1. Treg exert differential effects on the proliferation and differentiation of CD8 T cell subsets in chronic HIV-1 infection M. Nikolova 1, M. Muhtarova 1, M. Younas 2, J.D. Lelievre 2,3, H. Taskov 1, Y. Levy 2,3 1 National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria 2 Université Paris Est Créteil, Inserm U955, Créteil, France 3 Henri Mondor Hospital, APHP, Créteil, France XVIII International AIDS Conference | July 18-23 2010 | Vienna, Austria

2. CD45RA+ Central memory Effector memory 1 EffectorTerminal effector Effector memory 2 NaïveMemory Effector CD8 T cell differentiation and functional maturation CD45RA-/CD45RO+ CCR7+ CD28+ CD27+ CD45RA+ IFN  Cytotoxicity Proliferation Differentiation

3.  Treg are expanded in acute and chronic HIV-1 infection, and inhibit effector CD8 T cell responses in vitro (Weiss L. et al, Blood 2004; Kinter A. et al, J. Exp. Med 2004) Background & Rationale  CD8 T memory/effector subset balance determines the control of chronic viral infections  HIV infection is characterized by a decreased proliferative capacity of CM CD8 T cells and incomplete differentiation of HIV-specific effector T cells (Appay V. et al, J. Exp. Med 2000; Champagne P. et al, Nature 2001)  We have previously shown that Treg (CD4+CD25 high FoxP3+) influence M/E CD8 subset balance in HIV- donors: Treg inhibit the proliferation of effectors and the differentiation of memory CD8 T upon polyclonal and antigen-specific in vitro stimulation (Nikolova M. et al, Blood, 2009)

4. HYPOTHESIS AND OBJECTIVE We hypothesized that the expansion of Treg in HIV- infected patients might contribute to the dysbalance between effector and memory CD8 T cells Our objective was to investigate the effects of Treg on the proliferation and maturation of different CD8 T cell subsets in chronic HIV-1 infection

5. STUDY POPULATION HIV-1 + cART + subjects (n=14), CD4 >350 cells/  l, VL < 50 HIV RNA copies/ml STUDY DESIGN D5, flow cytometry (CFSE/CD45RA/CCR7/CD27/CD28/CD3/CD8) COMPARISON proportions and proliferation rates of CD8 T subsets PMNC Treg depletion, anti-CD25 DynaBeads PMNC, Treg+ CFSE staining, polyclonal stimulation: immobilized anti-CD3 (5  g/ml) D0, flow cytometry (CD45RA/CCR7/CD27/CD28/CD3/CD8) proportions of CD8 T subsets PMNC,Treg-

6. 1. CD27+CD45RA+ Naïve, N 2. CD27+CD45RA- Memory, M 3. CD27- CD45RA- Effector, E 4. CD27- CD45RA+ Terminal Effector, TE CD8 T SUBSETS PHENOTYPING : GATING STRATEGY CD8 T CELL SUBSETS: PROLIFERATION RATES CFSE low 68 % CFSE Treg+ Treg- Gated CD8 T subset CFSE-stained Treg+ and Treg- PMNC were stimulated with 5  g/ml immobilised anti-CD3 % CFSE low cells was determined on D5 N M E CD27 Gated CD3+CD8+ Ly CFSE low 81% TE CD45RA

7. Proliferation rates of Treg+ and Treg- CD8 T (n=14, D5 anti-CD3), mean 72 vs. 81 % CFSE low CD8 T ** p<0.01, Student’s T-test Polyclonal stimulation in the presence of Treg results in a decreased rate of CD8 T cell proliferation Treg+ CD8 Treg- CD8 % CFSE low CD8 T **

8. * Proliferation rates of Treg+ and Treg- CD8 T subsets (D5, anti-CD3); av. 46% vs. 74% E, (P< 0.05) and 48% vs. 85% TE, (P< 0.01) have proliferated N %CFSE low CD8 T Polyclonal stimulation in the presence of Treg results in lower proliferation rates within E and TE subsets, while M CD8 are not significantly affected M ETE E %CFSE low CD8 T * ** * p<0.05, **p<0.01, Student’s T-test

9. Distribution of CD8 T subsets before and after polyclonal stimulation in the absence or in the presence of Treg; 19 and 27 % E CD8 were observed in the presence and in the absence of Treg, respectively (* p<0.05, n=14, Student’s T-test) Treg inhibit the differentiation of polyclonally stimulated naive CD8 T cells into CD27-CD45RA- effectors N M E TE % of CD8 T cells * * ns

10. Analysis of memory CD8 T cells: CM/EM CD27 CD45RA M Gated CD3+CD8+ Ly Gated CD45RA-CD27+CD8 T CCR7 CD28 Analysis of CD28/CCR7 co-expression on M (CD27+CD45RA-) CD8 T cells after polyclonal stimulation (D5, anti-CD3) in the absence or in the presence of Treg CM EM1 EM2

11. Polyclonal stimulation in the presence of Treg results in a significant increase of CM (CCR7+) CD8 T cells Composition of the M CD8 T cell subset before and after polyclonal stimulation, in the absence or in the presence of Treg. (n=14, **p<0.05, **p<0.05 in comp. to D0, Student’s T-test) % of M CD8 T cells *** CM EM1 EM2

12. Proportions of CM cells within the memory (CD27-CD45RA+) CD8 T cell subset before and after 5 days anti-CD3 stimulation in the presence or in the absence of Treg: Average % of CM CD8 were 15.5 vs. 24 and 16 respectively. (n=14, *p<0.05, **p<0.01, Student’s T-test) Polyclonal stimulation in the presence of Treg results in a significant increase of CM (CCR7+) CD8 T cells NS * ** % of M CD8 T cells D0 Treg+ Treg- D5, anti-CD3

13. Central memory Effector memory 1 EffectorTerminal effector Effector memory 2 Naïve Memory Effector CD45RA-/CD45RO+ CD45RA+ CCR7+ CD28+ Treg CD8 T cell differentiation HIV+ specific CD8 T are mostly of EM2 phenotype IFNg Cytotoxicity Proliferation CD27+

14.  Polyclonal stimulation of CD8 T cells in the presence of Treg results in decreased differentiation of effectors and in accumulation of CM cells. Conclusions  Increased frequency of Treg in HIV infection significantly decreases the rate of CD8 T cell proliferation, affecting specifically E and TE subsets.  Globally, these results indicate that the expansion of Treg in the settings of HIV infection and generalized immune activation may contribute to the dysbalance between M and E antigen-specific CD8 T cells

15. The questions to answer  Subset-specific effects of Treg at the level of HIV-specific CD8 T cells  Subset-specific effects of Treg at the level of other virus-specific CD8 T cells  Mechanisms of Treg subset-specific effects in HIV infection… PD1/PDL1?

16. National Reference Laboratory of Immunology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria Henri Mondor Hospital, APHP, Université Paris Est Créteil, Inserm U955, Créteil, France Dr. Matthieu Carrière Dr. Mohammad-Ali Jenabian Dr. Christine Lacabaratz Pr. Jean-Daniel Lelièvre Pr. Yves Lévy Dr. Maria Muhtarova Dr. Draganka Stankulova Antoaneta Mihova Pr. Hristo Taskov ACKNOWLEDGEMENTS PHC Rila 2009 (Bulgarian Ministry of Education and Sience / Ministry of Foreign and European Affairs, France; Sidaction - France; ELTA’90 Ltd - Bulgaria