Short‐lived CD8 T cells, poor tissue infiltration‐ High doses of IL‐2 = systemic action of IL‐2 (side effects)
T cell adoptive transfer for cancer immunotherapy
Reinfuse post-lymphodepletionHigh dose IL-2
TCR
CD3
pMHCagonist
APC
Verdeil et al. Journal of Immunology 2006
1
TATA
Pol II
GASAP-1
NF‐B
STAT5
STAT5
P
c
JAK1 JAK3
2
Sustained expression of genes implicated in survival and cytolysis
CD8 T cells
P
Activation of IL2/STAT5 sustains CD8 T cells effector functions
Naïve TCR transgenicCD8 T cell (TCRP1A)
Cytotoxic CD8 T cells
Expansion / differentiation
APC
Retroviral transduction of TCR transgenic CD8 T
cells
Use of STAT5CA to increase tumor elimination by CD8 T cells
Safety
Day
Day 21 in Immunocompetent hosts
0 102 103 104 105
0
102
103
104
105
20
LN
Spleen
Lung
GFP
CD8
0 102 103 104 105
0
102
103
104
105
Liver
3.1Hostcells
TCRP1ASTAT5CA
0 102 103 104 105
0
102
103
104
105
60
0 102 103 104 105
0
102
103
104
105
71
Lymphoidorgans
Non‐lymphoidorgans
Preferential expansion and tissue‐homing of STAT5CA ‐T cells
STAT5CAGFP
GFP
Day 0 Day 18
WT B10D2(immunocompetent)
0.1
1
10
100
0 10 20 30 40 50 60 70 80 90
Days after transfer
% of C
D8 GFP+ am
ong PB
Ls
1001 00010 000
STAT5CA expressing T cells are maintained at a quiescent G0 state
Days after transferin Rag‐1°/° hosts
control TCRP1A effector
STAT5CA TCRP1A effector
STAT5CA TCRP1A effctor
68.74.2
20.8
24.21.1
71.6
98.10.1
1.2
d3 d3 d14 Days after transferin Rag‐1°/° hosts
7AAD
BrdU
S
G0/G1 G2/M
BrdU injected ip 24h before analysis
Rag-/-(immunodeficient)
Inducible melanoma model (TIRP mice)
Huijbers et al. Cancer research 2006;Soudja et al. Cancer research 2010
Amela Aggressive
P1A+
In melanocytesHRasG12V onP1A cancer‐germline Ag onInk4a/Arf off
36%143 days of latency
WT(immunocompetent)
The adaptive immune system controls tumor growth in TIRP mice.
72%82 days of latency
P1A+
In melanocytesHRasG12V onP1A cancer‐germline Ag onInk4a/Arf off
Rag-/- (immunodeficient)
Amela tumors are strongly immunosuppressive
Positive controlAmela tumor
Loss of functionnal properties
VEGF
IL‐10
IL‐6
CD11b Gr1
Arginase‐1+Cox‐2+
Myeloid Derived Suppressor Cells
G‐CSF
Endogenous CD8 T cells have an
exhausted phenotypeInside the tumor
Immunosuppressive environment
Expression of inhibitory receptors
Naïve TCR transgenicCD8 T cell (TCRP1A)
Cytotoxic CD8 T cells
Expansion / differentiation
APC
Retroviral transduction of CD8 T cells to express
STAT5CA
Use of STAT5CA to increase tumor elimination by CD8 T cells
P1A+
HRasG12V onP1A cancer‐germline Ag onInk4a/Arf off
Rag-/- (immunodeficient)
7 days post‐treatment
STAT5CA expressing T cells induce melanoma regression in Rag‐/‐ TiRP mice
Grange et al. Cancer research in press
STAT5CA‐T cells remain active in the melanoma immunosuppresive environment
TILs
CD8
FSC‐A
GzmB
TCRP1A‐STAT5CA1x106
TCRP1AGFP 1x106
TCRP1AGFP 7x106
Grange et al. Cancer research in press
TILs analysis
TCRP
1AeTC‐STAT
5CA
TCRP
1A eTC
TCRP
1A eTC
+ IL‐2c
STAT5CA T cells are more efficient than IL‐2 complexes to induce tumor regression
immunocompetent
Luc+ transplanted melanoma
Grange et al. Cancer research in press
EndogenousTCs
Transferred TCRP1A TCs
Conclusions
-STAT5CA transduced T cells show high cytolytic potential and capacity to migrate into tissues/tumors
- STAT5CA transduced T cells remain dependent on the presence of the antigen for restimulation
- STAT5CA transduced T cells induce strong regression of an autochthonous melanoma with high immunosuppressive properties
- What are the mechanisms involved in resistance of STAT5CA transduced T cells to immunosuppression?
CIMLAnne-Marie Schmitt-Verhulst’s Lab
Nathalie Auphan-AnezinMagali GrangeMichel Buferne
Marielle MelloMarilyn Giordano
Annick GuimezanesAmandine MasClaude Boyer
Pierre Mouchacca
Saïdi M’homa SoudjaMaria Wehbe
T cell adoptive transfer for cancer immunotherapy
STAT5-CA ?No need of IL‐2 injections= Targeted action on CD8 Te
Long‐lived CD8 Te (>d140) Tissue infiltrationFor safety: coupling to a suicide gene
NaïveCD8 TC
Antigenpriming
Early CD8 effector TC
Lymphoid organs Peripheral Tissues
Full CD8 effector TC
CD62L-
CCR7-
IL-7Rα-
IL-2Rα+CD44+IL-2Rβ+GzmBhi
KLRG1++
Central memory CD8 TC
CD62L+
CCR7+
IL-7Rα+
CD44 +IL-2Rβ +IL-2Rα _
GzmBlo
KLRG-1-
Inducible functionsHigh replicative potential
Lymphoïd organs
EffectorMemory CD8 TC
CD62L-
CCR7-
IL-7Rα-
CD44 +IL-2Rβ +IL-2Rα _
GzmBhi
KLRG-1+
Immediate functionsPoor replicative potential
Tissue Homing
+Stat5*
Memory CD8 eTC-STAT5CA
CD62L-
CCR7-
IL-7Rα-
CD44+IL-2Rβ+IL-2Rα +GzmBhi
KLRG-1-
Tissue Homing Immediate functionsHigh replicative potential
Immediate functionsShort-lived
Tissue Homing
Recipients = LysoM eGFP Ly5.1 B10.D2
D0: 106T429‐luc retro‐peritoneal
TCRP1A Te (106 cells)
TCRP1ASTAT5CA Te (106 cells)
Day 21
Adoptive TransferDay 24 Day 24+1 Day 24+7 Day 24+10
analysis
STAT5CA TCRP1A CD8 T cells also reject a P1A+ melanoma in immunocompetent hosts
STAT5CA expressing CD8 T cells show a higher expansion and tumor infiltration than non manipulated CD8 Te counterparts
TCRP1Aeffectors
TCRP1ASTAT5CAeffectors
Host CD8 TLGzmB
No Ph emission but small residual tumor
CRP1A CD8 eTC‐STAT5CAnduce regression of a1A+ transplanted melanoma in
immuno‐competent hosts
TCRP1A eTC‐STAT5CA i.v.Or TCRP1A eTCOr TCRP1A eTC + IL‐2c (day+1;+2;+3;+5;+7;+9)
Monitoring of tumor growth by bioluminescence
Day 0
Analysis day +11 post‐ adoptive transfer
LysoM‐eGFP B10.D2
TCRP1A CD8 eTC‐STAT5CA show a higher intra‐tumor accumulation than non manipulated CD8 eTC, even when coupled to IL‐2c infusions.
TILs analysisAdoptive therapy in mice bearing a transplanted melanoma
TCRP
1AeTC‐STAT
5CA
TCRP
1A eTC
TCRP
1A eTC
+ IL‐2c
Transferred TCs EndogenousTCs
TCRP1A eTC‐STAT5CA
TCRP1A eTCTCRP1A eTC + IL‐2c
Compared to endogenous CD8 TILs, TCRP1A eTC‐STAT5CA expressed higher levels of GzmB and responded efficiently to recall restimulation.
Transfer of TCRP1A eTC‐STAT5CA Transfer of TCRP1A eTC Transfer of TCRP1A eTC + IL‐2c
Current adoptive therapies use highly differentiated effector CD8 T cells
ex vivo expansion
T cell culture + IL‐2 + Ag High number of T cells
with high doses of IL‐2= systemic action of IL‐2/dise effects (including on Tregs)
Short‐lived CD8 Te (up to d14)Poor tissue infiltration
T cell culture +STAT5CA
STAT5CA based adoptive therapies promote less-differentiated and long-lived effector CD8 T cells
Low number of T cellsNo need of IL‐2 injections= Targeted action on CD8 Te
Long‐lived CD8 Te (>d140) Tissue infiltrationFor safety: coupling to a suicide gene
(From Rosenberg et al)
No further expansion
21
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STAT5CA expressing CD8 T cells have a T effector memory phenotype
MOMA B220 GFP
In correlation with CCR7 down‐modulation, STAT5CA transduced T cells are found in the red pulp(d6 after transfer in B10D2 syngenic mice, spleen)
100 101 10 2 10 3 1040
20
40
60
80
100
10 0 10 1 10 2 10 3 10 40
20
40
60
80
100
100 101 102 103 1040
20
40
60
80
100
CD122100 101 102 10 3 10 4
0
20
40
60
80
100
CD127100 101 10 2 10 3 10 4
0
20
40
60
80
100
CD25
CD44 GzmB
100 101 10 2 10 3 10 40
20
40
60
80
100
CD62L CCR70 10 2 10 3 10 4 10 5
0
20
40
60
80
100
CD8 Tn
TCRP1ASTAT5CA Te (d41)TCRP1ATe (d41)
Splenocytes
Inducible melanoma model (TiRP mice)
Inducible melanomaP1A+
TiRP mice
HRasG12V onP1A cancer‐germline Ag onInk4a/Arf off
Huijbers et al.2006; Soudja et al. 2010
Mela (pigmented)
Amela
(unpigmented)
Incidence
(Number/%)
29
(8.0 %)
94
(26.0 %)
Latency (days)
159 + 22
163 + 17
Growth
(mm3/day)
12 + 8
98 + 19
Amela
AggressiveMela Slow progressing
Les tumeurs Amela sont associées à l’ inflammation
Mela Amela
SérumMultiplex
TiRP-/-
traitées au 4OH
ELISA
MelaAmela
0
5
10
15
20
25
*
*
***
***
**
**ns
*
**
Ratio
compa
réau
x so
uris c
ontr
ôles
IL-1α IL-1βIL-2 IL-4 IL-5 IL-6 IL-10IL-12 IL-13 IL-17 FGFGM-CSF G-CSF VEGF KC MCP-1 MIP1γ MIG IP10 INFγ TNFα
*
M’homa Soudja S. Wehbe M. Mas A. 2009
Les tumeurs Amela sont associées à l’ inflammation
Mela Amela
SérumMultiplex
TiRP-/-
traitées au 4OH
ELISA
MelaAmela
0
5
10
15
20
25
*
*
***
***
**
**ns
*
**
Ratio
compa
réau
x so
uris c
ontr
ôles
IL-1α IL-1βIL-2 IL-4 IL-5 IL-6 IL-10IL-12 IL-13 IL-17 FGFGM-CSF G-CSF VEGF KC MCP-1 MIP1γ MIG IP10 INFγ TNFα
*
Mobilisation M’homa Soudja S. Wehbe M. Mas A. 2009
Les tumeurs Amela sont associées à l’ inflammation
Mela Amela
SérumMultiplex
TiRP-/-
traitées au 4OH
ELISA
MelaAmela
0
5
10
15
20
25
*
*
***
***
**
**ns
*
**
Ratio
compa
réau
x so
uris c
ontr
ôles
IL-1α IL-1βIL-2 IL-4 IL-5 IL-6 IL-10IL-12 IL-13 IL-17 FGFGM-CSF G-CSF VEGF KC MCP-1 MIP1γ MIG IP10 INFγ TNFα
*
RecrutementM’homa Soudja S. Wehbe M. Mas A. 2009
MelaAmela
0
5
10
15
20
25
*
*
***
***
**
**ns
*
**
Ratio
compa
réau
x so
uris c
ontr
ôles
IL-1α IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IL-17 FGFGM-CSF G-CSF VEGF KC MCP-1 MIP1γ MIG IP10 INFγ TNFα
*
Les tumeurs Amela sont associées à l’ inflammation
Mela Amela
SérumMultiplex
TiRP-/-
traitées au 4OH
ELISA
Polarisation de la réponse immunitaire (Th2/Th17) M’homa Soudja S. Wehbe M. Mas A. 2009