Fig 2: Multiple Cytotoxic CD4+ T Cell States Are Enriched and Clonally Expanded in Bladder Tumors and Possess Lytic Capacity against Tumors(A) Heatmap showing the expression of select cytotoxic or regulatory T cell marker genes (rows) for individual single cells (columns) within the cytotoxic CD4GZMB and CD4GZMK clusters compared with regulatory (CD4IL2RAHI and CD4IL2RLO) and CD4CM clusters. Cells were grouped based on their annotations by tissue (tumor or non-malignant), treatment, and patient. Log2-transformed expression of each gene was row scaled.(B) Flow cytometry staining of GZMB, perforin, or GZMK in CCR7- CD4+ FOXP3- T cells.(C) Percentage of cells expressing GZMB, GZMK, or perforin from CCR7- CD4+ FOXP3- T cells by flow cytometry (left) and the percentage of cells co-expressing perforin within GZMB+ or GZMK+ CCR7- CD4+ FOXP3- T cells (right) (N = 7 tumors, mean + SEM).(D) Representative flow cytometry staining of IFN? and TNF-a expression in GZMB+ or GZMK+ CCR7- CD4+ FOXP3- T cells stimulated with PMA and ionomycin. (E) Percentages of cells expressing IFN?, TNF-a, or both from GZMB+ or GZMK+ CCR7- CD4+ FOXP3- T cells with and without stimulation (N = 11 tumors, mean + SEM).(F) Multiplex immunofluorescent staining of DAPI (blue), CD4 (immunohistochemistry, red), GZMK (RNAscope probe, green), and GZMB (RNAscope probe, white) and overlay without DAPI from a cystectomy tumor region from a patient with parallel scRNA-seq and TCR-seq data (anti-PD-L1 C, top row) and from a corresponding tumor field with negative control staining (bottom row). CD4+ cells that co-express GZMK (arrows) or GZMB (arrowhead) are indicated. Scale bar, 10 µm.(G) The ratio of abundances of all regulatory T cell populations (CD4ILRAHI and CD4IL2RALO) to all cytotoxic CD4+ populations (CD4GZMB and CD4GZMK) across all tumor and non-malignant samples (mean + SEM shown; *p < 0.05 by unpaired t test, assuming unequal variance).(H) Gini coefficients for each of the cytotoxic CD4+ populations within tumor and non-malignant compartments across all samples (box and whisker plot is shown with formatting as in Figure 3C; *p < 0.05, **p < 0.01, exact permutation test, N = 7 tumor samples and 6 non-malignant samples).(I) Left panel: quantitation of Annexin V+ apoptotic cells over time from a time-lapse cytotoxicity experiment with tumor cells cultured alone or with bulk CD4+ TILs (CD4total) or CD4+ TILs depleted of regulatory T cells (CD4eff) at a 30:1 effector:target ratio. Right panel: CD4eff TILs and tumor cells (30:1 effector:target ratio) were co-cultured with a pan-anti-MHC class II antibody or isotype control. All traces were from the same culture and cytotoxicity assay from the same patient. All traces show relative change in cell death from time point 0. Cytotoxicity with CD4eff is representative of independent experiments from 4 different patients. Mean ± SEM from multiple technical replicates for each experiment is shown.

Fig 3: Flow Cytometry and Immunofluorescence Validation of T Cell Phenotypes in Bladder Tumors, Related to Figures 1, 2, 3, 4, and 5(A) Schematic of processing for paired tumor and adjacent non-malignant tissue from either anti-PD-L1-treated, or standard-of-care (untreated/chemotherapy-treated) cystectomy patients. FACS-sorted CD4+ or CD8+ T cells were subjected to droplet-based single-cell RNA sequencing (dscRNA-seq) with paired T cell receptor (TCR) sequencing as described in the text. (B) Parallel flow cytometry data from the same single-cell digest used for dscRNA-seq from 4 anti-PD-L1-treated tumors, showing the percentage of CD4+ or CD8+ T cells from total CD3+ cells. (C) Gating strategy for flow cytometric analysis of populations in CD4+ and CD8+ T cells from RNA-seq. CD4+ and CD8+ populations were gated out of CD3+ CD45+ single live cells. CD4+ cells were further gated as FoxP3- and FoxP3+. Treg cells are gated as FOXP3+ CD25+ cells. FOXP3- CD4+ and CD8+ cells were gated into central memory (CM, CCR7+ CD45RA-), and CCR7- cells (a combination of effector memory CCR7- CD45RA- and effector CCR7- CD45RA+). Boolean gating of CCR7- cells was used to obtain GZMK+, GZMB+ and Ki67+ populations for further marker analysis. Plots are shown here to demonstrate the presence of these populations. (D) Representative gates shown for each marker for CD4+ and CD8+ T cells were used for Boolean gating for the populations described above. (E) Flow cytometry staining of GZMB, GZMK, or perforin versus CD3 in CCR7- CD8+ T cells. Gates used for Boolean analysis are shown. (F) Flow cytometry staining of GZMB or GZMK co-expression with perforin in CCR7- CD8+ T cells. (G) Percentage of cells expressing GZMB, GZMK, or perforin from CCR7- CD8+ T cells by flow cytometry (left), and the percentage of cells co-expressing perforin within GZMB+ or GZMK+ CCR7- CD8+ T cells (right), are shown (N = 7 tumors, mean + SEM). (H) Percentages of cells expressing IFN?, TNFa, or both from GZMB+ or GZMK+ CCR7- CD8+ T cells with and without stimulation (N = 11 tumors, mean + SEM). (I) Multiplex immunofluorescent staining of DAPI (blue), CD4 (red), GZMK (green), GZMB (white) and overlay without DAPI are shown from a cystectomy tumor region from an additional patient with parallel scRNA-seq and TCR-seq data (anti-PD-L1 D). CD4+ cells that co-express GZMK (arrows) or GZMB (arrowhead) are indicated. Scale bar, 10 µm. (J) Percentage of cells co-expressing Ki67 and either GZMB or GZMK from CCR7- CD4+ FOXP3- T cells (left), or Ki67 and CD25 from CD4+ FOXP3+ T cells (right), by flow cytometry are shown, with dots for values from individual tumors (N = 7 tumors, mean ± SEM). (K) Flow cytometry staining showing co-expression of GZMB and Ki67, or GZMK and Ki67, from CCR7- CD8+ T cells.