Fig 2: 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 3: Kaplan Meier survival plots.Associations between high biomarker expression and survival in terms of PFS and OS. A, B, & E) High expression of CD3, CD4 & IDO1 respectively associated with better PFS. D) High expression of ICOS associated with better PFS & OS. F) Better OS associated with high tumour PD-L1 expression. C) Trend towards better PFS for high CD8 expression.

Fig 4: CD4+CXCR5+IL-4+ TFH cells are abundant around GCs in IgG4-RD and sometimes physically contact AID-expressing B cells.(A) CD4+CXCR5+IL-4+ T cells were enriched around TLOs in IgG4-RD SMGs. Immunofluorescence staining of CD4 (red), IL-4 (magenta), CXCR5 (green), and DAPI (blue) in IgG4-RD SMGs (patient G3). Quantification of CD4+CXCR5+IL-4+ TFH cells and CD4+CXCR5+ TFH cells comparing those outside and within GCs from each of five different areas in TLOs of 17 patients with IgG4-RD (G1–G17). The P-value is based on the Mann–Whitney U test. (B) IgG4+B cells were enriched outside GC, especially some these cells express AID. Immunofluorescence staining of CD4 (red), CD19 (blue), and Bcl6 (green) in IgG4-RD SMGs (patient G3). Immunofluorescence staining of AID (red), Bcl6 (magenta), IgG4 (green), and DAPI (blue) in IgG4-RD SMGs (patient G3). The numbers of IgG4+ cells (per square micrometer) outside and within GCs were quantified from each of five different areas in TLOs of 17 patients with IgG4-RD (G1–G17). The P-value is based on the Mann–Whitney U test. (C) AID-expressing B cells outside GCs in IgG4-RD lymph nodes were abundant. Immunofluorescence staining of Bcl6 (red), AID (green), and ICOS (orange) in a normal tonsil and IgG4-RD lymph node. (D) Immunofluorescence staining of ICOS (green), IL-4 (red), and AID (magenta) in IgG4-RD SMGs. (E) Immunofluorescence staining of IgG4 (green), ICOS (magenta), IL-4 (red), and DAPI (blue) in TLOs with an IgG4-RD patient (G3). Magenta arrows indicate ICOS+IL-4+ TFH cells. Green arrows indicate IgG4+ B cells. A number of IL-4–expressing TFH cells and IgG4+ B cells formed close and extensive intercellular plasma membrane contacts. The distance was measured between the edge of each IgG4+ B cell nucleus to the edge of the closest IL-4–expressing TFH-cell nucleus, and an internuclear distance of less than 0.4 μm was indicative of a T-B conjugate. (F) Immunofluorescence staining of ICOS (green), IgG4 (magenta), BATF (red), and DAPI (blue) in the lymph nodes of an IgG4-RD patient. Green arrows indicate ICOS+BATF+ TFH cells. Magenta arrows indicate IgG4+ B cells. A number of BATF+ICOS+ TFH cells and IgG4+ B cells formed close and extensive intercellular plasma membrane contacts. The distance was measured between the edge of each IgG4+ B-cell nucleus to the edge of the closest BATF+ICOS+ TFH-cell nucleus. All internuclear distances in this figure were <0.2 μm. LZ, light zone; DZ, dark zone.

Fig 5: CD4+ICOS+IL-4+ TFH cells are sparse in human SLOs.(A) Immunofluorescence staining of CD4 (red), ICOS (green), IL-4 (magenta), and DAPI (blue) in tissues from normal tonsils. The yellow broken line demarcates the area within GCs. The white broken line demarcates the area outside GC. (B) Immunofluorescence staining of CD4 (red), ICOS (green), IL-4 (magenta), and DAPI (blue) in tissues from normal tonsils. (C) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent antibody used to stain tissue from an IgG4-RD patient, normal tonsils, and normal mesenteric lymph nodes. (D) CD4+ICOS+IL-4+ (red) and CD4+ICOS+IL-4− (green) cells were quantified in tissue from 12 tonsils. (E) Scatter plots depict the mean fluorescence intensity per cell quantified using TissueQuest software for each fluorescent antibody used to stain normal tonsils and mesenteric lymph nodes.