engineering customized anti-tumor immune response: biology of mhc class i restricted tcr engineered...
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Engineering Customized Anti-Tumor Immune Engineering Customized Anti-Tumor Immune Response: Biology of MHC Class I Restricted Response: Biology of MHC Class I Restricted
TCR Engineered CD4 T CellsTCR Engineered CD4 T Cells
Active Specific Vaccination StrategiesActive Specific Vaccination Strategies Adoptive Immunotherapy ApproachesAdoptive Immunotherapy Approaches
TumorTumorCellsCells
DCDC DCDC
Synthetic Synthetic Peptide/Peptide/Tumor Tumor LysateLysate
Pulsed DCPulsed DC
Gene Gene ModifiedModified
DCDC
Synthetic Synthetic PeptidePeptide
Gene Gene ModifiedModified
Tumor CellsTumor Cells
A.A. B.B.
Ex-Vivo Generation of Large Scale Immune EffectorsEx-Vivo Generation of Large Scale Immune Effectors
LAKLAK TILTIL CD8+CD8+CTL CloneCTL Clone
TCR/CARTCR/CAREngineeredEngineered
T CellsT Cells
T Cell Based Cancer Immunotherapy ApproachesT Cell Based Cancer Immunotherapy Approaches
TCR Engineering Approach
CD4+
MH
C I
MH
C II
CD8+
Antigen Presenting CellAntigen Presenting Cell
CD4+CD8+
CD8+ CD8+
Engineered Anti-tumor T Cells
Antigen Specific TCR
Most active specific and adoptive immunotherapy approaches for cancer center around CD8+ cytolytic T lymphocyte (CTL)-based responses, generated through immunization or transferred adoptively with ex vivo generated CTL.
Generating a robust and long-lived CTL response, however, needs “help” from CD4+ T helper cells.
The task of simultaneously engaging CD4+ T helper cells and CD8+ CTL is quite difficult, especially in an antigen specific manner, since: (i). For most human tumor antigens HLA matched MHC class I and MHC class II antigenic epitopes are not characterized yet. (ii). In addition, even if HLA matched MHC class I and class II epitopes are available for a target antigen, since CD4 T cells recognize their target antigenic epitope in context to MHC class II molecules and most human tumors are MHC class II negative, engaging CD4 T cells in tumor immunity is technically a very challenging task.
Our working hypothesis was that the MHC class II-restricted natural CD4 T cells can be programmed to provide antigen specific “help” in tumor immunity by engrafting them with a MHC class I-restricted tumor epitope-specific T cell receptor (TCR), derived from the CD8+ anti-tumor CTL.
Creation of MHC Class I Restricted CD4 T Cells
93.8%6.9%
CD8
MA
RT
-1
Tet
ram
er
(ii).
(i).
Generation of MART-1 27-35 Epitope Specific Oligoclonal Line
A.
C.
5’ LTR TCRF2A TCR WPRE3’ LTRsr39tkUbi P2A
FUW-M14.2-TCR/sr39tk
ControlVα2.2 Vβ14 TCR (M14.2 )
Vα2.1 Vβ14 TCR (M14.1 )
0.9% 7.2% 12.7%
Ma
rt-1
Te
tra
me
rCD3
B.
0.07% 68.6%50.6%48%
V1
4
0.17% 49.8%32.5%30.9%
Mar
t-1
tetr
amer
CD3
CD3
Control 10 MOI 50 MOI 100 MOI
Cloning and Characterization of MART-1 27-35 Epitope Specific TCRFrom M1 Specific CTL Line
I- I- Biology of MHC Class I TCR Driven Human CD4 T CellsBiology of MHC Class I TCR Driven Human CD4 T Cells
A.
B. C.
A375 A375-M PT-M JL-M A375 A375-M PT-M JL-M T2
M1 Epitope Specific MHC Class I TCR Engineered CD4 T Cells Exhibit An Antigen Specific Effector Function
9.5%9.5%
83.8%83.8%
0.66%0.66%
97.3%97.3%
Control Control M14.2 TCRM14.2 TCRTransducedTransduced
M1-
Tet
r am
erM
1- T
e tra
mer
CD4CD4
Transgenic TCR Engineered CD4 T cells, Derived From Three Different Donors Exhibit A Th1 Biased Effector Function
0
500
1000
1500
2000
2500
Cy
tok
ine
(p
g/m
l)
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
IL-2IL-2 IFN-IFN-γγ IL-4IL-4 IL-10IL-10
Donor-3Donor-3
0
500
1000
1500
2000
Cy
tok
ine
(p
g/m
l)
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
IL-2IL-2 IFN-IFN-γγ IL-4IL-4 IL-10IL-10
Donor-2Donor-2
IFN-γIFN-γ IL-2IL-2 IL-4IL-4 IL-10IL-100500100015002000250030003500C
ytokine (pg/ml)
MediumT2T2+M1T2+M3MediumT2T2+M1T2+M3MediumT2T2+M1T2+M3MediumT2T2+M1T2+M3
Donor-1Donor-1
0
500
1000
1500
2000
2500
3000
3500
Cyt
oki
ne
(pg
/ml)
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
MediumT2T2+M1T2+M3
A.
IFN-γ IL-2 IL-10
IFN-γ IL-2 IL-10
(i).
(ii).
(iii).
A375A375-M A375A375-M A375 A375-M
IFN-γ IL-2 IL-10
Isotype
HLA-A2
MART-1
HLA-A2 MART-1
HLA-A2 MART-1
HLA-A2 MART-1
HLA-A2 MART-1
A375
A375-MART-1
PT-M
JL-M
B.
Transgenic TCR Engineered CD4 T cells Exhibit An Antigen Specific Th1 Biased Effector Response Against Human Melanoma Cells
PT-MPT-M
JL-MJL-M
A.
C.
1ng 10ng 100ng 1μg 10μg 1ng 10ng 100ng 1μg 10μg
1ng 10ng 100ng 1μg 10μg
CD8
Tet
ram
er
14.8%
B.
Transgenic TCR Engineered CD4 T cells Display High Functional Avidity
1:100 1:50 1:10 1:1 10:1E:T
1:100 1:50 1:10 1:1 10:1E:T
Transgenic TCR Engineered CD4 T cells Display High Functional Avidity
A.
B.
0
10
20
30
40
50
60
6.2/1 12.5/1 25/1 50/1
Per
cen
t S
pec
ific
Lys
is
Effector (Total Population):Target
T2
Transgenic TCR Engineered CD4 T cells Exhibit Antigen Specific Cytolytic Effector Function
0
20
40
60
80
100
120
6.2/1 12.5/1 25/1 50/1Effector (Total Population): Target
Per
cen
t S
pec
ific
Lys
is T2T2+M1T2+M3
A.
B.
Transgenic TCR Engineered CD4 T cells Kill Tumor Cells
010203040506070
6.2/1 12.5/1 25/1 50/1Effector (Total Population):Target
Perc
en
t S
pecif
ic L
ysis PT
PT+M1PT+M3
Isotype
Granzyme
Isotype
Perforin
A.
B.
C.
Isotype
Granzyme
Isotype
Perforin
Isotype
T2T2+M3
T2+M1
(i). (ii).
Cytolytic Machinery Of MHC Class I TCR Engineered CD4 T Cells
1.1 1.61.7.9
CD107CD107
CD
8C
D8
T2T2 T2+M1T2+M1 T2+M3T2+M3
CD107CD107
CD
4C
D4
B.
A.
Anti-MHCI Anti-MHCII EGTA
Anti-MHCI Anti-MHCII EGTA
Lytic Activity Exhibited by TCR Engineered CD4 T cells Is MHC class I Restricted And Granule Exocytosis Mediated Process
• MHC I restricted TCR transduced CD4+ T cells Exhibit An Epitope Specific MHC I restricted TCR transduced CD4+ T cells Exhibit An Epitope Specific ``Helper Cytokine Response``. ``Helper Cytokine Response``.
• Engineered CD4 T cells also exhibit an antigen specific, MHC class I restricted Engineered CD4 T cells also exhibit an antigen specific, MHC class I restricted and granule exocytosis-mediated cytolytic effector response.and granule exocytosis-mediated cytolytic effector response.
Functional Profile of CD4 T Cells Engineered With A Functional Profile of CD4 T Cells Engineered With A Different MHC Class I Restricted Transgenic TCRDifferent MHC Class I Restricted Transgenic TCR
MAR
T-1
MAR
T-1
a.a. b.b. c.c. d.d. e.e.
CFSECFSE
B.B.
A.A.Untransduced Untransduced
Baseline BaselineTransducedTransduced StimulatedStimulated
CD4CD4
MAR
T-1
MAR
T-1
0.1%0.1% 43.9%43.9% 85.1%85.1%
Transgenic TCR Engineered CD4 T cells Exhibit A Significant Epitope Transgenic TCR Engineered CD4 T cells Exhibit A Significant Epitope Specific Proliferation PotentialSpecific Proliferation Potential
DCDC DC+M3DC+M3 DC+M1DC+M1
+MART-1 +MART-1 PeptidePeptide
No PeptideNo Peptide
MAR
T-1
Tetr
amer
MAR
T-1
Tetr
amer
CFSECFSE
a.a. b.b. c.c. d.d. e.e.
Transgenic TCR Engineered CD4 T cells Undergo Epitope Specific Transgenic TCR Engineered CD4 T cells Undergo Epitope Specific Proliferation Against Human Melanoma CellsProliferation Against Human Melanoma Cells
A375-M1A375-M1 A375A375 PT-MPT-M
Transgenic TCR Engineered CD4 T cells Exhibit Transgenic TCR Engineered CD4 T cells Exhibit Polyfunctional Cytokine ProfilePolyfunctional Cytokine Profile
1.31.3
97.397.3
28.728.7
58.058.0
2.72.7
11.111.1
1.41.4
92.792.7
IVCIVC IVC+CD4M1IVC+CD4M1 IVC+IVC+CD4 ControlCD4 Control
2.22.2
85.085.0
2.32.3
46.946.9
34.234.2
16.716.7
1.61.6
95.695.6
CD8CD8
Donor-1Donor-1
Donor-2Donor-2
Donor-3Donor-3
2.52.5
83.583.5
26.826.8
49.049.0
6.06.0
18.518.5
3.53.5
88.888.8
Fold IncreaseFold Increase
Tet
ram
erT
etra
mer
25.325.3
9.39.3
2.72.7
Transgenic TCR Engineered CD4 T cells Facilitate ``Help`` For Transgenic TCR Engineered CD4 T cells Facilitate ``Help`` For The Generation Of A Robust CD8+ CTL ResponseThe Generation Of A Robust CD8+ CTL Response
+Cytokine+Cytokine -Cytokine-Cytokine
1.01.0 2.22.2 0.450.45 1.01.0
IVCIVC IVC+CD4M1IVC+CD4M1 IVCIVC IVC+CD4M1IVC+CD4M1
Transgenic TCR Engineered CD4 T cells Facilitate ``Help`` For Transgenic TCR Engineered CD4 T cells Facilitate ``Help`` For The Generation Of A Robust CD8+ CTL ResponseThe Generation Of A Robust CD8+ CTL Response
In An In-Vitro CTL Generation Assay iTreg Mediate Immune SuppressionIn An In-Vitro CTL Generation Assay iTreg Mediate Immune Suppression
iTregiTreg
CD8+CD8+
MH
C I
MH
C I
APCAPC
CD8+ CTL ResponseCD8+ CTL Response
MHC Class I MHC Class I Restricted Restricted InteractionInteraction
CD8+CD8+
CD8+CD8+
CD8+CD8+
CD8+CD8+
MHC Class I TCR Engineered CD4 T Cells Can Mitigate The iTreg MHC Class I TCR Engineered CD4 T Cells Can Mitigate The iTreg Mediate Immune SuppressionMediate Immune Suppression
iTregiTreg
CD8+CD8+M
HC
IM
HC
I
APCAPC
CD8+ CTL ResponseCD8+ CTL Response
MHC Class I MHC Class I Restricted Restricted InteractionInteraction
CD8+CD8+
CD8+CD8+
CD8+CD8+
CD8+CD8+
CD4M1CD4M1
A.A.
Donor-1Donor-1 Donor-2Donor-2B.B.
A.A.
(i).(i).
(ii).(ii).
B.B.
A.A.
B.B.
CD86CD86
DCDCDC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
DCDCDC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
CD80CD80
DCDC
DC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
DCDC
DC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
MHCIIMHCII MHCIMHCI
DCDC
DC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
DCDC
DC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
CD40CD40
DCDCDC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
DCDCDC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
DCDC
DC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
DCDC
DC+CD4-CDC+CD4-C
DC+CD4-M1DC+CD4-M1
Total Gated Total Gated PopulationPopulation
CD11c+veCD11c+veGated Gated PopulationPopulation
(i).(i).
(ii).(ii).
IsotypeIsotype
IsotypeIsotype
IsotypeIsotype IsotypeIsotype
IsotypeIsotype
C.C.
Total Gated Total Gated PopulationPopulation
CD11c+veCD11c+veGated Gated PopulationPopulation
iDCiDC
mDCmDC
iDCiDCmDCmDC
iDCiDC
mDCmDC
iDCiDC
mDCmDC
iDCiDCmDCmDC
iDCiDCmDCmDC
iDCiDC
mDCmDC
iDCiDC
mDCmDC
iDCiDC
mDCmDC
iDCiDC
mDCmDC
CD86CD86 CD80CD80 MHCIIMHCII MHCIMHCI CD40CD40
SUMMARYSUMMARY
• MHC I restricted TCR transduced CD4+ T cells Exhibit An Epitope Specific MHC I restricted TCR transduced CD4+ T cells Exhibit An Epitope Specific ``Helper Cytokine Response``. ``Helper Cytokine Response``.
• These MHC class I-restricted tumor epitope specific TCR engineered CD4+ T These MHC class I-restricted tumor epitope specific TCR engineered CD4+ T cells can facilitate the generation of a robust CD8+ CTL response.cells can facilitate the generation of a robust CD8+ CTL response.
• Engineered CD4 T cells also exhibit an antigen specific, MHC class I restricted Engineered CD4 T cells also exhibit an antigen specific, MHC class I restricted and granule exocytosis-mediated cytolytic effector response.and granule exocytosis-mediated cytolytic effector response.
expa
nsio
n
contraction
memory
Mag
nit
ude
of r
espo
nse
TimeTime
(PCD/AICD)
II. II. Are MHC Class I TCR Engineered CD4 and CD8 T Are MHC Class I TCR Engineered CD4 and CD8 T Cells Susceptible to Epitope Specific AICDCells Susceptible to Epitope Specific AICD??
&&Can Long Lasting Anti-Tumor T Cells Be Generated Can Long Lasting Anti-Tumor T Cells Be Generated
By Interfering With The Premature AICD By Interfering With The Premature AICD ??
MHC Class I Restricted CD4 T Cells Undergo Epitope Specific AICDMHC Class I Restricted CD4 T Cells Undergo Epitope Specific AICDJust Like the TCR Engineered CD8+ T CellsJust Like the TCR Engineered CD8+ T Cells
M1 M1 M1
DiOC6DiOC6
Annexin (CD4)Annexin (CD4)
DiOCDiOC66
DiOCDiOC66
T2T2 T2+M3T2+M3 T2+M1T2+M1
M1
Tet
.M
1 T
et.
M1
Tet
.M
1 T
et.
56.356.3 5.05.0 56.756.7 5.15.1 15.015.0 33.233.2
1.91.9 66.066.0 3.93.9 66.566.5 18.118.1 44.344.3
12.512.5 10.610.6 34.934.9
(A).(A). (B).(B).
M1 M1 M1
9.19.1 9.79.7 27.327.359.359.3 58.058.0 30.930.9
9.59.5 3.13.1 40.940.9
DiOCDiOC66
M1-
Te
t.M
1-T
et.
51.551.57.57.5 2.02.0 21.221.2
DiOCDiOC66
M1-
Te
t.M
1-T
et.
Annexin (CD8)Annexin (CD8)
73.773.7 79.679.6
T2+M3T2+M3 T2+M1T2+M1T2T2
MHC Class I Restricted CD4 T Cells Undergo Epitope Specific AICDMHC Class I Restricted CD4 T Cells Undergo Epitope Specific AICDIn A Dose Dependent MannerIn A Dose Dependent Manner
M1 M1 M1
8.98.9 11.011.0 43.643.6
M1 M1 M1 M1
38.938.9 43.543.5 35.535.5 24.524.5
DiOCDiOC66
T2T2 T2+M3T2+M3 T2+M1T2+M1
31.331.3 3.03.0 31.331.3 3.43.4 6.76.7 13.813.8 16.316.37.17.1 14.514.59.99.9 7.17.123.223.2 6.66.624.824.8
M1
Te
t.M
1 T
et.
AnnexinAnnexin
31.5±0.931.5±0.9 3.3±0.53.3±0.5 30.6±0.630.6±0.6 3.7±0.53.7±0.5 6.6±0.76.6±0.7 14.7±0.814.7±0.8
10.7%10.7% 69.0%69.0%
16.2±0.116.2±0.16.9±0.16.9±0.1 14.4±0.114.4±0.19.2±0.79.2±0.7 7.3±0.27.3±0.222.4±0.622.4±0.6 6.7±0.36.7±0.324.6±0.424.6±0.4
70.1%70.1% 62.3%62.3% 24.2%24.2% 21.4%21.4%
25ug/ml25ug/ml 10ug/ml10ug/ml 1ug/ml1ug/ml 0.1ug/ml0.1ug/ml 0.01ug/ml0.01ug/ml
Comparative AICD Susceptibility in MHC Class I TCR Engineered Comparative AICD Susceptibility in MHC Class I TCR Engineered CD4 & CD8 T CellsCD4 & CD8 T Cells
Freshly TransducedFreshly Transduced Antigen ExperiencedAntigen Experienced
CD4M1CD4M1Freshly TransducedFreshly Transduced Antigen ExperiencedAntigen Experienced
CD8M1CD8M1
AICD In MHC Class I TCR Driven AICD In MHC Class I TCR Driven CD4 T Cells CD4 T Cells Is DR-IndependentIs DR-Independent
T2T2 T2+M3T2+M3 T2+M1T2+M1 IFN-g RIIFN-g RI TRAIL-RITRAIL-RI TRAIL-RIITRAIL-RII Fas/FcFas/Fc TNF-sRI/FcTNF-sRI/Fc
34.034.0 5.25.2 35.035.0 5.25.2 7.77.7 14.014.0 8.98.9 14.714.7 8.28.2 15.415.4 8.88.8 14.814.8 9.09.0 15.115.1 9.89.8 14.114.1
Annexin (CD4 T Cells)Annexin (CD4 T Cells)
M1
Tet
.M
1 T
et.
M1 M1 M1 M1 M1 M1 M1 M1
19.519.5 33.833.8 56.156.1 51.951.9 55.855.8 56.656.6 55.055.0 50.550.5
DiOCDiOC66
13.2%13.2% 12.9%12.9% 64.5%64.5% 62.2%62.2% 65.2%65.2% 62.7%62.7% 62.6%62.6% 58.9%58.9%
AICD In MHC Class I TCR Engineered AICD In MHC Class I TCR Engineered CD8 T Cells CD8 T Cells Is Also DR-IndependentIs Also DR-Independent
M1 M1 M1 M1 M1 M1 M1 M119.519.5 33.833.8 56.156.1 51.951.9 55.855.8 56.656.6 55.055.0 50.550.5
DiOC6DiOC6
T2T2 T2+M3T2+M3 T2+M1T2+M1 IFN-g RIIFN-g RI TRAIL-RITRAIL-RI TRAIL-RIITRAIL-RII Fas/FcFas/Fc TNF-sRI/FcTNF-sRI/Fc
AnnexinAnnexin
M1-
Tet
.M
1-T
et. 34.034.0 5.25.2 35.035.0 5.25.2 7.77.7 14.014.0 8.98.9 14.714.7 8.28.2 15.415.4 8.88.8 14.814.8 9.09.0 15.115.1 9.89.8 14.114.1
AICD In MHC Class I TCR Driven CD4 T Cells Involves JNKAICD In MHC Class I TCR Driven CD4 T Cells Involves JNK
B.B.
p-cJunp-cJun
ActinActin
T2T2 M3M3 M1M1 SPSP PDPD
C.C. (i).(i). (ii).(ii).
A.A.
21.721.7 21.721.732.832.8 19.019.021.121.1 23.923.953.753.7 5.35.354.754.7 5.05.0
T2T2 T2+M3T2+M3 T2+M1T2+M1 SPSP PDPD
Annexin (CD4)Annexin (CD4)
22.2±0.822.2±0.8 21.2±0.621.2±0.631.6±1.431.6±1.4 19.9±0.819.9±0.820.8±0.520.8±0.5 23.3±0.623.3±0.652.8±0.852.8±0.8 5.3±0.35.3±0.353.7±0.853.7±0.8 5.4±0.35.4±0.3
M1
6.36.3M1
7.17.1M1
27.227.2M1
18.918.9
M1
29.329.3
9.8±4.09.8±4.0 7.3±0.97.3±0.9 25.6±2.025.6±2.0 21.9±3.321.9±3.3 30.1±1.830.1±1.8
DiOC6DiOC6
91.6%91.6% 90.8%90.8% 47.1%47.1% 61.3%61.3% 51.0%51.0%
M1-
Te
t.M
1-T
et.
AICD In MHC Class I TCR Eng. CD8 T Cells Also Involves JNKAICD In MHC Class I TCR Eng. CD8 T Cells Also Involves JNK
A.A.
B.B.
p-cJunp-cJun
ActinActin
T2 M3 M1 SP PDT2 M3 M1 SP PD
cJuncJun
SPSP PDPDSPSP PDPD
C.C.(i).(i). (ii).(ii).
T2T2 T2+M3T2+M3 T2+M1T2+M1 SPSP PDPD
Annexin (CD8)Annexin (CD8)
M1-
Te
t.M
1-T
et.
33%33%44%44%33%33%91%91% 89%89%
32.032.0 31.731.7 6.56.52.72.7 3.03.0 16.816.8 12.212.2 15.015.0 6.46.4 12.912.9
M1 M1 M1 M1 M1
DiOC6DiOC6
5.05.0 3.13.1 50.750.7 32.332.3 44.544.5
AICD In Human Tumor Antigen Specific AICD In Human Tumor Antigen Specific MHC Class I TCR Engineered CD4 & CD8 T Cells MHC Class I TCR Engineered CD4 & CD8 T Cells
Is a caspase-independent, DR-independent, JNK Activation Driven Process.
Intrinsic Death Signal
JNK
Caspase dependent death
(via cytochrome-c)
Caspase independent death
(via AIF, EndoG etc.)
??
S-100HMC M1 C M1
AIF
Cyto-C
Hsp-60
Actin
Smac
Endo-G
Htr-A2
AICD In MART-1 Epitope Specific CTL Involves Selective AICD In MART-1 Epitope Specific CTL Involves Selective Release Of Mitochondria Resident Death Executioner, AIFRelease Of Mitochondria Resident Death Executioner, AIF
Control
AICD
SP
PD
AIF
AIF
AIF
AIF
Cytox green
Cytox green
Cytox green
Cytox green
Overlay
Overlay
Overlay
Overlay
Mitochondrio-nuclear Translocation of AIF during AICD and Mitochondrio-nuclear Translocation of AIF during AICD and blockade of this translocation by SP600125 blockade of this translocation by SP600125
Mitochondrio-nuclear Translocation of AIF during AICDMitochondrio-nuclear Translocation of AIF during AICD
C-ssDNA C-DAPI C-ssDNA+DAPI
AICD-ssDNA AICD- DAPI AICD-ssDNA+DAPI
4 hr 24 hr
ss-DNA
M1
M1
M1
M1
M1
M1
16.1
26.4
39.3
9.1
12.9
20.4
Control CTL
CTL+ Control Peptide
CTL+ M1 Peptide
M1
M1
M1PI
6.1
19.5
38.2
24 hr
Control CTL
CTL+ Control Peptide
CTL+ M1 Peptide
C M1
50 Kb
C M1
4.0Kb2.3Kb2.0Kb
0.5Kb
C Stau
4.0Kb2.3Kb2.0Kb
0.5Kb
PFGE
AICD In MART-1 Epitope Specific CTL Involves Large Scale AICD In MART-1 Epitope Specific CTL Involves Large Scale DNA Fragmentation, Characteristic of AIF Mediated DeathDNA Fragmentation, Characteristic of AIF Mediated Death
A.A. B.B.
C.C.
ssDNA AIF AIF+ssDNA AIF+ssDNA+DAPI
AIF Is Localized At The ssDNA Breaks In CTL Undergoing AICDAIF Is Localized At The ssDNA Breaks In CTL Undergoing AICD
AIF release in TCR driven Death of Jurkat cellsAIF release in TCR driven Death of Jurkat cells
HM Fractions-CD3C 30` 1hr 4hr 8hr 24hr 48hr
Actin
AIF
Hsp60
Cyt-C
S-100 Fractions-CD3
C 30` 1hr 4hr 8hr 24hr 48hr
Actin
AIF
Hsp-60
Cyt-C
4h 24h
Effect of SP600125 on AIF Release in TCR Driven Death of JurkatEffect of SP600125 on AIF Release in TCR Driven Death of Jurkat
HM Fractions-CD3
C 4h 24h 4h 24h 4h 24h CD3 CD3+SP CD3+PD
S-100 Fractions-CD3
Cyto-C
AIF
Actin
C 4h 24h 4h 24h 4h 24h
CD3 CD3+SP CD3+PD
How Does JNK activation regulates How Does JNK activation regulates Mitochondria-Centric AICD In MHC Class I Mitochondria-Centric AICD In MHC Class I TCR Driven Human Primary Anti-Tumor T TCR Driven Human Primary Anti-Tumor T
Cells?Cells?
Bim-EL
Actin
C M1 M3 SP PD
short fragment
AICD-pBim OverlayAICD-Mito
C-pBim OverlayC-Mito
Mitochondrial Relocalization of Bim and Generation of a Mitochondrial Relocalization of Bim and Generation of a Small pBim Fragment During AICDSmall pBim Fragment During AICD
C M1 M3
S-100 Fraction
C M1 M3
Heavy Membrane
JNK
Actin
AICD-pBim AICD-JNK Overlay
C-pBim C-JNK Overlay
Co-localization of Mitochondrial JNK and phospho-Bim in Co-localization of Mitochondrial JNK and phospho-Bim in CTL undergoing AICDCTL undergoing AICD
Interaction of Mitochondrial JNK with Bcl Family proteins and Interaction of Mitochondrial JNK with Bcl Family proteins and VDAC in Jurkat and CTL VDAC in Jurkat and CTL
C 30` 1h 4h 8h 24h 48h
Actin
JNK
Control- IP JNK IP
JNK
Mcl-1
Bcl-2
VDAC
C 4h 24hC 4h 24h
Bid
JNK
VDAC
C C M1
Control-IP JNK IP
VDAC
JNK
Control-IP VDAC IP
C C M1
Jurkat CTL
A.A. T2T2 T2+M3T2+M3 T2+M1T2+M1
57.757.7 4.74.7 56.656.6 6.86.8 16.016.0 15.215.2 24.024.0 25.225.2 20.020.0 43.243.2 10.410.4
1uM1uM 10uM10uM 20uM20uM
P53 Inh.P53 Inh.
Annexin (CD4)Annexin (CD4)
M1
Tet
.M
1 T
et.
B.B.
46.446.4 4.24.2 46.846.8 4.24.2 15.015.0 21.921.9 39.239.2 6.16.1
T2T2 T2+M3T2+M3 T2+M1T2+M1P53 Inh.-P53 Inh.-
20uM20uM
Annexin (CD4)Annexin (CD4)
44.7±1.644.7±1.6 4.0±0.64.0±0.6 46.2±0.646.2±0.6 4.5±0.44.5±0.4 15.5±0.615.5±0.6 21.3±1.421.3±1.4 38.4±0.838.4±0.8 6.9±0.66.9±0.6
M1
5.15.1M1
7.67.6M1
38.738.7M1
16.916.9
DiOCDiOC66
4.7±2.34.7±2.3 8.3±2.38.3±2.3 35.5±5.635.5±5.6 19.7±2.819.7±2.8
7.5%7.5% 10.7%10.7% 61.4%61.4% 61.2%61.2% 44.2%44.2% 19.4%19.4%
25.525.5
8.2%8.2% 8.7%8.7% 57.8%57.8% 15.2%15.2%
C.C.(i).(i).
(ii).(ii).
AICD In MHC Class I TCR Driven CD4 T Cells Involves p53AICD In MHC Class I TCR Driven CD4 T Cells Involves p53
A.A.
B.B.
T2T2 T2+M3T2+M3 T2+M1T2+M1 1uM1uM 10uM10uM 20uM20uM
40.540.5 40.140.1 24.124.1 25.125.1 46.546.5 50.050.07.67.6 10.110.1 18.118.1 16.816.8 8.78.7 10.710.7
Annexin (TCR Engineered CD8)Annexin (TCR Engineered CD8)
P53 Inh.P53 Inh.
M1-
Te
t.M
1-T
et.
C.C.
p53-up53-u PDPD
T2T2 T2+M3T2+M3 T2+M1T2+M1 P53 Inh.P53 Inh.
M1 M1 M1 M1
Annexin (TCR Engineered CD8)Annexin (TCR Engineered CD8)
M1-
Te
t.M
1-T
et. 42.642.6 6.26.2 43.943.9 5.75.7 7.57.5 33.533.5 50.650.6 18.318.3
5.95.9 10.410.4 59.859.8 18.318.3
DiOC6DiOC6
AICD In MHC Class I TCR Eng. CD8 T Cells Also Involves p53AICD In MHC Class I TCR Eng. CD8 T Cells Also Involves p53
Biology Of MHC Class I TCR Engineered CD4 T CellsBiology Of MHC Class I TCR Engineered CD4 T Cells
CD4+CD4+ CD8+CD8+
MH
C I
MH
C I
MH
C II
MH
C II
Transgenic Transgenic TCRTCR
EndogenousEndogenous TCRTCR
Antigen Presenting CellAntigen Presenting Cell
MHC Class I restricted MHC Class I restricted ``Multi-Functional`` CD4+ ``Multi-Functional`` CD4+ Anti-Tumor Effector T cellsAnti-Tumor Effector T cells
CD4+CD4+
MHC Class I restricted
MHC Class I restricted
``Helper response``
``Helper response``
Robust CD8+ CTL ResponseRobust CD8+ CTL ResponseGenerationGeneration
Mitochondria-centric Mitochondria-centric Epitope Specific AICDEpitope Specific AICD
BimBaxJnk
AIF AIF
SP600125TCR
AIF-MediatedDNA Degradation
AIF
AIF
?Mitochondrial JNK
Ligand
Cytoplasmic JNK
Jnk
Nucleus
UCLA-CALTECH-CHLA-USC-UCHC Consortium on Engineered Immunity UCLA-CALTECH-CHLA-USC-UCHC Consortium on Engineered Immunity
UCLAJames Economou
Antoni Ribas
UCHCBijay Mukherji
AcknowledgementsAcknowledgements
CALTECHDavid Baltimore
Non stim. Restim. by T2
Restim. by T2 /M1
Restim. by T2/M3
CD8
Annexin V
Mar
t-1
Tet
ram
er
41.34 41.52 31.17
0.6540.76 1.6242.32 16.9115.56 2.4037.74
38.71
0.66 0.06c 1.86 0.20b 13.38 1.9a 2.41 0.2d
17.5 0.07
CD8
Base line 10 days after stimulation
Mar
t-1
Tet
ram
er
AICD In MART-1 Epitope Specific Human Primary CTLAICD In MART-1 Epitope Specific Human Primary CTL
A.A.
B.B.
AICD In MART-1 Epitope Specific CTL Does Not Involve AICD In MART-1 Epitope Specific CTL Does Not Involve Death Receptor EngagementDeath Receptor Engagement
0 50 100 150
Control
T2/M3
T2/M1
T2/M1 + Fas-Fc
T2/M1 + TNF R-Fc
T2/M1 + IFN R-Fc
T2/M1 + TRAIL R-Fc
T2/M1 + z-VAD-fmk
% MART-1% MART-127-3527-35 Tetramer Tetramer++AnnexinAnnexin--
Uncleaved PARP
Cleaved PARP
Co
ntr
ol
+
Stau
ros
po
rine +
Stau
ros
po
rine+
SP
600125
+ S
tauro
spo
rine+
z-VA
D-
fmk
+ S
tauro
spo
rine+
S
PS
600125+
z-VA
D-fm
k
M3
p
ep
tide
M1
p
ep
tide
+
SP
60
01
25
M1
p
ep
tide
M1
p
ep
tide
+
SB
20
35
80
CT
L
alo
ne
Uncleaved PARP
Cleaved- caspase-3
Pro-caspase-3
C M1 M3
C Stau
C M1 M3
Cleaved-caspase-8Pro-caspase-8
C Stau
AICD In MART-1 Epitope Specific CTL Is A Caspase-AICD In MART-1 Epitope Specific CTL Is A Caspase-Independent ProcessIndependent Process
A.A. B.B.
% MART-127-35 Tetramer+Annexin- CTLs
a
b
c
d
p-C-Jun
C-Jun
Actin
C M1 M3 SP
AICD In MART-1 Epitope Specific Human Primary CTL AICD In MART-1 Epitope Specific Human Primary CTL Involves JNK And Inhibition Of JNK Activation Blocks Involves JNK And Inhibition Of JNK Activation Blocks
Premature AICD OF CTLPremature AICD OF CTL
A.A. B.B.
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