Background:  HIV can persist in a latent state in resting CD4⁺ T cells despite highly active ART. Resting CD4⁺ T cells in lymphoid tissue ex vivo are relatively permissive to HIV infection while resting CD4⁺ T cells from peripheral blood are resistant to HIV infection. We investigated the role of the homeostatic chemokines and CCR7 ligands, CCL19 and CCL21, on HIV infection of resting CD4⁺ T cells.

Methods:  Highly purified resting CD4⁺ T cells were incubated with CCL19 or CCL21 (10 to 100 nM) and then infected with HIV NL4.3 or AD8. Resting CD4⁺ T cells were also infected following stimulation with phytohemagglutinin/interleukin-2 (PHA/IL-2) or without activation as positive and negative controls, respectively. Reverse transcriptase (RT) activity was quantified in culture supernatants and integrated HIV-1 DNA was quantified at day 4 and 7 post-infection using a nested Alu-LTR real-time polymerase chain reaction (RT-PCR) assay. Expression of HLA-DR, CD69, CD25, CXCR4, CCR5, CCR7, and Ki67 was determined by flow cytometry.

Results:  Stimulation with CCL19/CCL21 either alone or in combination prior to infection of resting CD4⁺ T cells with either NL4.3 or AD8 led to low level production of RT in culture supernatant in comparison with no significant increase in RT following infection of unactivated cells. Following stimulation with CCL19/CCL21, there was no change in the expression of markers of activation but expression of CCR5 was mildly increased and there was modest down-regulation of CCR7. We measured a significant frequency of integrated copies of HIV following infection of resting CD4⁺ cells stimulated with PHA/IL-2 or CCL19 compared with unactivated cells (n = 4; mean ratio of integrated copies of HIV following stimulation with PHA/IL2 and CCL19 compared with unactivated cells was 1800 and 320 respectively) at day 4 post-infection. The frequency of integrated HIV was on average 6 fold higher following stimulation with PHA/IL2 compared with CCL19. The high frequency of integrated HIV DNA and low production of RT was identified following stimulation with either CCL19 or CCL21 (alone or in combination) and following infection with either AD8 or NL4.3.

Conclusions:  Following stimulation with CCL19 and CCL21, resting CD4⁺ T cells were permissive to HIV infection with a high level of HIV integration and low level virus production. This study suggests that CCL19 and CCL21 may be critical chemokines in the lymph node environment that allow HIV infection of resting CD4⁺ T cells and the establishment of latency.