Fig. 2. Infusion of functional CD4 and CD8 T_{TCR-MA1-CD8αβ} are not associated with excessive toxicity in one patient and require enhancement to achieve tumor burden reduction.

A Contour flow plot of the patient’s CD4 T_{TCR-MA1-CD8αβ} (top) and CD8 T_{TCR-MA1-CD8αβ} (bottom) infusion product binding to CD28 (x-axis), CD62L (y-axis left panels) and CD127 (x-axis right panels). B IFNγ (x-axis) and TNFα (y axis) expression by the patient’s CD4 T_{TCR-MA1-CD8αβ} (top) and CD8 T_{TCR-MA1-CD8αβ} (bottom) infusion product after an 18-h exposure to 10 μM (left panels), 1 μM (middle panels), and no peptide stimulation (right panels). C Circulating T_{TCR-MA1-CD8αβ} in PBMCs identified with p/HLA multimer at indicated timepoints (x-axis) for the infused patient. D Schematic (left) and growth kinetics (right) of 1 ×10⁵ A375F tumors engrafted into NSG mice with subsequent transfer of 5 × 10⁶ total T cells (T_Irr.-CD8αβ, CD4⁺ T_{TCR-MA1-CD8αβ} alone, CD8⁺ T_{TCR-MA1-CD8αβ} alone, or a 1:1 ratio of combined CD4 and CD8 T_{TCR-MA1-CD8αβ}), 12 days after engraftment. (n = 3 mice/group, 2 tumors/mouse, data shown are representative of three independent experiments.) Image created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f. E A375F tumor volume from (D) assessed at sacrifice 8 days after T cell infusions for each indicated condition. Data shown are representative of three independent experiments. p value determined by Kruskal-Wallis with Dunn’s multiple comparison test. (n = 6 mice/group, 2 tumors/mouse averaged, from 2 separate experiments). All data are presented as mean ± SEM. F Quantification of Tim3⁺/PD-1⁺, Tim3⁺/CD39⁺ and 2B4⁺ binding across the experimental groups from (D) in CD4⁺ (top panel) and CD8+ (bottom panel) T_{TCR-MA1-CD8αβ} for the indicated conditions. Single cell suspensions were obtained at sacrifice, 8 days after T cell infusion. Graph represents mean ± SD. p values determined by unpaired two-tailed t-test. (n = 3 mice, 6 tumors/group, 2 tumors/mouse). G Quantification of IFNγ⁺ /TNFα⁺ binding after 1 μM cognate peptide stimulation for 18 h ex vivo across the same experimental groups from (D) in CD4 (left) and CD8 (right) T_{TCR-MA1-CD8αβ} for the indicated conditions. Single cell suspensions were obtained at sacrifice, 6 days after T cell infusion. Graph represents mean ± SD. p values determined by unpaired two-tailed t-test. (n = 3 mice/group, 2 tumors/mouse). H, I Heatmap showing the expression of curated co-stimulatory (green box) and inhibitory (red box) genes for lung cancer (H) and melanoma (I) patients across annotated cell subsets. Higher expression values are depicted in red, while lower expression values are shown in blue. J, K UMAP plot showing the two-dimensional distribution of a costimulatory gene score (CD80, CD86, CD40, TNFRSF4, TNFS9 and ICOSLG) in scRNAseq samples from lung cancer (J) and melanoma (K) patients. ICOSLG expression was present in the melanoma dataset but not in the lung cancer dataset. Dark red indicates higher expression, while light gray marks regions with low score expression.