Fig 2: Atypical phenotypes of ITK-deficient DN αβ and γδ T lymphocytes. PBMCs from P1 (obtained at the ages of 18 and 19 yr), P2 (obtained at the age of 16 yr, free from EBV viremia), P3 (at the age of 4 yr), heterozygous members of their families, one patient with a homozygous LOF (R29C) mutation of ITK with EBV viremia but no history of TB, and one FAS-deficient patient were studied by flow cytometric immunophenotyping. (A) A representative plot of CD38 and T-bet expression. (B and C) Phenotypes of (B) DN αβ T and (C) Vδ2− γδ T lymphocytes. Results from three batches of experiments are compiled. (D–G) FlowSOM-guided clustering analysis. (D and E) UMAP plots of (D) all DN αβ T lymphocytes and (E) 2,500 cells per group. Equal numbers of cells were randomly sampled from each individual in each group. (F) Heatmap of scaled median expression levels. (G) Cluster abundance. In B, C, and G, the bars represent the mean and SEM. In B and C, statistical significance was determined for differences between ITK-deficient patients and adult, pediatric, and familial controls combined. ***, P < 0.001, two-tailed Wilcoxon’s rank sum tests with FDR adjustment.

Fig 3: Development of a multispectral panel for the topological examination of B cells. (A) Representative example of antibody staining patterns for CD138 (yellow), Ki67(magenta), Bcl-6 (orange), CD38 (violet), CD20 (cyan), H2Ax (red), CD4 (green) and nuclar staining (JOPRO-1, blue) and distribution of each marker with respect to the MZ, LZ and DZ (middle panels). Orange circles in the lower row images denote H2Ax events. (B) Gating strategy used for the immunophenotyping of various B cell populations and CD38hiCD138hi cells in GC (CD20dimKi67hi), F (CD20hii/dim) and EF (CD20lo) areas. (C) Box plots showing the frequencies of various B cell populations (as defined by the expression of Ki67 and Bcl-6), H2Ax Volumehi CD20+ cells and CD38hiCD138hi cells in a representative tonsil tissue section. Each circle represents a distinct follicle and each color denotes a distinct subpopulation. p values were derived from ANOVA analysis (Tukey’s multiple comparisons test). Images were acquired at 40x (NA 1.3) with 1% magnification. Scale bars are 100um for all panels displaying an entire follicle), 50um for the middle panel close ups and 5um for the right panel single staining close ups. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: 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.

Fig 5: Functional Analysis of CD56dim and CD56neg NK Subsets and Gating Strategy for Analysis of NK:Tfh:B Cell Co-culture, Related to Figures 4 and 5(A) Spearman correlation of percentage of total NK cells undergoing degranulation (assessed by CD107a expression) on exposure to target cells with plasma HIV-1 neutralization breadth (PC1).(B and C) Summary boxplots for CD107a expression, IFN-? and TNF-a production from (B) CD56dim NK cells and (C) CD56neg NK cells following target cell stimulation in bnAb (red), control (blue) and HIV seronegative individuals (black). Each symbol represents data from an individual subject and the box-and-whisker plots show the median, quartiles and range. *p < 0.05, **p < 0.01. P values corrected for MCA and viral load.(D) Class-switched memory B cells were identified as live CD3-CD4-CD56-CD19+IgD-IgM-CD20+CD38+/- cells and plasmablasts as live CD3-CD4-CD56-CD19+IgD-IgM-CD20-CD38+ cells. Tfh cells were identified as live CD3+CD4+CD19-CD56- cells. The division index was calculated using Flowjo software. CountBright absolute counting beads were used to calculate absolute numbers. The staining shown is from a representative subject (Tfh+B cell only condition).