Efficient HIV latency reversal with immune checkpoint blockade in a primary T-cell latency model.

Authors: Van der Sluis RM1,2, Rachel D. Pascoe1, Jennifer M. Zerbato1, Kumar NA1, Evans VA1, Dantanarayana AI1, Jenny L. Anderson1, Sékaly RP3, Fromentin R4, Chomont N4,5, Cameron PU1,6 and Lewin SR1,6 1The Peter Doherty Institute for Infection and Immunity, The University of Melbourne and Royal Melbourne Hospital, Melbourne, Australia; 2Aarhus Institute of Advanced Studies (AIAS), Aarhus University, Aarhus, Denmark; 3Case Western University, Cleveland, OH; 4Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montreal, QC, Canada; 5Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montreal, QC, Canada; 6Department of Infectious Diseases, Alfred Health and Monash University, Melbourne, Australia. Introduction: In HIV-infected individuals on antiretroviral therapy (ART), HIV latency is the major barrier to a cure. HIV persists preferentially in CD4+ T-cells expressing multiple immune checkpoint (IC) molecules. Aim: To determine whether IC molecules have a role in maintaining HIV latency and if blocking IC molecules with antibodies (IC blockers: ICB) alone or in combination, can reverse latency. Methods: Resting CD4+ T-cells, isolated from blood of HIV-uninfected individuals, were stained with the proliferation dye eFluor670, co-cultured with syngeneic monocytes and infected with full length CCR5-tropic EGFP-reporter HIV. On day 5 post-infection, non-proliferating (eFLuorhi) and proliferating (eFluorlo) CD4+ T-cells that were not productively infected (EGFP-) and thus potentially latently infected were sorted. Expression of the IC molecules: cytotoxic T lymphocyte-4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), B- and T-lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or programmed death-1 (PD-1), were determined. To assess the ability of ICB to reactivate latency, sorted cells were cultured: 1. alone (background EGFP); 2. with anti-CD3/anti-CD28+IL-7+IL-2 (max EGFP/latency reactivation); 3. with one or more ICB / corresponding isotype control(s) in the presence of monocytes (mono), with or without SEB; or 4.with classic latency reversing agents including vorinostat and bryostatin. Raltegravir and T20 (an HIV integrase and fusion inhibitor, respectively) were added to all reactivations to prevent subsequent rounds of infection. Results: Latent infection was enriched in proliferating cells expressing PD-1 and in non-proliferating cells expressing CTLA-4, TIM-3, BTLA or PD-1. ICB reversed HIV latency in both proliferating and non-proliferating CD4+ T-cells, but only when multiple ICB were used or when an ICB was combined with an additional T-cell activating stimulus (ICB+mono+SEB). Latency reversal was higher following IC blockade compared to classical latency reversing agents. Conclusion: Combination IC blockade should be further explored as a strategy to reverse HIV latency.

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