Fig 2: Low percentages of differentiated Th cells in HEU newborns. Peripheral blood from HEU or umbilical cord blood from HUU newborns was stained with monoclonal antibodies and analyzed by flow cytometry. (A) Representative gating strategy. From singlet memory, CD4+ T lymphocytes, subsets of Th cells were identified with the following phenotypes: Th1 (CD183+CD194-CD196-/CXCR3+CCR4-CCR6-); Th2 (CD183-CD194+CD196-/CXCR3-CCR4+CCR6-); Th17 (CD183-CD194+CD196+/CXCR3-CCR4+CCR6+); and CD4+CD25++ T cells. The percentages of (B) Th1, (C) Th2, (D) Th17, and (E) CD4+CD25++ T cells among total CD4+ T lymphocytes from HEU and HUU newborns are shown. Error bars indicate the median and interquartile range. HEU, HIV-exposed uninfected; HUU, HIV-unexposed uninfected. p Values were calculated using the unpaired Mann–Whitney test. *p < 0.05, **p < 0.01, and ****p < 0.0001

Fig 3: T cell phenotype and senescence/exhaustion markers in MDD(A–D) PBMCs from patients with MDD and matched HC were examined by flow cytometry. CD4+ (upper panel) and CD8+ T cell subpopulations (lower panel) were analyzed for the expression of markers classifying differentiation (CD45RA, CCR7; n = 25) (A), chemokine receptors (CXCR3, CCR6, CCR4; n = 25) or regulatory T cell markers (CD25, CD127; n = 22) (B), markers indicative of exhaustion or senescence (PD-1, KLRG1, CD57; n = 19–20) (C) or inhibitory receptors (CTLA-4, LAG-3, Tim-3; n = 20) (D). Naive/Memory subpopulations were defined as TNaive (CCR7+/CD45RA+), TEM (CCR7-/CD45RA-), TCM (CCR7+/CD45RA-), and TEMRA (CCR7-/CD45RA+). T helper cell subsets were defined as Th1 (CXCR3+/CCR6-), Th2 (CXCR3-/CCR6-/CCR4+), Th17 (CXCR3-/CCR6+/CCR4+), and Th1/Th17 cells (CXCR3+/CCR6+) (n = 25). For the analysis of inhibitory receptors, PBMCs were stimulated with 10 µg/mL of a-CD3 and 1 µg/mL a-CD28 for 48 h. Median and interquartile ranges are shown. Wilcoxon signed-rank test was used to compare groups. All p values > 0.1 if not otherwise indicated.

Fig 4: Gating strategy and validation of immunophenotyping panel #1 (lymphocyte panel). (A) This gating strategy was used to define certain cell populations. A gate for main cells was used on all events to exclude debris and then cells were gated to obtain singlet cells. A viability dye was then used to exclude dead cells and thus the live cells were selected. The live cells were then gated with CD45. CD19+ gate was then used to define the B cell population within the CD45+ gate and the markers CD154 and CD25 were used to determine the activation statuses of the CD19+ population. The CD45+ gate was also used to then select the CD3+ cell population (T cells). This CD3+ population was then gated on CD8 and CD4 markers. CD8+/CD4− population defined cytotoxic T cells which were then further gated on naïve status (CD45RA+) and activation status (CD154/CD25). CD4+/CD8− population defined helper T cells and their activation status (CD154/CD25) and was also used to further gate on CCR4+, CD25+, CD127lo cells to define Tregs. The CD4−/CD8− gate was gated on γδ TCR to define this population of T cells. (B) Gating shown for unstimulated PBMC from one replicate of healthy donor M4W2. Red and blue overlays are the isotype controls while the black overlay is a replicate of the fully stained PBMC with labeling antibodies. Each gate is an inclusive gate (cells in gate are included in further gating) except the viability (live/dead) gate which is an exclusive gate (cells in gate are excluded from further gating) (C) PBMC from four healthy donors (N = 4, 2 technical replicates per donor) were activated with positive controls (LPS/PHA-M or PMA/Ionomycin) or left unstimulated/untreated for 24 h. The percentage of cells in each gate for each donor were determined following the same gating strategy as shown in A. The percentages indicate the percentage of selected cells in parent gate rather than in the total events. Each box plot represents data of 2 replicates from each of four donors (M4W2, F4Z5, J7X3, E3T5). The middle bar is the median and the whiskers are the 10th and 90th percentiles of the data.