Flow Cytometry Sorting of Memory CCR6⁺CD4⁺ T-Cells for HIV Reservoir Quantification

Antiretroviral therapy (ART) has transformed the deadly human immunodeficiency virus type I (HIV-1) epidemic into a manageable chronic condition. Current ART is not curative and treatment interruption leads to viral rebound in people living with HIV-1 (PLWH). The main cause of viral rebound is the persistence of HIV reservoirs in long-lived memory CD4⁺ T cells. Accurate techniques to identify and quantify viral reservoirs are required to monitor therapeutic approaches designed to cure HIV infection. Th17-polarized CD4⁺ T cells are located at mucosal sites of HIV entry and are preferentially targeted for infection and viral reservoir persistence. They constitute an important reservoir in both blood and colon. In this chapter we describe a step-by-step flow cytometry-based protocol to isolate a fraction of Th17-enriched cells from PBMC based on their expression of the Th17 surface marker CCR6. The isolation of memory CCR6⁺CD4⁺ T cells allows subsequent PCR/RT-PCR-based HIV DNA/RNA quantifications, as well as their culture for quantitative viral outgrowth assays (QVOA). This method can be adapted for the isolation of CCR6⁺CD4⁺ T cells from peripheral tissues, such as the colon.