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M. Nikolova 1 , M. Muhtarova 1 , M. Younas 2 , J.D. Lelievre 2,3 , H. Taskov 1 , Y. Levy 2,3

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

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M. Nikolova 1 , M. Muhtarova 1 , M. Younas 2 , J.D. Lelievre 2,3 , H. Taskov 1 , Y. Levy 2,3

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  1. Treg exert differential effects on the proliferation and differentiation of CD8 T cell subsets in chronic HIV-1 infection M. Nikolova1, M. Muhtarova1, M. Younas2, J.D. Lelievre2,3, H. Taskov1, Y. Levy2,3 1National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria 2Université Paris Est Créteil, Inserm U955, Créteil, France 3Henri Mondor Hospital, APHP, Créteil, France XVIII International AIDS Conference | July 18-23 2010 | Vienna, Austria

  2. CD8 T cell differentiation and functional maturation Differentiation IFNg Cytotoxicity Proliferation Effector Naïve Memory Effector memory 2 Central memory Effector memory 1 Effector Terminal effector CD45RA+ CD45RA-/CD45RO+ CD45RA+ CCR7+ CD28+ CD27+

  3. 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+CD25highFoxP3+) 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) • 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)

  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/ml, VL < 50 HIV RNA copies/ml STUDY DESIGN PMNC D0, flow cytometry (CD45RA/CCR7/CD27/CD28/CD3/CD8) proportions of CD8 T subsets Treg depletion, anti-CD25 DynaBeads PMNC,Treg- PMNC, Treg+ CFSE staining, polyclonal stimulation: immobilized anti-CD3 (5 mg/ml) D5, flow cytometry (CFSE/CD45RA/CCR7/CD27/CD28/CD3/CD8) COMPARISON proportions and proliferation rates of CD8 T subsets

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

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

  8. Polyclonal stimulation in the presence of Treg results in lower proliferation rates within E and TE subsets , while M CD8 are not significantly affected ** * * ** %CFSE low CD8 T %CFSE low CD8 T N E TE M TE E * p<0.05, **p<0.01, Student’s T-test 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

  9. Treg inhibit the differentiation of polyclonally stimulated naive CD8 T cells into CD27-CD45RA- effectors ns * * % of CD8 T cells ns M N TE E 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)

  10. Analysis of memory CD8 T cells: CM/EM Gated CD45RA-CD27+CD8 T Gated CD3+CD8+ Ly EM1 CM CD28 CD45RA M EM2 CM CD27 CCR7 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

  11. Polyclonal stimulation in the presence of Treg results in a significant increase of CM (CCR7+) CD8 T cells ** * % of M CD8 T cells EM1 CM EM2 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)

  12. Polyclonal stimulation in the presence of Treg results in a significant increase of CM (CCR7+) CD8 T cells ** * D0 Treg+ % of M CD8 T cells Treg- NS D5, anti-CD3 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)

  13. HIV+ specific CD8 T are mostly of EM2phenotype CD8 T cell differentiation Treg IFNg Cytotoxicity Proliferation Naïve Effector Memory Effector memory 2 Central memory Effector memory 1 Effector Terminal effector CD45RA+ CD45RA-/CD45RO+ CD45RA+ CCR7+ CD28+ CD27+

  14. Conclusions • Increased frequency of Treg in HIV infection significantly decreases the rate of CD8 T cell proliferation, affecting specifically E and TE subsets. • Polyclonal stimulation of CD8 T cells in the presence of Treg results in decreased differentiation of effectors and in accumulation of CM cells. • 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. ACKNOWLEDGEMENTS 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 PHC Rila 2009 (Bulgarian Ministry of Education and Sience / Ministry of Foreign and European Affairs, France; Sidaction - France; ELTA’90 Ltd - Bulgaria National Reference Laboratory of Immunology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria Dr. Maria Muhtarova Dr. Draganka Stankulova Antoaneta Mihova Pr.Hristo Taskov

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