2014 “Towards an HIV Cure” symposium Melbourne Following in vitro culture with myeloid dendritic cells, negative regulators of T cell activation are expressed preferentially on latently infected CD4 + T cells Vanessa A. Evans, Renée M. van der Sluis, Nitasha A. Kumar, Rafick-Pierre Sekaly, Remi Fromentin, Nicolas Chomont, Paul U. Cameron, Sharon R. Lewin

Infection of resting CD4 + T cells: a role for cell-cell interactions Unactivated resting cells Resting CD4 + T cell Dendritic Cells Evans et al. PLoS Pathogens 2013 Endothelial Cells Shen et al. J Virology 2013 Ex vivo tissue blocks Eckstein et al. J Virology 2001

Myeloid DC induce latency in resting memory CD4 + T cells  Not mediated by soluble factors: CCL19, CCL21, CXCL10, IL-10, IL-6  Close DC-T cell proximity required for induction of latency Non-proliferating CD4 + T cells Evans et al. PLoS Pathogens 2013

Negative regulators and latency  Negative regulators dampen the immune response and can be found on exhausted T cells Ahmed et al. J Immunol 2010; Day et al. Nature 2006  Latently infected T cells express negative regulators of T cell activation eg. PD-1, Tim-3 and TIGIT Chomont et al. Nat Med 2010; Fromentin et al. CROI 2014  Blocking PD-L1/PD-1 in vivo restores the SIV- specific cellular and humoral immune responses, improves viral control and reduces immune activation Velu et al. Nature 2009; Dyavar Shetty et al. J Clin Invest 2012; Finnefrock et al. J Immunol 2009

Hypothesis Expression of negative regulators during DC-T cell interactions may actively suppress viral replication and maintain latency

CD11c CD123 Plasmacytoid DC Myeloid DC PBMC Resting CD4 + T cells CD69CD25HLA-DR + mouse anti-human CD8, CD11b, CD14, CD16, CD19, CD69, HLA-DR Magnetic Bead Depletion CD4 CD3 >98% Resting CD4 + T cells Bulk DC PBMC + mouse anti-human CD3, CD11b, CD19 Magnetic Bead Depletion Lineage Cocktail HLA-DR Dendritic Cells

Resting CD4 + T cells eFluor 670 DC added 1:10 AND R5-EGFP-HIV-1 (2h pulse) 1 day Productive infection Latent infection >99% aCD3/aCD28 3 Days Activation + integrase inhibitor L8 Non-proliferating eFluor670 hi Not productively infected EGFP - EGFP eFluor670 eFluor670 hi EGFP - CD4 + T cells Day 5 p.i.

Expression of negative regulators following DC-T cell co-culture eFluor-resting CD4 + T cells + mDC OR R5-EGFP-HIV-1 (2h pulse) 1 day Baseline PD-1/Tim-3/CTLA-4 Phenotyping on days 1, 2, 3, 4, 5 post-infection ViableSingle cellsT cells eFluor hi EGFP - PD-1, Tim-3 and CTLA-4 EGFP eFluor HLA-DR CD3 SSC-H SSC-W FSC-A SSC-A

Days post-infection % Positive cells Expression of negative regulators following DC-T cell co-culture PD-1Tim-3 CTLA-4 % Positive cells Days post infection Mean fold change 24 No change n=3 Days post-infection Mean fold change 32 ()

Differential expression of negative regulator ligands on mDC and pDC Leitner PLoS Pathog 2013

Are PD-1 hi cells enriched for latency? eFluor hi EGFP - CD4 + T cells Anti-PD-1 Latent infection PD-1 lo/- cells PD-1 hi cells EGFP eFluor670 aCD3/28 +L8

HIV latency is enriched in PD-1 hi cells

HIV latency is enriched in Tim3 hi cells eFluor hi EGFP - CD4 + T cells Anti-Tim-3 Tim-3 lo/- cells Tim-3 hi cells

Summary  Myeloid DC facilitate establishment of HIV latency in resting memory CD4 + T cells via direct infection  Negative regulators PD-1 and Tim-3 but not CTLA-4 are up- regulated on resting CD4 + T cells upon co-culture with mDC  Negative regulator ligands are differentially expressed by mDC and pDC  mDC-induced HIV latency is enriched in PD-1 hi and Tim-3 hi cells

PD-L1 PD-1 PD-1/-L1 blockade Cell death  virus production  immune clearance Blocking interactions between negative regulators and their ligands EGFP + productively infected CD4 + T cells Latent Infection in sorted eFluor hi EGFP - CD4 + T cells

Implications  mDC may facilitate ongoing latent infection of resting CD4 + T cells leading to replenishment of the reservoir  Disrupting the function of PD-1 and/or Tim-3 could potentially be exploited to inhibit replenishment of the reservoir and/or reverse latency

Acknowledgements Monash University –Sharon Lewin –Paul Cameron –Renée van der Sluis –Nitasha Kumar –Suha Saleh –Candida da Fonseca AMREP Flow Cytometry –Geza Paukovicks –Michael Thompson –Jeanne Le Masurier –Phil Donaldson VGTI Florida –Rafick Sekaly –Nicolas Chomont –Remi Fromentin University of Melbourne –Damian Purcell