Fig. 3: ProCTLA-4 prefers to deplete super-inhibitory Treg in TME.

a, b MC38 tumor-bearing male mice were treated i.p. with CTR, 40 μg anti-mouse CTLA4 antibody (mCTLA-4), or equimolar mouse ProCTLA-4 (mProCTLA-4) (n = 5/group) on day 11 after tumor inoculation. 2 days later, Treg cells from the tumor, spleen, and dLN were analyzed by flow cytometry. The representative plot (a) and the frequency of Treg cells (b) from different treatment groups were shown. c–g Tumor-infiltrating CD45⁺ immune cells isolated from MC38 tumor-bearing male mice with different treatment (CTR, mCTLA-4, mProCTLA-4) were analyzed by scRNA-seq. c UMAP visualization of merged total CD45⁺ cells from three treatment groups. d Bar plot depicting the cell percentages of each cell type as a proportion of total cells. Feature plot illustrating gene expression of ICOS (e) and CTLA-4 (f) in Foxp3⁺ Treg cells. g Bar plot depicting the cell percentages of ICOS^high Treg cells within total CD3⁺ T cells. h–j MC38 tumor-bearing male mice were treated i.p. with CTR, 10 μg mCTLA4, or equimolar mProCTLA4 on day 11 after tumor inoculation. 2 days after the treatment, the total Treg cells and ICOS^high Treg cells (h) from tumors were analyzed by flow cytometry. The frequency and absolute number of total Treg cells (i, j) and ICOS^high Treg cells (k, l) was quantified. m Female Rag1^-/- mice (n = 5/group) were i.v. transferred with Tregs. 3 days post-transfer, the mice were challenged s.c. with MC38-OVA. Activated OT-Ι T cells were transferred 4 days later. 5 days after OT-Ι transfer, mice were treated with 100 μg mProCTLA-4 every three days for three times, and the tumor curve was monitored. Data in panels (b, i–m) representative of two independent experiments. Statistical analysis was performed using ordinary one-way ANOVA with multiple comparisons test (b, i–l), or two-way ANOVA with Tukey’s multiple comparisons test (m).