Fig 2: CD4+ TRM identification in cervix. a General gating strategy for phenotyping of CD4+ T cells obtained from cervicovaginal tissue of healthy donors. Gating strategy consisted of selecting hematopoietic CD45+ cells, followed by a double doublet exclusion, dead and CD19+ cells exclusion and finally a CD3+ CD4+ T cell gate from where CD69+/- cells were identified. b Representative flow cytometry plots of the expression of different cell-surface proteins and transcriptional factors in the CD4+CD69+/- T cell subsets from the cervical tissue of healthy donors (CD69- on the left column, CD69+ on the right column). c Frequency of different cell-surface proteins and transcriptional factors shown in b for CD4+CD69- T cells (empty circles) and CD4+CD69+ T cells (full circles; n = 5 for S1PR1; n = 6 for Eomes, T-bet, Hobit, CD49a; n = 7 for CCR7, PD-1). Lines and error bars represent median and interquartile ranges. Statistical comparisons were performed using Wilcoxon matched-pairs signed rank test to compare the two groups. Source data are provided as a Source Data file

Fig 3: Characterization of the Phenotype of Human Hemogenic Endothelial Cells on a Population Level(A) Schematic representation of blood-forming assay.(B) Representative photographs showing morphology of BFU-E erythroid colony, CFU-E erythroid colony, CFU-GM granulocyte-monocyte colony, and CFU-GEMM multipotent hematopoietic progenitor colony. Scale bars, 100 µm.(C) Among non-HSPC (CD45-), hematopoietic potential is enriched within the CD31+ population; n = 3 for all groups. Student’s t test: p = 0.03. Solid bars represent positive groups. Checkered bars represent negative groups. Striped bars represent parental groups.(D) Among CD31+CD45- cells, hematopoietic potential is enriched within the CDH5- and CD34- populations; n = 3 for all groups. Student’s t test: p = 0.005 (CDH5), p = 0.04 (CD34). Solid bars represent positive groups. Checkered bars represent negative groups. Striped bars represent parental groups.(E) Among CD31+CDH5-CD45- cells, hematopoietic potential is enriched within the KIT+ and CD34+ populations; n = 3 for all groups. Student’s t test: p = 0.03 (KIT), p = 0.003 (CD34). Solid bars represent positive groups. Checkered bars represent negative groups. Striped bars represent parental groups.(F) Among CD31+KIT+CD34+CDH5-CD45- cells, hematopoietic potential is enriched within the KDR+ population (~85% CFU-E, 2% BFU-E, 11% CFU-GM, 2% CFU-GEMM); n = 3 for all groups. Student’s t test: p = 0.03. Unless labeled otherwise, solid bars represent positive groups. Checkered bars represent negative groups. Striped bars represent parental groups.Data are represented as means ± SDs.

Fig 4: Identification of human liver myeloid cells, Kupffer cells and Kupffer cell subsets within the viable single cells harvested from collagenase-digested human liver tissue. Cells were first gated using FSC-A and SSC-A to exclude sub-cellular debris, then sequentially using FSC-H vs. FSC-A and SSC-H vs. SSC-A to exclude doublets. A live-dead stain was used to exclude cells with damaged membranes. Subsequent analysis involved gating on CD45+ cells, then on CD3-negative cells, then on CD11b+ CD68+ cells. Within the CD11b+ CD68+ subset, we invariably found CD14-high CD32-mid cells and CD14-mid, CD32-high cells. Analyses based on this gating scheme were performed on 23 separate patient tissue samples.

Fig 5: Impact of HIV infection on CD4+ TRM cell subsets from cervix. a Frequency of cervical CD4+, TRM, TRM CD103+, TRM CD32+, and HLA-DR+ CD4+ T cells from the total cervical CD45+ lymphoid cells was determined following the gating strategy described in Fig. 3 in normal donors (ND, blue circles, n = 9) and HIV infected patients (red circles, n = 18, corresponding to M01-M05, M11, M15-M23, and M25-M27). b HLA-DR expression in cervical TRM, CD103+ TRM and non-TRM in normal donors and HIV infected patients. c CD32 expression in cervical TRM, CD103+ TRM and non-TRM in normal donors and HIV infected patients. The median is shown as a solid line, the box indicates the 25–75 percentile range, while the whiskers show the range. Statistical comparisons were performed using two-tailed Mann–Whitney rank test to compare the two groups and using Friedman test with post hoc Dunn’s correction for multiple comparisons for intra-group comparisons. Source data are provided as a Source Data file