Figure 4.

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 CD4_GZMB and CD4_GZMK clusters compared with regulatory (CD4_IL2RAHI and CD4_IL2RLO) and CD4_CM 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 IFNγ and TNF-α expression in GZMB⁺ or GZMK⁺ CCR7⁻ CD4⁺ FOXP3⁻ T cells stimulated with PMA and ionomycin. (E) Percentages of cells expressing IFNγ, TNF-α, 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 (CD4_ILRAHI and CD4_IL2RALO) to all cytotoxic CD4⁺ populations (CD4_GZMB and CD4_GZMK) 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 (CD4_total) or CD4⁺ TILs depleted of regulatory T cells (CD4_eff) at a 30:1 effector:target ratio. Right panel: CD4_eff 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 CD4_eff is representative of independent experiments from 4 different patients. Mean ± SEM from multiple technical replicates for each experiment is shown.