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

2. 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

3. Processing and presentation of exogenous and endogenous antigens

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

5. (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)

6. 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)

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

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

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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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)

39. 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.