2014 “ Towards an HIV Cure ” symposium Melbourne

1. 2014 “Towards an HIV Cure” symposiumMelbourne The Immune Checkpoints PD-1, LAG-3 and TIGIT are Biomarkers of HIV Infected Cells During ART and Identify Distinct Cellular Reservoirs Remi Fromentin, Wendy Bakeman,Mariam B Lawani, Gabriela Khoury, Elizabeth Sinclair, Frederick M. Hecht, Steven G. Deeks, Sharon R. Lewin, Jean-Pierre Routy, Rafick P. Sékaly, Nicolas Chomont

2. Biomarkers of latently infected cells during ART Identifying biomarkers of latently infected cells is of primary importance to specifically target and eliminate the persistent reservoir Approach: Combining the “Where” with the “How” Where: HIV persists in discrete subsets of cells during ART How: Mechanisms driving the establishment and persistence of the HIV reservoir Biomarkers of latently infected cells could be: - surrogate markers of higher susceptibility to HIV infection - markers of persistence providing selective advantages (latency maintenance, immune escape, replenishment)

3. Why immune checkpoints (ICs) could be biomarkers for HIV persistence during ART ? • ICs, negative regulators of T cell activation, regulate T cell proliferation and cytokine production. • Several of these molecules are associated with T cell dysfunction in chronic HIV infection (PD-1, CTLA-4, TIM-3, CD160). • (Trautmann et al, NatMed 2006; Kaufmann et al, NatImm 2007; Jones et al, JEM 2008, Peretz el al, PLoSPath 2012) • Immune dysregulations persist during ART (residual immune activation, incomplete CD4 T cell restoration, T cell dysfunction). • (Hatano et al, JID 2012; Kelley et al, CID 2009) Chen L, Nat Rev Imm. 2013

4. Hypothesis By inhibiting T cell activation, negative regulators (Immune Checkpoints, ICs) may actively maintain viral latency and identify reservoir cells during ART. A B ART APC ICs ICs Inhibitory signals silencing HIV - + - + Activated/Productively infected CD4 T cell Persistent/Latently infected CD4 T cell ICs = selective advantage for latently infected cells ICs silence HIV

5. Association between the expression of ICs and virological markers of HIV persistence Study population: 48 HIV infected subjects virally suppressed for at least 3 years with CD4>350 c/µL Methods: - Multiparametric flow cytometry analysis of the expression of 8 ICBs (PD-1, LAG-3, TIGIT, CTLA-4, BTLA, CD160, 2B4, TIM-3) in PBMCs - ultrasensitive qPCRs to measure the frequency of CD4 T cells harboring virological markers of HIV persistence The frequency of CD4 T cells expressing PD-1, LAG-3 and TIGIT are positively correlated with the frequency of CD4 T cells harboring integrated HIV DNA during ART.

6. PD-1 identifies TCM and TTM CD4 T cells enriched in integrated HIV DNA A B TCM TTM TEM Differentiation The frequency of cells harboring integrated HIV DNA is significantly higher in PD-1 expressing TCM and TTM when compared to their PD-1 negative counterparts.

7. LAG-3 identifies TCM and TTM CD4 T cells enriched in integrated HIV DNA A B TCM TTM TEM Differentiation The frequency of cells harboring integrated HIV DNA is significantly higher in LAG-3 expressing TCM and TTM when compared to their LAG-3 negative counterparts.

8. TIGIT identifies TEM CD4 T cells enriched in integrated HIV DNA TCM TTM TEM A B Differentiation The frequency of cells harboring integrated HIV DNA is significantly higher in TIGIT expressing TEM when compared to their TIGIT negative counterparts.

9. Can we further enrich in the reservoir by combining multiple ICs? A B Means +/-SD from 5 subjects Memory CD4 T cells expressing multiple ICs are highly enriched for integrated HIV DNA

10. Is the virus carried by latently infected cells expressing ICs functional ? A Principle of “Tat/Rev Induced Limiting Dilution Assay” (TILDA) B Nested RT-PCR For msHIV RNA (24+40 cycles) Maximum likelihood method PMA/Ionomycin (12h) 18000 cells/well Frequency of cells with inducible msHIV RNA 9000 cells/well 3000 cells/well 1000 cells/well Memory CD4 T cells expressing LAG-3 and/or PD-1 and/or TIGIT are highly enriched for inducible HIV latently infected cells. Sorted CD4+ T cells

11. Conclusions 1. 2. Enrichment for HIV infected cells Altogether, our data suggest that blocking ICs may reactivate HIV from latency and paves the way for the development of novel strategies to cure HIV infection.

12. Acknowledgements • VGTI Florida Wendy Bakeman Amanda McNulty Mariam B. Lawani Rafick-Pierre Sekaly Nicolas Chomont • UCSF Steven Deeks HiroyuHatano Rick Hecht Rebecca Hoh Elisabeth Sinclair Lorrie Epling • Burnet Institute Gabriela Khoury Sharon R. Lewin • McGill University Jean-Pierre Routy • Merck DariaHazuda Mike Miller Richard J.O. Barnard Dan Gorman The study participants