Myeloid dendritic cells and HIV latency in resting T-cells Nitasha 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

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

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

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

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

DC induce latent infection in non- proliferating (SNARF hi ) cells

SNARF hi EGFP - T-cells express CD69

mDC specifically mediate HIV latency Days 05 +pDC Productive +mDC + + Alu-LTR PCR 5 days Latent PHA SNARF hi EGFP - CD4 + T-cells PBMC

SNARF hi EGFP - T-cells express PD1 Chomont et al., Nature Med 2010

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

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

Cytokines/chemokines in mDC-T- cell co-cultures Days 05 +pDC +mDC + + Bead arrays

Neutralization of DC-derived chemokines does not inhibit latency

Neutralisation of select cytokines does not inhibit latency

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

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

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

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.

Acknowledgements  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  Flow Cytometry Facility, Monash University –Geza Paukovics –Michael Thomson –Jeanne Le Masurier  Vaccine Gene Therapy Institute –Rafick Sekaly –Nicholas Chomont