Fig 2: CD20dim CD4+ T cells involve memory phenotypes with more activation than CD20- cells. PBMC from uninfected donors, ART-suppressed patients and viremic patients were stained with CD20, activation markers (HLA-DR, CD69, CD25), and T-cell differentiation markers (CCR7, CD45RO, CD27, CD95). a Gating strategy used to identify CD20dim CD4+ T cells. Previous sequential gates are represented in Supplementary Fig. 9. Viable CD4+ T cells (identified by CD3 and CD4 expression) were first selected for the analysis of CD20 expression, defined by its Fluorescence Minus One (FMO) control. T-cell memory subsets were selected as follows: CD4+ TNA (CCR7+, CD45RO-, CD27+, CD95-), CD4+ TSCM (CCR7+, CD45RO-, CD27+, CD95+); CD4+ TCM (CCR7+, CD45RO+), CD4+ TTM (CCR7-, CD45RO+, CD27+); CD4+ TEM (CCR7-, CD45RO+, CD27-), CD4+ TTD (CCR7-, CD45RO-). b, c Representative bright-field and pseudo-color fluorescence images of CD20dim CD4+ T cells (b) and B cells (c) from two ART-suppressed patients (#9 and #22) using the Amnis imaging flow cytometer technology. Scale bar 10 µm. d Percentage of CD20dim expression within CD4+ T cells in uninfected controls and the two patient cohorts. Mann–Whitney comparison was used to compare n = 6 uninfected controls, n = 21 ART-suppressed patients, n = 20 viremic patients. e Expression of the activation markers CD25, CD69 or HLA-DR in CD20dim and CD20- CD4+ T cells in different cohorts of HIV+ patients. Comparisons were performed using the two-tailed Wilcoxon test (n = 21 ART-suppressed patients and n = 20 viremic patients). f Distribution of CD20dim in CD4+ T-cell subsets in ART-suppressed (orange) and viremic (red) HIV-infected patients. NA, naive; SCM, memory stem cell; CM, central memory; TM, transitional memory; TD, terminal differentiated. ANOVA and Dunn’s multiple comparison test were performed, n = 10 ART-suppressed and n = 9 viremic patients. Median values and min and max ranks are represented in panels d–f. g Correlation of CD20dim CD4+ T cells with time on suppressive ART (left), CD4+ T-cell counts (middle) and plasma viral load (right) are shown. ART-suppressed patients in orange and viremic patients in red. Spearman’s nonparametric correlation coefficients and associated P values are shown. Panels (d) and (e) included patients #2–10, 15–26, 60–65, 67–74, and 76–81. Panel (f) patients #1–10, 60–65, and 67–69. Panel (g) patients #2–10, 15–26, 60–65, 67–74, and 76–81. Data underlying this Figure are provided as Source Data file

Fig 3: Macrophages differentiated with IFN-?3 induce lymphocyte chemotaxis and NK cell activation. Transwell inserts containing autologous PBMCs were placed into wells containing 6-day differentiated M?s ± IFN-?3 to examine immune cell chemotaxis (n = 6). Migrated cells were analyzed by flow cytometry using CD14, CD19, CD3, and CD56 antibodies to identify the number of migrated monocytes, B, NK, and T cells (A). The addition of IFN-?3 to GM-CSF M?s alone, stimulated lymphocyte migration, with significant increases in NK, NKT, and T cell migration (B). Consistent with migration data, GM-M? CCL2, CCL3,CCL4, and CXCL10 secretion were significantly increased by IFN-?3, with no significant effect on M-M?s (C) (n = 7). To assess the role of IFN-?3 on M? activation of NK cells, autologous NK cells were incubated with M?s for 16 h a ratio of 1:1, removed and incubated with K562 target cells for a further 6 h to measure cytotoxicity by degranulation and IFN-? expression (n = 4). GM-M?s differentiated with IFN-?3 significantly increased NK cell cytotoxicity as measured by CD107a expression, as well as IFN-? production (D). Data are representative of two (B,D) and three (C) independent experiments. Paired t-test *p < 0.05, **p < 0.01 (mean ± SE). NE, No expression.

Fig 4: Graft-infiltrating, recipient-derived T cells take on a TRM cell phenotype over time, with CD103+ and CD103- CD8+ T cells displaying distinct phenotypes(A) Fluorescence microscopy chip cytometry image of small intestinal transplant mucosa from a single subject 3 months after transplant. The donor was HLA-A3+, and the recipient was HLA-A3-. False-color fluorescence imaging for cytokeratin (gray), CD3 (purple), CD8 (red), CD103 (blue), and HLA-A3 (green). Donor-derived CD8+ CD103+ and CD103- T cells are indicated by white and yellow arrows, respectively.(B) Phenotypic analysis of CD8+ T cell populations in healthy small intestinal epithelium and lamina propria. Proportion of positive cells (CD161) or MFI (CD7, CD127, ß2-integrin, and KLRG1) of intraepithelial lymphocyte (IEL) or lamina propria lymphocyte (LPL) CD8+ T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 4). Mean percentage or MFI represented by bars. Connecting lines represent populations from the same subject.(C) Fluorescence microscopy chip cytometry of representative CD103- (cells 1 and 2) and CD103+ (cells 3 and 4) donor-derived CD8+ T cells, showing the expression of CD18 (ß2-integrin), KLRG1, and granzyme K.Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 5: Frequency and phenotype of CD3+T-cell subsets in HCC patients. (A) Box plots showing the frequency of CD4, CD8, DN, MAIT, and TCR ?d cells in TILs vs NTILs and PBMCs. Each dot represents an individual sample. (B) Box plots showing the phenotype of CD3+ cells. The percentage of cells expressing the indicated markers is indicated for each sample. (C) tSNE map of total CD3+ cells in TILs (yellow dots), NTILs (purple dots), and PBMCs (green dots). *P = .05, **P = .01, ***P = .001, and ****P = .0001, 2-tailed paired test for multiple comparison (Tukey test). ns, not significant.