Extended Data Fig. 5: αPD1-mEry activates the splenic immune microenvironment and subsequently induces systemic anti-tumor responses in tumor models.
a-b, Quantification of MDSCs (a) and effector CD8⁺ T cells (b) at multiple time points in spleens and tumors from the indicated treatment groups, as shown in Fig. 2i, j, assessed by flow cytometry. n = 3 mice per group for each time point; for day 22, one animal each in the anti-PD1 and αPD1-mEry groups was excluded because the tumors were too small to assess, resulting in n = 2 for that time point. c, Lymph nodes and bone marrow were harvested from MC38 tumor–bearing C57BL/6-hPD1 mice treated with IgG4-mEry, anti-PD1 or αPD1-mEry at day 22 for flow cytometry analysis (n = 3 mice per group). d-e, Spleens were isolated from B16F10-OVA tumor-bearing mice when tumors reached ~50 mm³. The presence of OVA-reactive T cells was evaluated by IFN-γ ELISpot assay (d), and PD1/PDL1 expression in splenic immune cells was measured by flow cytometry (e). n = 3 tumor-free mice; n = 5 tumor-bearing mice. Fold change in IFN-γ⁺ cells was calculated as the ratio of OVA-stimulated (SP + OVA) to unstimulated (SP only) splenocytes. f, B16F10-OVA tumor-bearing C57BL/6 mice received a single dose of IgG-mEry (mouse erythrocytes conjugated with IgG2a isotype), anti-PD1 (anti-mouse PD1 antibody) or αPD1-mEry (mouse erythrocytes conjugated with anti-mouse PD1 antibody) when tumors reached ~500 mm³. Splenocytes were isolated on day 17 (3 days post-treatment), pulsed with OVA protein, and incubated for 48 h prior to IFN-γ ELISpot assay (created by BioRender and used under license). g, The presence of tumor-reactive T cells was evaluated by IFN-γ ELISpot assay. n = 3 mice per group (mean of two technical replicates per mouse). h, B16F10-OVA tumor-bearing C57BL/6 mice received adoptive transfer of unactivated OT-I cells 0.5 h prior to the first treatments of IgG-mEry, anti-PD1 or αPD1-mEry, when tumors reached ~500 mm3. The spleens and tumors were isolated at day 19 (2 days after the 2nd treatment) for subsequent analysis (created by BioRender and used under license). i, Proportions of effector OT-I cells (CD44+ CD62L− OT-I+) in spleens (left) and tumors (right), analyzed by flow cytometry (n = 3 mice per group). Data are presented as mean ± s.e.m. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test (a-c, i), two-tailed unpaired t-test (d) or two-way ANOVA with Sidak’s multiple-comparison test (e, g).
