Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4 + T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4 + T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4 + T cells during normal chemokine-directed recirculation of CD4 + T cells between blood and tissue.

Objective In HIV-infected individuals on antiretroviral therapy (ART), latent HIV is enriched in CD4+ T-cells expressing immune checkpoint molecules (ICs), in particular programmed cell death-1 (PD-1). We therefore assessed the effect of blocking PD-1 on latency, both in vitro and in vivo. Methods HIV latency was established in vitro following co-culture of resting CD4+ T-cells with myeloid dendritic cells. Expression of PD-1 was quantified by flow cytometry, and latency assessed in sorted PD-1high and PD-1low/− non-proliferating CD4+ memory T-cells. The role of PD-1 in the establishment of latency was determined by adding anti-PD-1 (pembrolizumab) to co-cultures before and after infection. Additionally, a single infusion of anti-PD-1 (nivolumab) was administered to an HIV-infected individual on ART with metastatic melanoma, and cell-associated (CA) HIV DNA and RNA, and plasma HIV RNA were quantified. Results HIV latency was significantly enriched in PD-1high compared to PD-1low/− nonproliferating, CD4+ memory T-cells. Sorting for an additional IC, T-cell immunoglobulin domain and mucin domain-3 (Tim-3), in combination with PD-1 further enriched for latency. Blocking PD-1 prior to HIV infection, in vitro, resulted in a modest but significant decrease in latently infected cells in all donors (n=6). The administration of anti-PD-1 to an HIV-infected individual on ART, resulted in a significant increase in CA HIV RNA in CD4+ T-cells, without significant changes in HIV DNA or plasma HIV RNA, consistent with reversal of HIV latency. Conclusions PD-1 contributes to the establishment and maintenance of HIV latency and should be explored as a target, in combination with other ICs, to reverse latency.

Latently infected resting CD4+ T cells are a major barrier to HIV cure. Understanding how latency is established, maintained and reversed is critical to identifying novel strategies to eliminate latently infected cells. We demonstrate here that co-culture of resting CD4+ T cells and syngeneic myeloid dendritic cells (mDC) can dramatically increase the frequency of HIV DNA integration and latent HIV infection in non-proliferating memory, but not naïve, CD4+ T cells. Latency was eliminated when cell-to-cell contact was prevented in the mDC-T cell co-cultures and reduced when clustering was minimised in the mDC-T cell co-cultures. Supernatants from infected mDC-T cell co-cultures did not facilitate the establishment of latency, consistent with cell-cell contact and not a soluble factor being critical for mediating latent infection of resting CD4+ T cells. Gene expression in non-proliferating CD4+ T cells, enriched for latent infection, showed significant changes in the expression of genes involved in cellular activation and interferon regulated pathways, including the down-regulation of genes controlling both NF-κB and cell cycle. We conclude that mDC play a key role in the establishment of HIV latency in resting memory CD4+ T cells, which is predominantly mediated through signalling during DC-T cell contact.