Resting CD4 + T cells: a cellular reservoir of latent HIV-1

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

2. Resting CD4+ T cells: a cellular reservoir of latent HIV-1 • Memory – considered major reservoir • Observed early in infection • Long half life ~ 6 - 40 months • Resistant to • Antiretrovirals • Host immune recognition IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 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

3. OR Post-integration Resting CD4+ T cell latency Pre-integration Resting CD4+ T cell Blood IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Lymphoid blocks Possible role for cellular secretions and/or cell-cell interactions within lymphoid tissues Eckstein et al., Immunity 2001, 15: 671; Kreisberg et al., J Exp Med 2006, 203:865; Saleh et al., Blood 2007, 110:416

4. High frequency of integrated HIV-1 with low level of productive infection in resting cells incubated with CCL19 1000 RT CPM/ul IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 100 0 2 3 6 4 5 7 1 Days after infection Unconditioned PHA/IL-2 CCL19 Saleh et al., Blood 2007; 110:4161

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

6. +/- SEB 10ng/mL DC added 1:10 +/- SEB 10ng/mL In vitro model SNARF 24 hrs R5 EGFP-HIV-1 - 1 0 Resting CD4+ T cells IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Resting CD4+ T cells + DC

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

8. Unstimulated SEB Stimulated p<0.05 DC induce latent infection of SNARFhiEGFP- CD4+ T cells SNARFhi EGFP-CD4+ T cells Days post sort 0 5 PHA-PBMC IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Unstimulated Amplification of HIV-1 from latently NOT productively infected cells

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

10. Is DC-T cell contact required? DC DC EGFP HIV-1 p=0.03 Resting CD4+ T EGFP+ cells /104 cells (Latently infected cells) IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 24 hrs Soluble factors play a role in DC-induced latency

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

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

13. Do pDC or mDC induce latency? Unstimulated SEB Stimulated p=0.02 p=0.02 p=0.02 p=0.01 EGFP+ cells /104 cells (Latently infected cells) CD4+ T CD4+T CD4+T (post pDC) (post mDC) CD4+ T CD4+T CD4+T (post pDC) (post mDC) CD4+ T + pDC ± HIV-1 EGFP + mDC SNARF-resting CD4+ T cells Days -1 0 5 5 Days SNARFhiEGFP- CD4+ T cells + PHA-PBMC IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Myeloid DC induce HIV-1 latency in non-proliferating CD4+ T cells

14. Unstimulated SEB Stimulated Alu-LTR copies /106 cells CD4+ T pDC: CD4+T mDC: CD4+T CD4+ T pDC: CD4+T mDC: CD4+T mDC induce post-integration latency in non-proliferating CD4+ T cells Integrated HIV-1 DNA (Alu-LTR nested PCR) SNARFhiEGFP- CD4+ T cells IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA CD4+ T CD4+T CD4+T (post pDC) (post mDC) CD4+ T CD4+T CD4+T (post pDC) (post mDC)

15. 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

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

17. Non-proliferating CD4+ T cells co-cultured with DC p<0.05 Latent Mock 461 Genes - + 0 IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

18. 17 Downregulated 11 Upregulated CHKA – G0/G1 transition CDC20/AURKB – mitosis IL-16/BIRC5 – cell cycle GADD45A – growth arrest IFI16 – growth arrest 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) IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Latently infected non-proliferating CD4+ T cells

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

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

21. 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