Engineering Customized Anti-Tumor Immune Response: Biology of MHC Class I Restricted TCR Engineered CD4 T Cells.

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.