Fig 2: Molecular toolbox of site-specific functionalizable anti-PD-L1 antibody formats and imaging peptides. A Application of the CRISPR/HDR strategy to anti-PD-L1 hybridoma MIH5 created a sortaggable Fab fragment and chimeric mouse IgG1 monoclonal antibody against PD-L1. B Molecular structure of imaging peptides IH20 and IH18

Fig 3: Thiostrepton promoted maturation and function of BMDCs from TBM. BMDCs were treated with Thiostrepton 1 µm for 3 days. (A and B) FACS analysis for the quantitative percentage of CD86, MHC-II, CCR7, and PD-L1 on gated CD11c+ cells in DCs from wild-type mice, TBM. The amounts of CD11c+ cells set at 10 000, A is from C57BL/6J, B is from BABL/c mice. (C and D) T cells were co-cultured with DCs at the ratio of 10 : 1 for 24 h. The proliferation on gated CD3+ T cells was then determined by CFSE assay. (E and F) The secretions of IL-12 p70 from LPS primed BMDCs were quantified by ELISA assay. Data represented mean ± SD from at least 3 independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001 compared with control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the model.

Fig 4: PD-L1 blockade partially restores Th1 cell secretion and improves ob-ASC mediated Th17 cell activation without any effect on pro-inflammatory cytokine secretion by accessory cells. MNC were activated by PHA in the presence or absence of ob-ASC and anti-PD-L1mAbs at concentrations of 1 or 10 µg/mL. Cytokine mRNA expression levels were measured by RT-qPCR. Results were expressed as a ratio relative to the TBP housekeeping gene. The one-way ANOVA with Fisher’s LSD comparison tests were used. Data are the mean ± SD of n = 4. The p-value shown in the figure is from an ANOVA test, whereas ****, ***, **, or * represents p-values of <0.0001, <0.001, <0.01, <0.05 respectively, from Fisher’s LSD tests.

Fig 5: Lymphatic PD-L1 induces apoptosis in tumor-specific CD8+ central memory T cells in tumor-draining LNs. A–C, Representative FACS plots [pre-gated for tetramer-specific (A), ova-specific TCM and naive (B) or pmel-specific TCM and naive (C) CD8+ T cells] and frequency of apoptotic cells [pooled early (Zombie-) and late (Zombie+) apoptotic] among all ova- and pmel-specific CD8+ T cells (A) or within TEM, TCM and naive ova-specific (B) and pmel-specific (C) CD8+ T cells in B16-ova–draining LNs (N = 10 Cre-/9 Cre+ mice). D and E, Representative FACS plots [pre-gated for all ova-specific (D) or ova-specific TCM and naive (E) CD8+ T cells] and frequency of apoptotic cells among all ova-specific CD8+ T cells (D) or within TEM, TCM and naive ova-specific (E) CD8+ T cells in MC38-ova–draining LNs (N = 5 mice/group). F–G, Apoptosis among endogenous and transferred CD45.1+ OT-1 T cells (F) and among CD45.1+ TEM, TCM and naive T cells (G) in B16-ova–draining LNs after adoptive transfer of freshly isolated, unstimulated OT-1 cells (N = 3 mice/group). *, P < 0.05; **, P < 0.01, Student t test.