1. Memory CCR6+CD4+ T-Cells are Selectively Imprinted with a Transcriptional Program Favorable to Productive HIV-1 Infection Patricia Monteiro, Jean-Philippe Goulet, Annie Gosselin, Vanessa Sue Wacleche, Mohamed-Rachid Boulassel, Jean-Pierre Routy, Nathalie Grandvaux, Elias Haddad, Rafick-Pierre Sekaly, and Petronela Ancuta Université de Montréal CHUM-Research Center, Saint-Luc Hospital Montréal, Quebec, Canada IAS2011, Rome, Italy, July 20, 2011

2. HIV-1 is a deadly but very selective virus Fact: –HIV-1 productively infects and persists in a very small fraction of memory CD4+ T-cells (<10%) Goals: –To identify phenotypic and functional markers of discrete CD4+ T-cell subsets permissive vs. resistant to HIV infection –To identify the molecular mechanisms of HIV permissiveness vs. resistance in primary T-cells

3. Th17: mucosal immunity and HIV infection CCR6 Th17 cells  Mucosal homeostasis  Bridge between innate and adaptive immunity  Immune activation via the production pro- inflammatory cytokines  Sites of HIV replication  Microbial translocation  Chronic immune activation

4. Preferential HIV-DNA Integration in Memory CCR6+ T-Cells of HIV-Infected Patients Gosselin/Monteiro et al., J Immunol, 2010 CCR6: –Marker for Th17 polarization –Regulates T-cell migration into Peyer’s Patches and other organs (spleen, brain)

5. Imprinting of CD4+ T-cells with gut-homing potential: Mucosal dendritic cells → Retinoic acid (RA) production → up-regulation of integrin  4  7 → Migration into the gut (Mora et al., Immunity, 2004 ; Manicassamy et al., Nat Immunol, 2009) Integrin  4  7 and HIV / SIV: –New binding receptor for HIV gp120 (Arthos et al., Nat Immunol, 2008) –Identifies a subset of memory CD4+ T-cells producing IL-17 that is preferentially infected and depleted during acute SIV infection (Kader et al., Mucosal Immunol, 2009; Wang et al., Mucosal Immunol., 2009) –Peripheral blood  4  7+ CD4+ T-cells are depleted during primary HIV infection (Krzysiek et al, Blood, 2001) The Gut-Homing Molecule Integrin  4  7

6. We investigated: –whether memory T-cells co-expressing CCR6 and integrin  7 are selective HIV targets –whether RA-induced imprinting for gut- homing selectively increases CCR6+ T- cell permissiveness to infection

7. Increased Expression of the HIV Coreceptor CCR5 on CCR6+  7+ and CCR6+  7- T-Cell Subsets Monteiro et al. J. Immunology 2011

8. Increased Permissiveness to R5 HIV Replication in CCR6+  7+ and CCR6+  7- T-Cell Subsets Monteiro et al. J. Immunology 2011

9. ATRA Upregulates Integrin  4  7 and CCR5 Expression on CCR6+ and/or CCR6- T-Cells Monteiro et al. J. Immunology 2011 CD3/CD28 Abs ±ATRA (10 nM)

10. ATRA Treatment Selectively Increases CCR6+ T-Cell Permissiveness to HIV at Entry and Post-Entry Levels Monteiro et al. J. Immunology 2011

11. Enhanced TNF-  Production and NF-  B p65 DNA-Binding Activity in CCR6+ Compared to CCR6- T-Cells Monteiro et al. J. Immunology 2011

12. A Systems Biology Approach Toward the Identification of New HIV Permissiveness and Restriction Factors Ancuta et al., unpublished data CCR6+ ATRA CCR6+ Medium CCR6- ATRA CCR6- Medium TCR signaling and cell activation (Lck, ZAP-70, PTPN13, MAP3K4, TANK) Lineage polarization profiles (IL-22, IL-26, CCL20, IL-5, IL-9) Regulation of gene transcription (RORC, RORA, PPARG, ARNTL, KLF2, NLF2, ATF5, E2F2, RUNX1) Immunological synapse formation (CXCR6, TNFRSF18)

13. CCR6+ memory T-cells expressing or not the gut-homing integrin  7 are highly permissive to HIV replication. However, CCR6+ T-cells co-expressing integrin  7 and CCR5 might have an extraordinary ability to disseminate HIV from the portal sites of entry ATRA selectively enhances permissiveness to HIV-replication in CCR6+ T-cells via entry (CCR5 upregulation) and yet unidentified post-entry mechanisms CCR6 is a marker for memory CD4+ T-cells imprinted with a transcriptional program favorable to HIV replication The identification of HIV dependency factors in CCR6+ T-cells will open the path for the design of new therapeutic strategies to limit HIV replication in these cells while maintaining their role in mucosal immunity Conclusions

14. Annie Gosselin, MSc Research Assistant Patricia Monteiro, PhD Postdoctoral fellow Vanessa Sue Wacleche PhD Student Hanane Touil MSc Student Ancuta Lab Petronela Ancuta, PhD Aurélie Cleret, PhD Postdoctoral fellow Annie Bernier MSc Student

15. Acknowledgements CRCHUM and VGTI Rafick-Pierre Sekaly, PhD Nicolas Chomont, PhD Mohamed El-Far, PhD Jean-Philippe Goulet, MSc Elias Haddad, PhD CRCHUM Nathalie Grandvaux, PhD Flow Cytometry Core Facility – CHUM-Research Center Annie Gosselin Laurence Lejeune Sylvain Gimmig Primo infection cohort - McGill University Jean-Pierre Routy, MD Mohamed-Rachid Boulassel, PhD Slow Progresors Cohort Cécile Tremblay, MD - CRCHUM Nicole Bernard, PhD - McGill University FRSQ-AIDS Infectious Diseases Network Anne Vassal and Mario Legault HIV-infected and uninfected subjects for their gift of leukapheresis and essential contribution to this work

16. Superior Ki67 Expression in CCR6+ Compared to CCR6- T-Cells ex vivo Monteiro et al. J. Immunology 2011

17. ATRA does not increase proliferation of CCR6+ T-cells Monteiro et al. J. Immunology 2011

18. CCR6+  7- and CCR6+  7+ T-Cells are Enriched in Cells with a CCR7-CD27- (EM) and CCR7-CD27+ (TM) Phenotype Monteiro et al. J. Immunology 2011

19. Decreased Frequency of Circulating CCR6+  7- and CCR6+  7+ T-Cells in HIV-Infected Subjects Monteiro et al. J. Immunology 2011 HIV+ (n=14) median CD4: 506 cells/µl median VL: 6,500 HIV-RNA copies/ml Median time of infection: 5.5 months ART for 1-3 months: 3 of 14 subjects