Background:
CD4+ T cells are the main cellular targets of both HIV and SIV. While the phenotype of HIV-infected cells has been extensively reported, the cellular subsets in which SIV actively replicates in blood and tissues from non-human primates (NHPs) remain largely unidentified. We developed a novel flow-based approach to identify the phenotype of SIV-infected cells during untreated SIV infection. By using two different antibodies specific for the p27 SIV protein (2F12 and 4B7) we enumerated and phenotyped productively infected cells with high specificity. The assay (SIV-Flow) can detect a single infected cell in a million CD4+ T cells and has a large dynamic range (1 to 100,000 infected cells/106 CD4+ T cells).
Methods:
We analyzed infected cells in multiple tissues from 4 SIVmac239-infected ART-naïve rhesus macaques, including blood, spleen, axial, mesenteric and inguinal lymph nodes. CD4+ T cells were isolated by negative magnetic selection and stained with a combination of antibodies to obtain the phenotype of p27+ cells (CTLA-4, PD-1, Ki67, CD69, CD127, CD45RA, CCR7, CD27, CD95, CD3, CD4, CD8).
Results:
In all animals, the highest frequencies of p27+ cells were measured in the spleen (mean 1887 cells/106 CD4+ T cells), whereas blood and lymph nodes displayed similarly lower frequencies (842 and 848 cells/106 CD4+ T cells, respectively). p27+ were under represented in the naïve compartment in both blood and tissues (fold enrichment of 0.6 and 0.3, respectively). In the blood, p27+ cells were highly enriched in the central memory subset (fold enrichment of 3.8), whereas p27+ cells frequently displayed a transitional and effector memory phenotype in lymphoid tissues (fold enrichment of 2.3 and 2.9, respectively). Overall, when compared to their uninfected counterparts, p27+ cells expressed higher levels of several cellular markers including PD-1 (68% vs 32%), CD69 (47% vs. 23%), Ki67 (38% vs. 12%) and CTLA-4 (21% vs. 12%). Of note, enrichment of p27+ cells subsets expressing these activation and exhaustion markers was more pronounced in lymphoid tissues.
Conclusions:
In untreated SIV-infected NHPs, productively-infected cells are found at higher frequencies in the spleen, which can be explained by the high percentages of PD-1+ and effector memory cells and the relatively low proportion of naïve cells in this tissue. Our results suggest that activated CD4+ T cells in the spleen are important contributors to SIV replication in chronically infected NHPs.