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The BLCs and PBMCs were counted and assessed for viability using trypan blue (Sigma-Aldrich) and compound microscopy to ensure >90% lymphocyte viability. To assess distribution of CD4⁺ and CD8⁺ T cells, immune regulation and functionality of CD8⁺ T cells in HIV infection, cells from the two compartments were subjected to two panels of fluorescently labeled antibodies. Panel 1 (phenotypic panel): Live/dead-amcyan (Life Technologies, Carlsbad, CA, USA), CD3-BV650, CD4-BV711, CD14-APC-Cy7, PD-1-BV711, TIM-3-BV785 (BioLegend, San Diego, CA, USA), and CD8-PE Texas Red (Invitrogen, Carlsbad, CA, USA). Panel 2 (intracellular cytokine staining panel): Live/dead-amcyan (Life Technologies), CD3-Alexa700, CD4-BV711, CD8-APC-Cy7, granzyme b-Alexa647, IFNγ-Dazzle 549 (BioLegend). Panel 2 staining was done after stimulation with PMA/Ionomycin (25/500 ng/mL). Acquisition was performed on BD FACS Aria III. Flowjo v10.5 (Flowjo, LLC) was used for flow cytometry analysis.

To monitor the impact of ART on major immune cell populations with emphasis on CD4⁺ T cell restoration and immune activation, the immune cells were immunophenotyped by flow cytometry. First, a two-step TruCount technique was used to enumerate CD4⁺ and CD8⁺ T cells in blood, as previously described [113 ]. The number of CD45⁺ cells was quantified using 50 μl of whole blood stained with antibodies in TruCount tubes (BD Biosciences) that contained a defined number of fluorescent beads to provide internal calibration. The numbers of CD4⁺ and CD8⁺ T cells were then calculated based on the ratio of CD4⁺ and CD8⁺ T cells to CD45⁺ cells in whole blood at the same time point. Whole peripheral blood was stained with fluorescently-labeled antibodies (all antibodies from BD Bioscience, San Jose, CA, USA unless otherwise noted), CD4 (APC), HLA-DR (PE-Cy7), CD45 (PerCP), CD25 (PE), CD69 (APC-Cy7), CD20 (APC-H7), CD8 (PE-Texas Red) (Invitrogen) and CD38 (FITC) (Stemcell). For intracellular staining, cells were fixed, permeabilized and stained for Ki-67 (PE). Flow cytometry acquisitions were performed on an LSR II flow cytometer (BD Biosciences) and flow data were analyzed with FlowJo software (Treestar, Ashland, OR, USA).