Myeloid dendritic cells induce HIV-1 latency in non- proliferating CD4 + T cells IAS WORKSHOP, 16 & 17 JULY 2010

Resting CD4 + T cells: a cellular reservoir of latent HIV-1  Memory – considered major reservoir  Observed early in infection  Long half life ~ months  Resistant to  Antiretrovirals  Host immune recognition Finzi et al., Science 1997; 278:1295; Wong et al., Science 1997; 278:1291; Chun et al., PNAS 1997; 94:13193; Siliciano et al., Nat Med 2003; 9:727; Ramratnam et al., JAIDS 2004; 35:33 IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

Resting CD4 + T cell latency Resting CD4 + T cell Blood Lymphoid blocks Possible role for cellular secretions and/or cell-cell interactions within lymphoid tissues Pre-integration Post-integration OR Eckstein et al., Immunity 2001, 15: 671; Kreisberg et al., J Exp Med 2006, 203:865; Saleh et al., Blood 2007, 110:416

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA High frequency of integrated HIV-1 with low level of productive infection in resting cells incubated with CCL19 Saleh et al., Blood 2007; 110: RT CPM/ul Days after infection Unconditioned PHA/IL-2 CCL19

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Hypothesis Establishment of latency occurs as a result of signalling by DC during the recirculation of CD4 + T cells through lymphoid tissue DC are found within T-zones of lymphoid tissues

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA In vitro model R5 EGFP-HIV-1 24 hrs SNARF - 10 Resting CD4 + T cells +/- SEB 10ng/mL DC added 1:10 Resting CD4 + T cells + DC +/- SEB 10ng/mL

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA R5 EGFP-HIV-1 (2h pulse) Non-proliferating SNARF hi Not productively infected EGFP - EGFP SNARF PHA 5 Days SNARF hi EGFP - CD4 + T cells Amplification in PBMC SNARF EGFP 5 Days Detection of replication competent latent infection PBMC EGFP + feeder PBMC a surrogate for latently infected cells

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA SNARF hi EGFP - CD4 + T cells 05 Days post sort Unstimulated PHA-PBMC DC induce latent infection of SNARF hi EGFP - CD4 + T cells Unstimulated Amplification of HIV-1 from latently NOT productively infected cells SEB Stimulated p<0.05

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Activation state of the SNARF hi EGFP - CD4 + T cells Unstimulated Sorted SNARF hi EGFP - cells were non-proliferating CD4 + T cells Percent positive cells CD69 HLA-DR Ki67 SEB Stimulated

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 24 hrs EGFP HIV-1 Resting CD4 + T DC Soluble factors play a role in DC-induced latency Is DC-T cell contact required? EGFP + cells /10 4 cells (Latently infected cells) p=0.03

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Blocking CD18 inhibits DC-T cell adhesion Cell-cell contact AND soluble factors are required for maximal DC-induced latency

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 24 hrs Resting CD4 + T DC EGFP HIV-1 Latency inducing soluble factors are secreted by DC as a result of HIV-1 stimulation Do DC condition resting CD4 + T cells towards latency without HIV-1 exposure?

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Do pDC or mDC induce latency? Myeloid DC induce HIV-1 latency in non-proliferating CD4 + T cells Days 0 CD4 + T± HIV-1 EGFP 5 + PHA-PBMC SNARF hi EGFP - CD4 + T cells SNARF-resting CD4 + T cells + pDC + mDC 5 Days Unstimulated CD4 + T CD4 + T CD4 + T (post pDC) (post mDC) EGFP + cells /10 4 cells (Latently infected cells) p=0.02 p=0.01 SEB Stimulated CD4 + T CD4 + T CD4 + T (post pDC) (post mDC) p=0.02

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA mDC induce post-integration latency in non-proliferating CD4 + T cells SNARF hi EGFP - CD4 + T cells Integrated HIV-1 DNA (Alu-LTR nested PCR) Unstimulated Alu-LTR copies /10 6 cells CD4 + T pDC: CD4 + T mDC: CD4 + T SEB Stimulated CD4 + T pDC: CD4 + T mDC: CD4 + T CD4 + T CD4 + T CD4 + T (post pDC) (post mDC ) CD4 + T CD4 + T CD4 + T (post pDC) (post mDC )

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Summary  mDC induce post-integration latency  Mediated by  DC-T cell contact  Soluble factors (HIV-1 exposure)  Partial activation?  Enhanced by SEB

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Days post infection 0 ± DC HIV-1 EGFP MOI 1 5 Non-proliferating (SNARF hi )Not productively infected (EGFP - ) Lysed for microarrays MOCK Gene profiles of DC-induced latently infected CD4 + T cells

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA MockLatent p<0.05 Non-proliferating CD4 + T cells co-cultured with DC Genes

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 65 genes involved in cell cycle  28 genes  Arrest in cell division (24)  Delay in cell division (9)  Arrest in cell cycle progression (13)  Arrest G0/G1 phase transition (2) GADD45A – growth arrest IFI16 – growth arrest 11 Upregulated CHKA – G0/G1 transition CDC20/AURKB – mitosis IL-16/BIRC5 – cell cycle 17 Downregulated Latently infected non-proliferating CD4 + T cells

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA  TRIM22  OAS1  TXNRD1 Upregulation Genes that inhibit HIV-1  IKBE  ITGAL  PRKCA  TNFRSF14 Downregulation Genes involved in NFkB activation Latently infected CD4 + T cells

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA  Suboptimal activation – ↑CD69  Enhanced integration  Productive infection prevented:  Blocks in HIV-1 transcription  Blocks in cell proliferation mDC-induced post-integration latency in non-proliferating CD4 + T cells In vitro model to study the establishment and/or reactivation of latency in primary resting CD4 + T cells A mechanism for the establishment of latency in vivo within lymphoid tissues

Acknowledgements MONASH UNIVERSITY Sharon Lewin Paul Cameron Suha Saleh Geza Paukovics UNIVERSITY OF MONTREAL Rafick-Pierre Sekaly Elias Haddad Nadia Kettaf Jean-Philipe Goulet UNIVERSITY OF MELBOURNE Damian Purcell IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA