Extended Data Fig. 2: αCD25^NIB depletes Tregs and drives effector immune responses.

C57BL6 mice were injected with 500,000 MC38 tumor cells. Once tumors were palpable, on day 7, mice were injected IP with αPC61/αCD25^NIB/αCD25^NIB + αIL2 (200μg). Tumors and LN were harvested on day 15 post-tumor inoculation and processed as described in materials and methods section. (a) Graph showing % FoxP3⁺ cells of total CD4⁺ cells. (b) Absolute number of Tregs shown as number of Tregs/g of tumor. p-value=0.0003 between No Tx and αCD25^NIB group. ****=p-value <0.0001 (c) Ratio of effector T cells over Tregs. For CD8/Treg ratio, P-value between No tx versus αCD25^NIB=0.0008, and between No tx and αCD25^NIB + αIL2 =0.0045. For CD4 Eff/Treg ratio, p-value between no tx and /αCD25^PC61 = 0.0056, between No tx and αCD25^NIB=0.0002, between no tx and αCD25^NIB + αIL2=0.0001. For NK/Treg ratio, p-value=0.0001 for no tx versus αCD25^PC61, 0.0010 for no tx versus αCD25^NIB and 0.0004 for No tx versus αCD25^NIB + αIL2. (d) Representative FACS plots showing Granzyme B expression versus Ki67 expression in CD8, CD4 effectors and NK cells. (e) Graph showing percentage of Granzyme B⁺ cells in different effector subsets. (f) Graph showing the Mean Fluorescence Intensity of Granzyme of the effector cells plotted in (e). For CD8 cells, p-value between No tx group and αCD25^NIB =0.0001. For CD4 Eff, p-values between No tx versus αCD25^NIB =0.0007, for αCD25^NIB versus αCD25^PC61=0.0009, and between αCD25^NIB and αCD25^NIB + αIL2 group= 0.0002. For NK cells, p-value between No tx group and αCD25^NIB group=0.0164 and between αCD25^NIB and αCD25^NIB + αIL2 group=0.0280. Quantification plots: mean ± SEM, 1-way ANOVA, Tukey’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

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