Fig 2: Co-expression of PD-1 and PD-L1 in a Lung Cancer Patient(A) Gating strategy for analyzing PD-1 and PD-L1 expression in human patient samples where CD45+/CD3+ cells were classified as T cells, CD45+/CD3- cells as B cells, CD45+CD11c+ cells as DCs, CD45+CD11b+ cells as macrophages (Macs) and MDSCs, and CD45- cells within the tumor site as tumor cells.(B) Expression of PD-1 and PD-L1 on the indicated cell types derived from PBMCs from a healthy human individual, a lung cancer patient, and the tumor site of the same patient.See also Figures S1 and S2 and Table S1.

Fig 3: LTX-315 downregulates PD-L1 expression in pancreatic cancer. (A) Representative images and further quantification of PD-L1 IHC staining in the immunocompetent mice treated with LTX-315. (B–F) Representative images and further quantification of flow cytometry analysis of PD-L1 expression in tumor cells (B), dendritic cells (C), granulocytic-MDSCs (D), monocytic-MDSCs (E), and macrophages (F) in tumors collected from immunocompetent mice. (G–I) LTX-315 inhibition of PD-L1 expression in a dose-dependent manner. PD-L1 expression in BxPC-3 (G), SW1990 (H), and KPC (I) cell treated with LTX-315 at increasing concentrations, individually analyzed by western blotting. (J–L) Inhibition of PD-L1 expression by LTX-315 in a time-dependent manner. PD-L1 expression in BxPC-3 (J), SW1990 (K), and KPC (L) cell treated with LTX-315 at increasing time points, individually analyzed by western blotting. (M–O) Inhibition of PD-L1 expression by LTX-315 regardless of the effect of cell death. PD-L1 expression in BxPC-3 (M), SW1990 (N), and KPC (O) cells treated with LTX-315 and CDDP, individually analyzed by western blotting. Results are presented as mean±SD of one representative experiment. *p<0.05, **p<0.01, ***p<0.001 by a two-tailed t-test; DC, dendritic cell; IHC, immunohistochemical; MDSCs, myeloid-derived suppressor cells; NS, not significant; PD-L1, programmed cell death ligand 1.

Fig 4: Compromised Affinity Maturation in the Absence of PD-1-PD-L1 Interactions between GC Tfh and B CellsMixed BM chimeras were constructed using 50% CD45.2 Cd274+/+ or Cd274-/- BM cells and 50% CD45.1 BM cells. Chimeric mice were immunized with NP-KLH.(A) Gating strategies for total B cells, plasma cells, GC B cells, NP-specific GC B cells, and memory B cells (top row) and for CD45.1 and CD45.2 cells within each indicated population (bottom row). The example is from Cd274-/-:CD45.1 chimera.(B) Scatterplots of competitive competencies of CD45.2 cells in the two sets of mixed chimeras contributing to GC B cells, NP-specific GC B cells, NP-specific memory B cells and plasma cells, and BM plasma cells at day 8, day 13, or day 21 after immunization. The competitive competency is defined as the CD45.2/CD45.1 ratio in the indicated compartment normalized against the same ratio in the total B cell compartment or the GC compartment in the case of NP-specific GC cells. Data are pooled from three independent experiments involving 3–4 mice per group per experiment. Each symbol denotes one mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.(C) Total mutation rates and frequencies of affinity-enhancing W33L mutation in NP-specific, VH186.2-carrying GC B cells isolated on day 13 after NP-KLH immunization. Numbers in the center of the pie chart indicate total numbers of clones analyzed, and numbers of W33L+ or W33L- cells are indicated. Numbers of total mutation are presented as mean ± SEM. Frequencies of W33L mutations were compared by the Fisher’s exact test. Data are pooled from three independent experiments, each involving 3–4 mice for each group.

Fig 5: PD-1 and PD-L1 Promote Tfh Cell Concentration toward the GC Area(A and B) GFP-transduced wild-type or Pdcd1KI/KI OT-II T cells were transferred into B6 mice (5 × 105 sorted GFP+ cells per mouse) together with 5 × 105 dsRed-expressing MD4 B cells. OT-II distribution patterns (A) and density ratios between the GCs and the follicle (B) in draining lymph nodes 5 days after subcutaneous HEL-OVA immunization.(C and D) Wild-type or Cd274-/- mice received 5 × 105 GFP-transduced wild-type or Pdcd1KI/KI OT-II T cells together with 5 × 105 dsRed-expressing MD4 B cells. OT-II distribution patterns (C) and density ratios between the GCs and the follicle (D) in draining lymph nodes 5 days after subcutaneous HEL-OVA immunization.Scale bar, 100 µm. All data are pooled from three independent experiments involving 3–4 mice per condition per experiment. ****p < 0.0001; ns, not significant.