Fig 2: TRM are preferentially infected in the cervical explant model of HIV infection. a General gating strategy for phenotype analyses and intracellular p24 detection in CD4+/- TRM and non-TRM obtained from cervicovaginal tissue infected ex vivo with HIV-1BaL strain. Gating strategy consisted of a lymphocyte gate based on FSC vs. SSC, followed by doublet exclusion, hematopoietic CD45+ cells, live CD3+ T cells with CD19+ cells exclusion, further B cell exclusion and a CD4+/- T cell gate from where the different gates shown were quantified: CD4+/- non-TRM (purple) or CD4+/- TRM (orange) or HLA-DR/CD32dim expression. An example of p24 expression from an infected and a non-infected sample is shown. b Frequency of p24 viral antigen in total CD4+/- cells (green), CD4+/- non-TRM (purple), CD4+/- TRM (orange), CD4+/- CD103+ TRM and CD4+/- CD32+ TRM detected after 10–12 days of ex vivo infection and measured by flow cytometry (n = 9, except for CD4+/- CD32+ TRM where n = 8). c Number of HIV-DNA molecules per cell in non-TRM and TRM detected after 10–12 days of ex vivo infection and measured by qPCR (n = 7). d Frequency of non-TRM and TRM from the total p24 positive cells measured by flow cytometry (n = 9). e Frequency of HLA-DR expression in different subsets based on their expression of p24 viral antigen after 10–12 days of ex vivo infection (n = 9, except for CD4+/- CD32+ TRM where n = 8). f Number of HIV-DNA molecules per cell in TRM CD32+ and TRM CD32- detected after 10–12 days of ex vivo infection and measured by qPCR (n = 4). g CD32 frequency in p24+ TRM compared to p24- TRM, measured by flow cytometry (n = 8). For b and e the median is shown as a solid line, the box indicates the 25–75 percentile range, while the whiskers show the range. Statistical analysis consisted of a Wilcoxon matched-pairs signed rank test. Source data are provided as a Source Data file

Fig 3: Individual variation in myeloid cell subsets. (A) Among the CD45+ CD3– liver cells, the most abundant populations were lacking CD68. Around half of those were other myeloid cells, based on their expression of CD11b. Putative Kupffer cells, CD11b+ CD68+ were variable in abundance from 2% up to 40% of the total. Cells with CD68 but no CD11b were present in most samples but always 5% or less. (B) A frequent pattern of staining within the CD45+ CD11b+ CD68+ cells was that CD14-high, CD32-mid cells were in excess over CD14-mid, CD32-high cells. (C) Variation among 22 individual samples. The CD14-high, CD32-mid cells were most often between 60 and 100% of the total, and the CD12-mid, CD32-high cells correspondingly between 40% and none. The reproducibility of this imbalance was highly significant (p < 0.0001, paired Wilcoxon test). The data in A and C represent 22 separate patient samples.

Fig 4: Reconciliation of our staining plus qRT-PCR data from FACS-sorted CD32-mid and CD32-high Kupffer cells with data from two published scRNAseq studies reveals broad agreement. Genes highlighted in blue seem to be diagnostic of a subset optimized for endocytosis and immune suppression, which genes highlighted in red are diagnostic for a subset optimized for inflammation and anti-microbial activity. Genes that are disparate between the different studies are highlighted in purple. Note in particular that CD163 expression in our study was discordant with the two single cell RNA sequencing studies.

Fig 5: 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.