Fig 2: Single-cell RNA sequencing identified CD4+T cell subsets in patients with IgAN. (A) Single-cell RNA sequencing on PBMCs of patients with IgAN (n = 3) and healthy controls (n = 4). (B) tSNE dimensional reduction analysis of CD4+T cell based on single-cell transcriptomes. Each dot represents a single cell; dots in same colors indicate same cluster. Violin plot showing marker gene expression in each cluster. For naïve CD4+T cell (CCR7+SELL+), Th1 (TBX21+CXCR3+), Th2 (GATA3+CCR4+), Th17 (RORC+CCR6+), Tfh (CXCR5+ICOS+), Treg (IL2RA+FOXP3+) (t-SNE, t-distributed stochastic neighbor embedding).

Fig 3: IL-4+BATF+ TFH cells are rare in normal SLOs but abundant in IgG4-RD.(A) Immunofluorescence staining of CD4 (red), Bcl-6 (green), IL-4 (magenta), and DAPI (blue) in tissues from normal tonsils. Quantification of CD4+Bcl-6+IL-4–positive TFH and CD4+Bcl-6+IL-4–negative TFH in 12 tonsils. (B) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD SMGs (G1) and normal tonsils. Quantification of CD4+CXCR5+IL-4+ and CD4+CXCR5+ TFH cells in 17 IgG4-RD SMGs, 12 tonsils, and 7 SS salivary glands. The P-value is based on the Mann–Whitney U test. (C) Almost all IL-4–expressing CD4+ T cells in IgG4-RD lymph nodes were CXCR5+ TFH cells. Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD lymph node (LN1). The quantification of CD4+IL-4+CXCR5–positive TFH and CD4+IL-4+CXCR5–negative non-TFH cells in lymph nodes from two patients with IgG4-RD (LN1 and LN2) is shown. (D) CD4+IL-4+BATF+ T cells were enriched in IgG4-RD SMGs. Immunofluorescence staining of CD4 (red), IL-4 (green), BATF (magenta), and DAPI (blue) in IgG4-RD SMGs (G12). White arrows indicate CD4+IL-4+BATF+ T cells. (E) Quantification of CD4+BATF+IL-4+ T cells and CD4+ T cells in 17 IgG4-RD SMGs and 12 normal tonsils. The P-value is based on the Mann–Whitney U test. (F) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent antibody used to stain normal tonsils and IgG4-RD lymph node (LN1). (G) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD SMGs (G3). (H) Immunofluorescence staining of ICOS (green), BATF (magenta), and IL-4 (Red) in IgG4-RD LNs (LN1). (I) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD LNs (LN1). The quantification of ICOS+BATF+IL-4–positive T and ICOS+BATF+IL-4–negative T cells in lymph nodes from six patients with IgG4-RD (LN1–LN6). (J) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent immunostain in IgG4-RD SMGs (G3).

Fig 4: DP CD4+ Th cells are in the tumor stroma proximal to MHC class II+ cells.mIHC for CD3, CD8, FOXP3, ICOS, MHC class II, and cytokeratin on FFPE tissue sections from HNSCC and CRC tumors. Representative ROIs (left) from an HNSCC tumor (A) and a CRC tumor (B) and the corresponding cellular spatial relationship maps (right). Scale bars: 200 µm. The maps show cytokeratin+ cells (tumor) in black, MHC class II+ cells in blue, and CD3+CD8–FOXP3–ICOS+ (DP CD4+ Th cells) in red. (C) Analysis of the distribution of CD3+CD8–FOXP3–ICOS+ (DP CD4+ Th cells) in the tumor stroma and tumor epithelium in 14 tumors (n = 8 HNSCC and n = 6 CRC). (D) Neighbor’s cell analysis was performed on 11 tumors (n = 6 HNSCC and n = 5 CRC). The proportion of MHC class II+ cells in the stroma and cytokeratin+ cells detected within a 15 µm radius from CD3+CD8–FOXP3–ICOS+ (DP CD4+ Th) cells is indicated as the frequency of total cells. Circles represent HNSCC tumors, and triangles represent CRC tumors. Horizontal lines indicate the mean ± SEM. Data for patients with MHC class II–expressing tumor cells are shown in red in D.

Fig 5: PD-1 and ICOS identify distinct subsets of CD4+ Th TILs.(A) t-SNE analysis of tumor-infiltrating CD3+CD4+ T cells isolated from 22 patients with HNSCC. The gate identified PD-1+ cells and gate was applied to all plots. (B) Flow cytometric analysis of CD4+ TILs and the gating strategy are shown for a representative patient with HNSCC. Mem, memory. (C) Subsets of CD4+ Th cells (DN, SP, and DP) were defined on the basis of differential expression of PD-1 and ICOS. (D) A summary of the frequency (Freq.) of DN, SP, and DP CD4+ Th cells in the blood and tumor of patients with HNSCC (left, n = 40) or CRC (right, n = 31) is shown. The DN (teal), SP (gray), and DP (orange) CD4+ Th cell subsets for each patient are highlighted on the graphs in C and D. Small horizontal lines indicate the mean ± SEM.