Fig. 5: ProCTLA-4 promotes Teffector and memory-like CD8⁺ T cell responses.

a MC38 tumor-bearing male C57BL/6 mice were i.p. treated with PBS (CTR), 40 μg mProCTLA-4 (n = 5) on days 12, 15, and 18. For CD4⁺ T or CD8⁺ T cell depletion, mice were i.p. injected with 200 μg α-CD4 or α-CD8 antibodies since day 11 post tumor inoculation in every 3 days for four injections in total. b–e scRNA-seq of CD8⁺ T cells isolated from total CD45⁺ cells for subset analysis. b tSNE visualization of CD8⁺ T cells colored by subsets. c Heatmap of average gene expression of marker genes in different CD8⁺ T cell subsets. d Bar plot depicting the cell number of each CD8 T cell subsets and their percentages as a proportion of total CD8 T cells. e CD8 T cells were ordered into a branched pseudotime trajectory using Monocle3. The UMAP embedding showed cells colored by subsets (left) or by their estimated pseudotime values (right). Black lines indicated the inferred branched trajectory. f B16-OVA tumor-bearing male mice were i.p. treated with PBS, 40 μg mCTLA-4, or equimolar mProCTLA-4 (n = 5/group) on days 10 and 13. Lymphocytes from the draining LN were collected at the endpoint for the ELISpot assay. g MC38 tumor-bearing male hCTLA-4 KI mice were i.p. treated with CTR, 40 μg Ipi, or equimolar ProCTLA-4 (n = 5/group) on days 12 and 15. Cured mice were rechallenged with MC38 tumor cells on the opposite flank at 8 weeks post the initial inoculation. Rechallenged tumors reached 40 mm³ in volume was regarded as present tumors, and the percentage of tumor recurrence was calculated. h–i, Tetramer⁺ CD8⁺ central memory T cells (CD44⁺ CD62L⁺) in dLN were analyzed for frequency and absolute numbers (n = 5/group). j–l, Dual-flank MC38 tumor experiment was conducted (j), the tumor growth curve on the right flank (k) and the left flank (l) were monitored. Data in panels (a, f, h–l  are representative of two independent experiments. Data in panels g are pulled from two independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test (a, k, l), or ordinary one-way ANOVA with multiple comparisons test (f, i). The figure in panel (j) was created with BioRender (https://BioRender.com/90qugj2).