Infection of resting CD4 + T-cells

1. Myeloid dendritic cells and HIV latency in resting T-cellsNitasha A Kumar, Vanessa A Evans, Suha Saleh, Candida de Fonseca Pereira, Paula Ellenberg, Paul U Cameron, Sharon R Lewin. International AIDS Conference, Washington DC, July 2012

2. Infection of resting CD4+ T-cells Resting CD4+ T-cell In vitro Unactivated resting cells chemokines Eckstein et al, Immunity 2001; 15: 671; Kreisberg et al., J Exp Med 2006; 203:865; Saleh et al., Blood 2007;110:416; Cameron et al., ProcNatlAcadSci 2010;107(39):16934-9 Ex vivo tissue blocks CCR7 (CCL19/21) CXCR3 (CXCL9/10) CCR6 (CCL20) Swiggard et al., J Virol 2005; 79(22):14179-88; Lassen et al., Plos One 2012; 7(1):e30176

3. Infection of resting CD4+ T-cells Resting CD4+ T-cell Unactivated resting cells Eckstein et al, Immunity 2001; 15: 671; Kreisberg et al., J Exp Med 2006; 203:865; Ex vivo tissue blocks In vitro Dendritic cells Swiggard et al., J Virol 2005; 79(22):14179-88; Lassen et al., Plos One 2012; 7(1):e30176 Saleh et al., Blood 2007;110:416; Cameron et al., ProcNatlAcadSci 2010;107(39):16934-9

4. DC added 1:10 AND PBMC + Mouse-anti-human CD3, CD11b, CD19 Bulk DC Plasmacytoid DC Myeloid DC SNARF or Efluor Resting CD4+ T-cells HLA-DR Lineage cocktail CD123 CD11c

5. DC added 1:10 AND 1 day SNARF Non-proliferating SNARFhi Not productively infected EGFP- R5-EGFP-HIV-1 (2h pulse) 5 days EGFP Latent infection SNARF PBMC EGFP Productive infection SNARF or Efluor Resting CD4+ T cells SNARFhiEGFP- CD4+ T-cells PHA Amplification in PBMC 5 Days

6. DC induce latent infection in non-proliferating (SNARFhi) cells

7. SNARFhiEGFP- T-cells express CD69

8. Alu-LTR PCR 5 days Productive Latent mDC specifically mediate HIV latency SNARFhiEGFP- CD4+ T-cells + +pDC Days PHA -1 0 5 + +mDC PBMC

9. SNARFhiEGFP- T-cells express PD1 Chomont et al., Nature Med 2010

10. Establishment of latency not related to degree of productive infection MDDC = monocyte derived DCs

11. Myeloid DC induce latency in memory not naïve CD4+ T-cells

12. Cytokines/chemokines in mDC-T-cell co-cultures + Days +pDC Bead arrays -1 0 5 + +mDC

13. Neutralization of DC-derived chemokines does not inhibit latency

14. Neutralisation of select cytokines does not inhibit latency

15. DC DC EGFP HIV-1 DC CD4+ T CD4+ T DC-T cell proximity required for DC-induced latency 24 hrs DC CD4+ T

16. CD4+ T DC-T-cell culture supernatant doesn’t facilitate latency n=3 DC Supernatant CD4+ T CD4+ T

17. Conclusions • Myeloid DC facilitate establishment of HIV latency in resting memory CD4+ T-cells via direct infection. • Close proximity (± cell contact) of mDC and T-cells required • Role for negative regulators in the establishment/maintenance of DC-induced latency

18. Implications • Efficient infection of resting T-cells in close contact with mDC and HIV may explain the rapid early establishment of the latent reservoir. • If infectious virus persists in tissues such as LN in patients on cART, mDC may facilitate ongoing infection of resting T-cells leading to replenishment of the reservoir.

19. Department of Infectious Diseases, Monash University Sharon Lewin Paul Cameron Vanessa Evans Suha Saleh Paula Ellenberg Candida de Fonseca Pereira Ajantha Solomon University of Melbourne Damian Purcell Acknowledgements • Flow Cytometry Facility, Monash University • Geza Paukovics • Michael Thomson • Jeanne Le Masurier • Vaccine Gene Therapy Institute • RafickSekaly • Nicholas Chomont