Aqua viability stain

Half a million BAL cells or PBMCs were stained with a cocktail of antibodies for DN T-cell phenotyping. To eliminate dead cells from the analysis, we stained cells with Aqua viability stain (Invitrogen) and define DN T cells as Aqua-CD3⁺ CD4⁻ CD8αα⁻ CD8αβ⁻ cells. Anti-HLA-DR was used as a marker of activation. Anti-CD28, anti-CD45RA, and anti-CD57 were used to identify naive, CM, EM, TD, and senescent DN T cells. Anti-CCR6 and anti-CXCR3 were included to identify previously described cellular HIV reservoirs, as well as T-cell homing in lung tissue using the latter (65 (link)). Intracellular stainings were performed using anti-perforin and anti-granzyme B to assess cytotoxicity phenotype of DN T cells. References for all antibodies used for this study are described in Table 2.

Frequency of different CD4+ and CD8+ T cell subsets were defined via multi-parameter flow cytometry (BD Fortessa X-20) in total BAL cells or PBMCs as we previously described [7 (link),16 (link)]. To eliminate dead cells from the analysis, we stained cells with Aqua viability stain (Invitrogen, Waltham, MA, USA). Expressions of CD8+, CD4+ and/or CD8+, CD45RA+, CD28+ were used to enumerate naïve (CD45RA⁺ CD28⁺), central memory (CD45RA^- CD28⁺), effector memory (CD45RA^- CD28^-) and terminally differentiated (CD45RA⁺ CD28^-) cells. Levels of CD8+ and CD4+ T cell immune activation (CD38+ HLADR+), and senescence (CD28- CD57+) were assessed on CD4+ and CD8+ T cell subsets.