1. www.ias2015.org Molecular Determinants of HIV-1 Permissiveness and Persistence in Gut-Homing CD4+ T-Cells Expressing the Th17 Marker CCR6 Delphine Planas Ancuta’s Lab. Université de Montréal Centre de Recherche du CHUM Montréal, Québec, Canada

2. www.ias2015.org The Chemokine Receptor CCR6, Th17 Cells, and HIV-1 Marker for Th17-polarized CD4+ T-cells Marker for Th17-polarized CD4+ T-cells CCR6 Marker for CD4+ T-cells permissive to HIV infection in vitro and in infected subjects Signalling via CCR6 facilitates establishment of HIV-1 latency in resting CD4+ T-cells Gosselin et al., JI, 2010 Monteiro et al., JI, 2011 El-Hed et al., JID, 2010 Alvarez et al., JVI, 201X Cameron et al., PNAS, 2010 Regulates T-cell trafficking into the Peyer’s Patches Regulates T-cell recruitment into the brain Reboldi et al., Nature Immunology, 2009 Wang et al., Mucosal Immunology, 2009 Lugering et al., Am J. Pathol, 2005 Salazar-Gonzalez et al., Immunity, 2006 Marker for HIV-specific CD4+ T-cells Lécureuil et al., Blood, 2007 Wacleche et al. PlosOne, 2012 Inhibition of the CCR6- CCL20-regulated cell traficking via glycerol monolaurate prevents SIV dissemination upon mucosal challenge Qingsheng Li et al., Nature, 2009

3. www.ias2015.org The Retinoic Acid, a Gut Homing Elixir Produced by Intestinal DC Gut homing of T and B cells Dendritic cells from the GALT Vitamin A Retinoic Acid CCR9, α 4 β 7 Integrin Regulation of Gene Expression RARE RAR RXR RA Mora et al, Immunity, 2006 Islam et al, Nature Medicine, 2012 HIV LTR Transcription Balmer et al., Journal of Lipid Research, 2002 Lee et al., PNAS, 1994 RARE site on HIV LTR RARE, retinoic acid responsive elements

4. www.ias2015.org ATRA Increases HIV Permissiveness by Increasing CCR5 Expression Specifically in CCR6+ T-cells Monteiro et al., JI, 2011 CCR6-CCR6+ MedATRAMedATRA Infection with R5 NL4.3-BaL virus CCR5 expression

5. www.ias2015.org ATRA Increases HIV Permissiveness in CCR6+ but not CCR6- cells via Post-Entry Mechanisms Monteiro et al., JI, 2011 Infection with single-round VSVG-pseudotyped HIV

6. www.ias2015.org Study Objectives To identify and functionally characterize new molecular determinants of HIV permissiveness versus restriction in CCR6+ versus CCR6- T- cells with gut-homing potential

7. www.ias2015.org Genome-Wide Transcriptional Profiling of ATRA-Treated CCR6+ versus CCR6- T-cells CCR6- CCR6+ CD3/CD28 Abs ± ATRA Microarray BeadChips TM Illumina HT 12v4 BeadChip, >46,000 sondes CCR6-ATRA vs CCR6- 369 genes CCR6+ATRA vs CCR6+ 283 genes 103180266 FC=|1.3|

8. www.ias2015.org Homing Molecules Modulated by ATRA in CCR6+ vs. CCR6- T-cells FC threshold 1.3 CCR6-CCR6+ MedATRAMedATRA CXCR6 expression

9. www.ias2015.org Differential Modulation by ATRA of Transcripts Associated with T-cell Activation and HIV Permissiveness

10. www.ias2015.org Gene Set Variation Analysis (GSVA): Canonical Pathways Modulated by ATRA in CCR6+ cells Th17 polarization Akt, PI3K Autophagy mTORC1 pathway

11. www.ias2015.org ATRA Preserves the Th17 Polarization Program in CCR6+ T-cells

12. www.ias2015.org RNA Interference Against RORC Cleret-Buhot, Zhang, Planas et al., manuscript in preparation, 2015 CD3/CD28 AbsRT-PCR RORC HIV exposure NucleofectionHIV-p24 ELISA Day -3Day 0Day -1Day 6 IL-2 HIV-DNA PCR IL-17A ELISA GFP FACS Day 3 IL-2 Day 5 Viability FACS Proliferation FACS

13. www.ias2015.org RORC, a Master Regulator of HIV-1 Permissiveness in Th17 Cells CD3/CD28 AbsRT-PCR RORC HIV exposure NucleofectionHIV-p24 ELISA Day -3Day 0Day -1Day 6 IL-2 HIV-DNA PCR IL-17A ELISA GFP FACS Day 3 IL-2 Day 5 Viability FACS Proliferation FACS Cleret-Buhot, Zhang, Planas et al., manuscript in preparation, 2015

14. www.ias2015.org ATRA Increases mTOR Phosphorylation in CCR6+ T-Cells β-actin p-mTOR mTOR 42 kDa 80 kDa 289 kDa R6+R6-R6+R6- ATRA-+-+-+-+ Donor #1Donor #2

15. www.ias2015.org Conclusion Memory CCR6+ T-cells Memory CCR6- T-cells R5 HIV ATRA Differential regulation of gene expression Gut homing and increased permissiveness to HIV infection upon ATRA treatment Increased expression of HIV positive regulators: e.g., CCR5, CXCR6, KLF2 Increased expression of HIV restriction factors: e.g., ABCA1, NFIL3 Gut-homing but preserved HIV resistance upon ATRA treatment Increased expression of RARRES3/RIG-I Decreased expression of HIV negative regulators: e.g., PPARG, CCL3, CCL3L1 Decreased expression of autophagy negative regulators: e.g., mTOR No induction of CCR5 expression

16. www.ias2015.org Take-Home Message Gut-homing CCR6+ T-cells are major HIV targets and may play a critical role in viral dissemination from the portal sites of entry Take-Home Message

17. www.ias2015.org Acknowledgements CRCHUM Petronela Ancuta, Ph.D. Yuwei Zhang, Ph.D. Annie Gosselin, M.Sc. Tévy-Suzy Tep, B.Sc. Sylvia Pouvreau, M.Sc. Vanessa Sue Wacleche, M.Sc. Mohamed El-Far Ph.D. Nicolas Chomont, Ph.D. Past lab members: Patricia Monteiro.Ph.D. Aurelie Cléret-Buhot, Ph.D. Sandrina DaFonsceca, Ph.D. Caprion Jean-Philippe Goulet, M.Sc. Flow Cytometry CRCHUM Laurance Lejeune Dominique Gauchat, Ph.D. Primo infection cohort Université McGill Jean-Pierre Routy, M.D. HIV/AIDS and Infectious Diseases Network of the FRQ-S HIV-infected and uninfected donors for their gift of leukapheresis.

18. www.ias2015.org Take-Home Message Superior Proliferation of CCR6+ vs. CCR6- T-cells in the Presence or Absence of ATRA

19. www.ias2015.org Take-Home Message Inflammation GSK3 pathway Metabolisme Glycolyse pathway Gene Set Variation Analysis (GSVA): Canonical Pathways Modulated by ATRA in CCR6- cells