Fig 2: 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 3: Transcriptional analysis identifies circulating counterparts of GC-Tfh cells. (A and B) Flow cytometric plots showing the gating strategy of the four populations of CD4+CD3+CD45RA- cells sorted from iliac LNs (left) and peripheral blood (right) for RNA-sequencing and the expression of BCL6 protein in these populations, with representative plots shown; n = 6 individuals. (C and D) Hierarchical clustering (C) and PC analysis (D) of the most highly variable transcripts (standard deviation, >0.7) from RNA-sequencing data from four populations sorted in A from blood and LN samples. Three peripheral blood samples from the blood population no. 4 were excluded from the analysis as they did not pass quality control. In D, each symbol represents one individual. (E) Supervised analysis of the RNA-sequencing data from the four populations isolated from the blood using a gene signature for the LN GC-Tfh cells (LN 4); n = 254 genes. (F) Expression of IL7R, ASCL2, IL21, ICOS, and S1PR2 mRNA from RNA-sequencing data of the populations sorted in A; cell types are indicated by the colors shown in A. RNA-sequencing data were standardized relative to a library size of 1 million reads, and then read counts were log2 transformed; a value of -5 represents the limit of detection. P values were determined using ANOVA and the Tukey multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (G) Quantification of the number of TCRß CDR3 amino acid sequence clonotypes found in each blood cell population that are shared with the different LN populations (for each cell type n = 6 donors, except blood population no. 4 where n = 3; shared numbers of clonotypes were converted to normalized pseudocounts as described in Statistical analyses and Materials and methods). PBMC1, P = 0.055; PBMC2, P = 0.59; PBMC3, P = 0.0062; PBMC4, P = 0.029. The P values were determined using Kruskal-Willis tests. Data are from six individuals in one experiment.

Fig 4: CD103+ and CD103- CD4+ T cells display analogous phenotypic and functional differences to their CD8+ counterparts(A) MFI (CD161, CD7, CD127, ß2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4+ T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69-CD103- cells; red, CD69+CD103- cells; black, CD69+CD103+ cells.(B) MFI of ß2-integrin on recipient-derived CD4+ T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars.(C–H) Cytokine production by small intestinal CD4+ T cells. Representative histograms of expression, and group summaries of proportion of CD4+ T cells expressing IL-17A (C), TNF-a (D), IFN-? (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10).(I) Mean proportion of CD4+ T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-a, IFN-?, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10).Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 5: Phenotypic characterization of CD4 and CD8 T cells from SARS-CoV-2 naïve HIV positive individuals according to their neutralization levels.(A) viSNE map of FlowSOM metaclusters of CD4 T cells from HIV positive SARS-CoV-2 naïve subjects after two vaccine doses (nab-/low= no neutralization or low level of neutralization, nAbhigh=high neutralization level; n=9 in each group). Each point on the high-dimensional mapping represents an individual cell, and metaclusters are color-coded.(B) Cell count of each FlowSOM metaclusters out of total CD4 T cells (20,000 cells/group).(C) Representative flow plots from a nAb-/low and nAbhigh SARS-CoV-2 naïve HIV-positive donor showing expression of CXCR5 and CXCR3 within CD4 T cells.(D) Summary analysis of the percentage of CXCR5+CXCR3+CD4 T cells (n=9 for each group). Statistical test: Mann-Whitney U-test (MWU).(E) Correlation between frequency of CXCR5+CXCR3+CD4 T cells and ID50 neutralization level in nAb-/low and nAbhigh SARS-CoV-2 naïve HIV-positive individuals after two vaccine doses. Statistical test: Spearman’s rank correlation coefficient.(F) viSNE map of FlowSOM metaclusters of CD8 T cells from nAb-/low and nAbhigh HIV-positive SARS-CoV-2 naïve subjects after two doses of the vaccine (n=9 in each group).(G) Cell count of each CD8 FlowSOM metaclusters out of total CD8 T cells (20,000 cells/group).(H) Representative flow plots from a nAb-/low and nAbhigh SARS-CoV-2 naïve HIV-positive donor showing expression of CXCR3, CD127, and CD38 within naïve CD8 T cells.(I) Summary analysis of the percentage of CD127+CXCR3+CD38+niave CD8 T cells (n=9 for each group). Statistical test: MWU.(J) Correlation between proportion of CD127+CXCR3+CD38+niave CD8 T cells and SARS-CoV-2 specific T cell responses in nAb-/low HIV-positive SARS-CoV-2 naïve subjects. Statistical test: Spearman’s rank correlation coefficient.