Life and Death of CD8 T Cells: A Role of IL-12 Jun Chang Research Institute, National Cancer Center

Immune Surveillance and Tumors Tumor growth Immune Boost (Breaking Tolerance/evasion) Immune surveillance Tumor growth Immune evasion Immune selection Protective immune responses and surveillance to tumors definitely exist Many tumors have been immune-selected to evade immune elimination T cells are a critical mediator of tumor immunity Successful immunotherapy requires immune boost to overcome tumor-induced immune evasion

Processing and presentation of exogenous and endogenous antigens

Life cycle of CD8 T cells CTL tumor cell CD8 T cell stimulation Proliferation and differentiation CTL tumor cell Memory development Effector function

(adoptive cell transfer) Cancer Immunotherapy: dream can come true? Dendritic cells mAb DC Cancer Immunotherapy Cell therapy Non-cell therapy TH TC Vaccines, Cytokines T cells (adoptive cell transfer)

Dudley and Rosenberg, Nature Reviews Cancer, 3:666 (2003) Adoptive T-cell transfer as immunotherapy 13 HLA-A2 melanoma patients Expansion of selected T cells with autologous APC+Ag and aCD3 ab Chemotherapy + High dose IL-2 6 partial response (Greater than 50% reduction in all lesions) 4 partial response in some legion with progression at other sites 3 no objective response 5+/8- autoimmunity (Vitiligo/Uveitis) Dudley and Rosenberg, Nature Reviews Cancer, 3:666 (2003)

3 signals required for the generation and maintenance of CD8 T cells? APC B7 TCR CD28 MHC I naïve CD8+ IL-7R IL-15 Other cytokines? IL-15R IL-7 Signal 1 : MHC I  TCR signal Signal 2 : Costimulatory signal Signal 3 : Cytokine signal? ? TC Memory CD8+ CTL

The biological functions of IL-12 Pathogen DC/MΦ : (p40) : (p40)2 : IL-12 (p70) : (p35) (p40),(p40)2 IL-12 (p70) + + stimulation inhibition CD8+ (Tc) CD4+ (Th0) Th1 immunity IFN-g CTL response

STRATEGY: Mutation that reduces p40 but not p70 production STRATEGY: Mutation that reduces p40 but not p70 production? (N-glycosylation sites) potential N-glycosylation sites DC/MΦ hp40 IL-12p40 125 135 222 303 hp40-N125Q NQ (p40),(p40)2 IL-12m (p70) hp40-N135Q NQ hp40-N222Q NQ hp40-N303Q NQ + + stimulation hp40-N135,222Q NQ NQ CD8+ (Tc) CD4+ (Th0) hp40-N135,303Q NQ NQ hp40-N222,303Q NQ NQ CD8+ (Tc) CD4+ (Th0) hp40-N135,222,303Q NQ NQ NQ

N222 mutation indeed alters p40 secretion IL-12p40 125 135 222 303 IL-12p40 Western blot hp40-N135,222,303Q hp40-N135,222,303Q hp40-N135,222Q hp40-N135,303Q hp40-N222,303Q hp40-N135,222Q hp40-N135,303Q hp40-N222,303Q hp40-N125Q hp40-N135Q hp40-N222Q hp40-N303Q hp40-N125Q hp40-N135Q hp40-N222Q hp40-N303Q hp40 (tu) hp40 (tu) hp40 hp40 neo neo 49 kDa 43.1 40 37.1 35 33.2 kDa Cell Lysate Supernatant

Secretion profile and bioactivity of IL-12 mutant IL-12 ELISA 150 100 IL-12p40 IL-12p70 120 100 98 Relative secretion (%) 90 60 30 2.2 IL-12wt IL-12mut (N222) IFN-g ELISA

Does IL-12 mutant bind its receptor equivalently? QUESTION: Does IL-12 mutant bind its receptor equivalently? CD4 IL-12 or mutant Incubation on ice CD4 aIL-12 Ab

IL-12 mutant induces stronger CTL response in vivo? QUESTION: IL-12 mutant induces stronger CTL response in vivo? p40 IRES p35 HCV E2 BALB/c mice p40-N222L IRES p35 HCV E2 CTL assay i.m. DNA injection & boosting TC TC TC Intracellular IFN-g staining CTL in spleen

Quantitation of antigen-specific T cells using intracellular IFN-g staining CD8 IFN-g Cytokine production Secretion inhibitor Anti-IFN-g staining Permeabilize

E2-specific IFN-g CD8+ T cells E2-specific IFN-g CD8+ T cells The IL-12 mutant induces long-term memory 0.8 0.7 0.6 E2-specific IFN-g CD8+ T cells / total CD8+ T cells (%) 0.5 0.4 0.3 0.2 0.1 3 6 10 14 G I : mock DNA G II : E2 DNA G III : E2 DNA + IL-12wt DNA G IV : E2 DNA + IL-12mut DNA Weeks after booster immunization 0.6 0.5 0.4 E2-specific IFN-g CD8+ T cells / total CD8+ T cells (%) 0.3 0.2 0.1 4 8 14 Weeks after single immunization

Effect of IL-12 mutant on E2-specific CTL responses G I G II G III G IV Target cells : CT26-E2t 100 a 0 week d 10 weeks 80 60 40 20 100 b 3 weeks e 14 weeks 80 Specific lysis (%) 60 40 20 100 c 6 weeks f 14 weeks 80 60 40 Target cells : CT26-neo 20 10 : 1 30 : 1 10 : 1 30 : 1 E : T ratio E : T ratio

Frequency kinetics of E2-specific CTLs (Limiting dilution assay) Frequency of precursor CTL / 107 spleen cells 10 20 30 40 50 60 70 80 3 6 14 Weeks after booster immunization G I : mock DNA G II : E2 DNA G III : E2 DNA + IL-12wt DNA G IV : E2 DNA + IL-12mut DNA 10 20 30 40 50 60 4 8 14 Weeks after single immunization Frequency of precursor CTL / 107 spleen cells

Protection of the immunized mice against challenge with lethal dose of tumor expressing E2 G I : mock DNA G II : E2 DNA G III : E2 DNA + IL-12wt DNA G IV : E2 DNA + IL-12mut DNA * 2000 4000 6000 8000 12 15 18 21 24 27 30 Days after tumor challenge Mean tumor volume (mm3) 20 40 60 80 100 10 30 50 Days after tumor challenge Survival (%) CT26-E2 tumor challenge CT26-E2 tumor challenge

IL-12 acts as an “adjuvant” for memory CTL? DC/MΦ : (p40) : (p40)2 : IL-12 (p70) : (p35) (p40),(p40)2 IL-12m (p70) + + stimulation CD8+ (Tc) CD8+ (Tc) CD8+ (Tc) IFN-g longer CTL response

HYPOTHESIS: IL-12 is required for the sustained memory CTL response? APC naïve CD8+ TCR costimulation MHC I IL-7R +Ag Memory CD8+ CTL Exogenous IL-12 Prolonged memory CTL response? ? TC

Experimental scheme: in vitro stimulation and adoptive cell transfer OVA peptide (SIINFEKL) 72h i.v. 2 x 106 naïve OT-I APC Va2 H-2Kb MACS to purify CD8+ Vb5 IL-12 Activated OT-I transfer into C57BL/6 mice OT-I TCR transgenic CD8+ T cells + peptide ± IL-12 (5 ng/ml) Primary (day 3) and Memory (day 70) response Flow cytometry Confocal Microscopy CTL assay

MHC I tetramer technology easily distinguishes Antigen-specific CD8 T cells OT-I TCR Transgenic model Before transfer (day 0) OT-I OVA tetramer-PE OVAp (SIINFEKL) Other CD8+ After transfer (day 3) OT-I CD8 After transfer (day 70) Other CD8+ OVA tetramer-PE

IL-12 priming increases CD8 T-cell effector and memory populations Spleen LN OT-I cell number (10-6) 5 10 15 20 25 30 35 1 2 3 4 6 CD8 naive OVAp OVAp /IL-4 OVAp /IL-12 OVAp /IL-15 Day 3 Day 70 Day 3 Day 70 Day 3 Day 70 OT-II cell number (10-6) 0.5 1 1.5 2 2.5 3 0.1 0.2 0.3 0.4 CD4

IL-12 priming increases CD8 T-cell effector and memory populations naive OVAp OVAp/IL-12 Anti-Vb5-PE 2.09% 30.01% 77.35% Anti-Va2-FITC Day 3 OT-I Anti-Vb5-PE 1.37% 11.52% 52.38% Day 32 Other CD8+ OT-I Anti-Vb5-PE 0.34% 0.95% 23.31% Day 70 Other CD8+ Anti-Va2-FITC Anti-Va2-FITC

Surface marker expression on IL-12-primed OT-I 24h~72h after activation (CD8+ gated) CD25 CD44 CD94 OVAp OVAp/IL-12 NKG2 Ly49C/I

IL-12-primed cells exhibit down-regulation of CD69 CD69, CFDA stain (CD8 gate) 6h 24h 48h 60h 72h 53.9 60.6 68.2 64.7 50.0 CD69 7.2 3.7 0.9 2.5 19.4 OVAp 55.4 55.2 63.3 25.7 7.9 6.0 4.8 8.7 43.7 63.1 CFSE OVAp/IL-12

IL-12 priming reduces TCR avidity OVAp OVAp/IL-12 MHC I Tetramer stain (CD8+ gate) IL-12Rb1-/- 6 h 24 h 48 h 48 h 145 159 114 182 56 266 119 123 OVA tetramer-PE % Max Activation marker stain (CD8+Tet+ gate) 48 h CD11a CD44 CD43

IL-12 induces diffusion of lipid raft? HYPOTHESIS: IL-12 induces diffusion of lipid raft? Lipid raft microdomain

IL-12-primed cells exhibit diffused distribution of lipid raft Cholera Toxin b-FITC stain (48 h) OVAp OVAp/IL-12

IL-12 priming modifies activation-induced cell death? Hypothesis : IL-12 priming modifies activation-induced cell death? APC APC Memory CD8+ CTL Memory CD8+ CTL TC TC IL-12 Strong signal moderate signal More activation-induced Cell death (AICD) Less AICD More effector and memory Less effector and memory

IL-12 priming decreases death rate of CD8 T cells Annexin V and PI staining Naïve OT-I 48h stimulation ± IL-12 CD8 MACS Resting w/o IL-12 Annexin V and PI staining Hours 20 40 60 80 100 3 6 9 21 24 27 30 36 42 48 % Cell death OVAp OVAp/IL-12

IL-12 priming decreases death rate of CD8 T cells Day 3 Day 70 61.7% 8.82% OVAp Counts 38.4.% 2.59% OVAp/IL-12 Caspase-3

IL-12-primed cells exhibit lower cytolytic activity but higher IFN-g production Direct CTL assay Intracellular IFN-g stain OVAp OVAp/IL-12 52.8 91.2 CD8 IFN-g

IL-12 priming induces better protective immunity against tumor challenge Naïve OT-I spleen + LN 48 h Stimulation CTL assay Tumor challenge d70 CD8 MACS A.T. (1 x 106) Direct ex vivo CTL assay Tumor challenge at day 70 2 4 6 8 10 12 14 16 11 22 44 Day 70 E:T ratio Specific lysis (%) 20 40 60 80 100 2 4 6 8 10 12 14 16 Weeks Survival rate (%) naive OVAp OVAp/IL-12

The effect of in vivo IL-12 priming Splenocytes CD8 MACS E2 peptides ELISPOT rAd infection i.m. Day 9 IFN-g SFU/million cells Day 30

Endogenous IL-12 is not critical for CD8 T-cell responses? Naïve OT-I spleen + LN Wild type C57BL/6 IL-12p40-/- KO A.T. 1 x 106 rAd-OVA i.n. infection Lung or spleen Day 30 Day 42 WT p40-/- WT p40-/- 0.3 0.5 0.9 0.9 0.6 0.2 0.7 0.5 spleen 91.3 89.1 34.8 53.6 64.6 0.8 58.4 0.7 39.9 0.8 51.4 0.5 H-2Kb/OVAp Tetramer lung 9.8 17.9 45.6 33.7 CD62L

Purification of naïve and memory OT-I cells before MACS after MACS 16.8% H-2Kb/OVAp Tetramer after MACS Day70 LN Day70 Spleen CD8 96.9% 88.6% 96.1% H-2Kb/OVAp Tetramer CD44

What messages can we get? Novel IL-12 mutant induces long-term CTL memory In vitro IL-12 priming increases effector and memory CTL IL-12 priming increases protective immunity against tumor Possible application to tumor immunotherapy (adoptive-cell-transfer therapy)

Acknowledgement Sang-Jun Ha, Ph.D. Jae-Ho Cho, Ph.D. So-Young Choi Man-Ki Song, Ph.D. Other members of Cellular Immunology Lab, POSTECH And Young-Chul Sung, Ph.D.