In spite of the considerable body of evidence that CD28 provides a critical costimulatory signal for T cell responses,

1. In spite of the considerable body of evidence that CD28 provides a critical costimulatory signal for T cell responses, not all T cell-mediated responses are CD28-dependent.

2. Kunding T.M. et al. Immunity 5:41, 1996

3. VSV does not replicate extensively in vivo, in contrast to LCMV. Continued presence of signal 1 alone, either through prolonged viral replication or repeated injection of peptide, generates a functional T cell response in vivo in absence of CD28. The duration of TCR stimulation determines the costimulatory requirement of T cells.

4. Schweitzer, A.N. and Sharpe A.H. J. Immunol. 61:2762, 1998. 4 days T cells + WT APC pulsed with peptide T cells

5. London C.A. et al. J. Immunol. 164:265, 2000. TCR Trsg T cells + WT APC pulsed with peptide 4 days T cells Transferred into mice 6-7 months Memory T cells Stimulated in vitro with WT APC B7 KO APC

6. In the presence of strong or prolonged signal 1, T cells don’t need costimulation. Costimulation is not required for effector and memory T cells. Now, In the presence of strong signal 1, T cells don’t need CD28-mediated costimulation. CD28-mediated costimulation is not required for effector and memory T cells. These responses may involve additional costimulatory pathways.

7. The B7 family and its receptors

8. B7h/ICOS costimulatory pathway A new molecule with structural characteristic similar to the B7 molecules was identify in 1999, and was named B7h (B7-related protein 1; also GL-50 or B7RP-1 or ICOS-L). B7h does not bind to CD28 or CTLA-4, but bind to ICOS (inducible costimulatory molecule). ICOS shares 30-40% sequence similarity with CD28 and CTLA-4. ICOS expression: ICOS is not constitutively expressed on naïve T cells but is induced on CD4+ and CD8+ T cells following stimulation through the TCR and is further enhanced by CD28-mediated costimulation. FIGURE 2. Expression of ICOS on activated T cells. Dissociated splenocytes from wild-type or B7-1/2-/- 129/SvS4Jae mice were incubated with anti-CD3, anti-CD3 and CD28, or no Ab. The thick line shows ICOS expression on T cells from wild-type splenocyte cultures, the dotted line shows ICOS expression on T cells from B7-1/2-/- splenocyte cultures, and the thin line represents a negative staining control (rat IgG-FITC). McAdam A.J. et al. J. Immunol. 165:5035, 2000.

9. ICOS is expressed on memory T cells (CD45RO+) and on T cells in germinal centers. b, Localization of ICOS+ cells in the apical light zone of germinal centres, as determined from histochemical stains of frozen human tonsillar sections with monoclonal antibody F44 using the APAAP technique30. LZ, light zone; DZ, dark zone; MZ, mantle zone (original magnification 69). Hutloff A. et al. Nature 397:263, 1999. More highly expressed on Th2 than Th1 clones. ICOS expression decreases during the differentiation of Th0 cells to Th1. McAdam A.J. et al. J. Immunol. 165:5035, 2000. Coyle A.J. et al. Immunity 13:95, 2000.

10. B7h expression • Strongly expressed in the B-cell area of the lymph nodes and spleen and in germinal centers. • Induced on DCs and monocytes • Induced on non-lymphoid cells (kidney, lungs and testis) by inflammatory stimuli The expression of B7RP-1 is strong in the lymph nodes from both normal and sensitized mice (Fig. 2a), particularly in the cortex (a B-cell area) and in both primary and secondary follicles. In other lymphoid tissues, B7RP-1 expression is primarily localized in B-cell areas of the spleen, the follicles in the Peyer's patches and the medulla of the thymus (Fig. 2b). Yoshinaga S.K. et al. Nature 402:827, 1999.

11. Roles for the B7h/ICOS costimulatory pathway

12. Activation of naive and recently activated CD4+Tcells 2- Second activation OVA peptide + + CD4+ OVA-specific TcR Tg APC CTLA-4 Ig ICOS Ig hIg Ig ICOS Ig CTLA-4 Cytokine production (ELISA) Experimental procedure Naive T cells Recently activated T cells 1- First activation of CD4+ T cells with APC-peptide + + OVA peptide CD4+ OVA-specific TcR Tg APC hIg Proliferation IL-2 production (ELISA) Coyle A.J. et al.(2000). Immunity, 13: 95-105.

13. Activation of naive and recently activated CD4+Tcells in vitro ICOS-Ig ICOS-Ig CTLA-4Ig CTLA-4Ig hIg • ICOS is not implicated in the activation of naive CD4+ T cells and production of IL-2. • ICOS pathway is important for cytokine production from recently activated CD4+ T cells.

14. Cytokine production by fully differentiated Th1 and Th2 effectors cells Experimental procedure Threerounds of repetitive stimulations with OVA peptide CTLA-4 Ig + + ICOS Ig CD4+ T cells APC hIg Cytokine production (ELISA) Th1 Th2 Coyle A.J. et al.(2000). Immunity, 13: 95-105.

15. Cytokine production by Th1 and Th2 effectors cells Coyle A.J. et al.(2000). Immunity, 13: 95-105. • B7RP-1/ ICOS pathway contributes to the cytokine production from Th2 but not Th1 cells in vitro.

16. ICOS +/+ ICOS +/- ICOS -/- T cell-dependent Ab production and germinal center formation in ICOS -/- miceExperimental procedure Primary Immunization with KLH/ CFA Antibody response (ELISA) GC formation (Immunohistochemistry) Second Immunization with KLH/ CFA Cytokine production (Immunostaining and cytofluorimetry) Tafuri A. et al (2001). Nature, 409: 105-109.

17. Antibody response and germinal center formation in ICOS -/- mice ICOS +/+ ICOS +/- ICOS -/- Tafuri A. et al (2001). Nature, 409: 105-109. ICOS is required for antibody responses and GC formation.

18. ICOS +/+ T cells ICOS -/- T cells Cytokine production after restimulation in vitro Tafuri A. et al (2001). Nature, 409: 105-109. ICOS is required for Th2 differentiation.

19. Additional in vivo evidences that Th2 responses are primarily regulated by B7h/ICOS pathway Mucosal model of inflammation in the lung (Th2-mediated) Gonzalo JA et al (2001). Nature Immunol, 2: 597-604. Blocking B7h/ICOS during priming: no effect Blocking B7/CD28 during priming:  inflammation Blocking B7h/ICOS during effector phase:  inflammation Blocking B7/CD28 during effector phase: no effect EAE (Th1-mediated disease) EAE in ICOS deficient animal : Exacerbation of the disease Dong C et al (2001). Nature, 409: 97-101. Blocking B7h/ICOS during antigen priming : Increased disease severity Rottman JB et al (2001). Nature Immunol, 2: 605-611. augmentation of Th1 polarization by inhibition of Th2 polarization

20. In vivo evidences that B7h/ICOS pathway also regulatesTh1 responses Th1 cells express lower levels of ICOS compared to Th2 cells. In vitro studies have indicated that ICOS is not relevant in Th1 polarized T cells. However, in vivo studies indicate that expression of ICOS by Th1 cells appears to be relevant EAE (Th1-mediated disease) Rottman JB et al (2001). Nature Immunol, 2: 605-611. Blocking B7h/ICOS during antigen priming : Increased disease severity augmentation of Th1 polarization by inhibition of Th2 polarization Blocking B7h/ICOS during effector phase: Limits disease progression by inhibiting Th1 functions (IFNg). Th1-mediated cardiac allograft rejection: Ozkaaaynak E et al (2001). Nature Immunol, 2: 591-596. Blocking ICOS prolongs allograft survival by preventing both acute and chronic rejection. Thus, these results show that ICOS is also important in regulating Th1 responses in vivo.

21. Role of B7h/ICOS in CD8 T cell responses • B7h/ICOS interactions are not relevant in CD8 T cell responses to LCMV • Kopf M et al (2000)J. Exp. Med 192: 53-62. • New studies indicate that ICOS may be relevant for effective CD8 T cell mediated • anti-tumor responses: • Wallin JJ et al (2001) J. Immunol. 167: 132-139. • Liu X et al (2001)J. Exp. Med 194: 1339-1348. • In both studies, expression of B7h on tumor cells results in efficient CD8 • mediated tumor rejection. (Delayed kinetic compared with rejection of • B7.2-expressing tumors).

22. PD-L1, PD-L2 and their receptor PD-1 • PD-L1 (programmed death 1 ligand),PD-L2 (programmed death 2 ligand) and do notbind to CD28, ICOS, or CTLA-4, but bind to PD-1 (programmed death 1 molecule). • PD-1 shows 24 % of sequence similarity to CD28 and CTLA-4. • An ITIM motif (Immunoreceptor Tyrosine-based Inhibitory Motif) is present in the cytoplasmic tail of PD-1. The ITIM motif is known to be present in receptors that have an inhibitory function on lymphocyte responses.

23. Expression patterns of PD-L1, PD-L2 - PD-L1 and PD-L2 are expressed on DCs and monocytes. - They are constitutively expressed on several non-lymphoid tissues. Dong H. et al (1999). Nature Med, 5(12): 1365-1369 Latchman et al. (2001). Nature Immunol., 2(3):261-268 Coyle A.J. et al (2001). Nature Immunology, 2(3): 203-209

24. Expression pattern of PD-1 • PD-1 is constitutively expressed on T cells following activation. • (Chambers, C. (2001) Trends Immunol., 22(4): 217-223). • C57Bl/6 PD-1 -/- mice develop lupus-like arthritis and glomerulonephritis with an increase in serum IgG3 and B cell proliferation.(Nishimura, H. el al (1999). Immunity, 11: 141-151). • BALB/c PD-1 -/- develop an autoimmune dilated cardiomyopathy, with IgG deposites on cardiomyocytes and an increase of IgG in blood.(Nishimura, H. el al (2001). Science, 291: 319-322). • CTLA-4 has a stronger negative effect than PD-1 on T cell activation. All Ig isotypes are elevated in CTLA-4 -/- mice independently of the genetic background. The lymphoproliferation is more severe, and the onset of the phenotype and the lethality occur faster in CTLA-4 -/- mice. (Chambers, C. (2001) Trends Immunol., 22(4): 217-223).

25. PD-L1-Ig hIg  CD3  CD3 CD4+ T cells T cell proliferation and cytokine production after PD1- PD-L1 engagement Experimental procedure + Proliferation Cytokine production (ELISA) Freeman et al (2000). J. Exp. Med, 192 (7): 1027-1034.

26. T cell proliferation and cytokine production after PD-1/ PD-L1 engagement Freeman et al (2000). J. Exp. Med, 192 (7): 1027-1034 PD-L1/ PD-1 interactions can inhibit TcR-mediated proliferation and cytokine production.

27. Proliferation and cytokine production after PD-L2/ PD-1 engagement Experimental procedure OVA peptide + APC First activation OVA-specific TcR Tg CD4+ T cells CHO.I-Ad CHO.I-Ad.PD-L2 OVA peptide Second activation + Proliferation Cytokine production (ELISA) Latchman et al. (2001). Nature Immunol., 2(3):261-268

28. Cytokine production after PD-L2/ PD-1 engagement Latchman et al. (2001). Nature Immunol., 2(3):261-268 PD-L2/ PD-1 interactions can inhibit TcR-mediated proliferation and cytokine production.

29. Contradicting Results as to the function of the PD-L2/PD-L1/PD-1 pathway

30. Costimulation of T cells by PD-L2 Tseng, SY et al. (2001). J. Exp. Med., 193 (7): 839-845. • PD-L2 costimulates : • a T cell proliferative response to a greater level than B7.1. • mainly CD4+ T cells. • greater levels of IFN- than B7-1, and fails to promote IL-4 or IL-10 production. • PD-L2 appears to drive Th1 responses.

31. Contradicting results • T cell proliferation and IL-10 secretion increased with PD-L1 stimulation. Dong et al (1999). Nature Medecine, 5(12): 1365-1369). Tamura et al (2001). Blood, 97(6): 1809-1816. • It is unclear whether the PD-L1/L2 costimulation reported by these groups is PD-1 dependent or whether it could be mediated by an alternative receptor for PD-L1: Parallel of CD28/ CTLA-4 receptors- B7.1/ B7.2 ligands. Freeman et al (2000). J. Exp. Med, 192 (7): 1027-1034

32. B7-H3 B7-H3 • Expression pattern: • B7-H3 (B7 homolog 3)binds to an unknown receptor on activated T cells that is distinct from CD28, CTLA-4, ICOS or PD-1. Ctrl serum • B7-H3 is an inducible molecule on the surface of DCs and monocytes. • Not restricted to lymphoid tissue but also observed in heart, kidney, testis, colon and human tumor lines Transient expression of the unknown receptor of B7-H3 on activated T cells. B7-H3 Ig Ctrl Ig Chapoval et al (2001). Nature Immunology, 2(3): 269-274

33. Costimulating T cell responses by B7-H3Experimental procedure ImmobilizedB7-H3 Ig B7-1 Ig control Ig Melanoma cell line transfected with B7-H3 or CT vector Coated  CD3 + T cells T cells CTL activity Proliferation Cytokine production Chapoval et al (2001). Nature Immunology, 2(3): 269-274

34. Costimulating T cell responses by B7-H3 Chapoval et al (2001). Nature Immunology, 2(3): 269-274 • B7-H3: • increases T cell proliferation but is less potent than B7.1. • enhances proliferation of both CD4+ and CD8+ T cells. • B7-H3- transfected cells induces CTL activity. • selectively enhances IFN  production with modest effects on TNFa and IL-8

35. Resting T cell CD28 CTLA-4 Primed T cell PD-1 Primed T cell Summary + Initiation of T cell activation. APC B7.1 B7.2 - Inhibition of T cell activation APC (B) +  T cell activation in 2nd responses, differentiation in Th2 B7RP-1 ICOS Primed T cell DC, M PD-L1 - Negative regulation of T cell activation. PD-L2 + ? Th1 responses (INF ). DC, M + Th1 (INF) and CTLs. B7-H3 ? Primed T cell

36. Conclusion • Regulation of T cell activation by costimulation is more complex than originally envisioned. • None of the newly discovered pathways appears to be completely redundant with CD28 in terms of naïve T cell activation but rather regulate the fate of primed and memory T cells. • The spirit of the two-signal model of T-cell activation remains intact, but a second activation step process called « step two of signal 2» can be add to this model. Coyle A.J. et al (2001). Nature Immunology, 2(3): 203-209

37. Complementation in functions: ICOS/B7h: Th2 ?/B7-H3: Th1 and CTL • Spatial differences in expression: The novel B7 family members have a broad distribution in non-lymphoid tissues and are therefor uniquely position to regulate antigen-specific T cell functions at sites of inflammation.