MHC T cell Receptor Antigen Antigen-presenting cell Antigen-reactive T cell CD28 B7 Activation (proliferation Cytokine production Cytotoxicity) Antigen-presenting cell Antigen-reactive T cell Dormant State (unresponsiveness) Antigen engagement without inflammation(costimulation) Unresponsiveness to subsequent stimulation

Molecular dissection of tolerance-related molecules/negative regulators of T cells (Spry1, Ndr1, Cbl-b, TGF-beta, CTLA-4, etc.) Development of reagents modulating regulators (Dominant negative mutants / sirna in lentivirus/adeno-associated virus) Tumor-bearing mouse Stimulation with Tumor antigen Modulated T cells

anti-tcr anti-tcr anti-cd28 Rest for 5-7 days Proliferation IL-2 production IL-2(pg/ml) 8000 7000 6000 5000 4000 3000 2000 1000 Effect of various reagents on anergy induction Effect of various reagents on mrna induction Sprouty1 Average difference 6000 5000 4000 3000 2000 1000 0 700 600 Mock Signa l 1 Only 1 + Cs A 1 + 6976 Combined 0 Mock Signa l 1 Only 1 + CsA 1 + 6976 Combined Ndrg1 Average difference 500 400 300 200 100 0 Mock Signal 1 Only 1 + Cs A 1 + 6976 Combined

Ndrg1-overexpressed A.E7 Anergic A.E7 4500 4000 Buffer TAT-Ndr1 10000 9000 Anergized, 10ug/ml Unanergized 3500 TAT-GFP 8000 IL-2(pg/ml) 3000 2500 2000 1500 IL-2 (pg/ml) 7000 6000 5000 4000 3000 1000 2000 500 1000 0 0 0.1 0.3 1 3 10 30 100 0 0 0.1 0.3 1 3 10 anti-cd3(ug/ml) anti-cd3 (ug/ml) 25000 Buffer TAT-Ndr 10000 9000 Anergized, 10ug/ml Unanergized 20000 TAT-GFP 8000 7000 cpm 15000 10000 5000 IL-2 (pg/ml) 6000 5000 4000 3000 2000 1000 0 0 0.1 0.3 1 3 10 30 100 anti-cd3 0 0 0.1 0.3 1 3 10 anti-cd3 (ug/ml)

160000 140000 120000 TAT-GFP TAT-Ndr1 Buffer 100000 cpm 80000 60000 40000 20000 0 0 0.3 1 3 10 30 100 IL-2 (ng/ml)

(A) Effect of TAT-sprouty1 on IL-2 production 60000 8000 7000 50000 6000 40000 IL-2 (pg/ml) 5000 4000 3000 IL-2 (pg/ml) 30000 20000 2000 1000 10000 Buffer TAT-Spry1 0 0.0 0.1 0.3 1.0 3.0 10.0 0 0 0.1 0.3 1 3 10 30 TAT-GFP Anti-CD3 Ab (μg/ml) Anti CD3 Ab (ug/ml) AE7 T cell clone F1.A2 T cell clone (B) TAT-Spry1 inhibits activation of MAPK and NFAT Un CD3 Ab (10ug/ml) + CD28 Ab Buffer TAT-Spry1 TAT-GFP CD3 Ab (1ug/ml) + CD28 Ab CD3 Ab (10ug/ml) + CD28 Ab IB: p-erk Un Buffer TAT-Spry1 TAT-GFP Buffer TAT-Spry1 TAT-GFP IB: NFATc2 IB: Total Erk

50000 45000 40000 Unstimulated Stimulated 35000 Luc. activity 30000 25000 20000 15000 10000 5000 0 Ndr1 0ug Ndr1 2ug Ndr1 5ug Ndr1 10ug 3500 3000 unstimulated acd3 alone acd3+acd28 120000 100000 unstimulated acd3 alone acd3+acd28 Luc. activity 2500 2000 1500 1000 Luc. activity 80000 60000 40000 500 20000 0 NFAT/Luc. phm6 NFAT/Luc. Ndr1 0 AP-1/Luc. phm6 AP-1/Luc. Ndr1 800 700 600 unstimulated acd3 alone acd3+acd28 Luc.activity 500 400 300 200 100 0 NFkB/Luc. phm6 NFkB/Luc. Ndr1

900 800 phm6 10ug Ndr1 10ug 700 600 500 400 300 200 100 0 unstimulated 1ug/ml acd3 10ug/ml acd3 1ug/ml acd3 + 10ug/ml acd28 10ug/ml acd3 + 10ug/ml acd28 PMA+Iomontcin

With bait With vector 1 2 3 4 5 + 1 2 3 4 5 - + : Positive control - : Negative control lacz expression Filter assay Master plate SD-LW HIS3/ADE2 expression SD-LWHA With bait With vector 1 2 3 4 5 6 + 1 2 3 4 5 6 - + : Positive control - : Negative control lacz expression Filter assay Master plate SD-LW HIS3/ADE2 expression SD-LWHA

T cell Receptor Zeta-chain PLC-γ Lck ZAP-70 LAT? Spry1 PIP 2 Ca 2+ IP 3 + DAG Ras-GRP Ras NF-AT MAPK NF-AT AP-1 IL-2 luciferase activity 3000 2500 2000 1500 1000 unstimulated stimulated with acd3 + acd28 500 0 v ector-2ug v ector-5ug sprouty1-2ug sprouty1-5ug

250 unstimulated stimulated uninduced induced 200 anti-flag (Spry1) anti-actin IL-2(pg/ml) 150 100 50 0 uninduced induced

Master plate With bait With vector With bait With vector With bait With vector 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 8 9 10 11 12 13 14 15 16 17 18 19 20 + 15 16 17 18 19 20 - - : Negative control + : Positive control SD-LW lacz expression URA3 expression ADE2 expression Filter assay SD-LWU SD-LWA

Sprouty1 7000 6000 unstimulated acd3+acd28 luciferase activity 5000 4000 3000 2000 1000 0 Empty vector WT-Spry1 WT-Spry1+dRBM Sprouty2 Sprouty4 IL-2 Luciferase activity 4500 4000 3500 3000 2500 2000 1500 1000 500 0 unstimulated stimulated empty vector Spry 2 Spry 2 + drbm IL-2 Luciferase activity 6000 5000 4000 3000 2000 1000 0 unstimulated stimulated empty vector Spry4 Spry4 + drbm

Transgenic Construct of DN-Spry1 4401 bp Transgene to be injected 4401bp Not I Sal I Sal I Not I 2700bp CD4 promoter DN-Spry1 p37.1 HA tag PolyA tail

IL-2 IFN-r IL-2 secreting population /transfected population (% positive) 16. 00 14. 00 12. 00 10. 00 8. 00 6. 00 4. 00 2. 00 Empty vector Spry1 drbm IFN-r secreting population /transfected population (% positive) 14. 00 12. 00 10. 00 8. 00 6. 00 4. 00 2. 00 Empty vector Spry1 drbm 0. 00 0hr 1hr 2hr 4hr 18hr Duration of stimulation 0. 00 0hr 1hr 2hr 4hr 18hr Duration of stimulation