Fig 2: HIV infected patients lack PD-L1 expression in mucosa. Immunohistochemistry was performed on a selection of nine patients to assess the expression of PD-L1 in both sigmoid colon and terminal ileum. (A) Representative stainings from both anatomical sites in each patient group. (B) Hematoxylin-eosin (left and middle panel) and anti-PD-L1 (right panel) staining of a biopsy from the terminal ileum. This sample also contained tissue from a lymphoid follicle.

Fig 3: Single-cell RNA sequencing analysis of islets and CyTOF of pancreas in NOD-Cre+ and NOD-?ß mice(A–E) Islets or pancreata were isolated from NOD-Cre+ and NOD-?ß mice at 8 weeks of age, dissociated into single cells, and subjected to single-cell RNA sequencing (A, B, and E; N = 2 individual animal replicates) or CyTOF (C and D, N = 3–4 individual animal replicates), respectively.(A) Uniform manifold approximation and projection (UMAP) embeddings of merged single-cell RNA sequencing profiles from islets colored by cell clusters that were identified.(B) UMAP gene expression patterns and violin plots of Cd274 and Socs1 in a combined macrophage cluster (cluster 12) and a dendritic cell cluster (cluster 13) (false discovery rate [FDR] < 0.05).(C) Number of PD-L1-positive cells expressed by CD11b+, CD11c+, and F4/80+ cells identified by CyTOF of whole pancreatic cells.(D) Frequency of CD11b+PD-1+ cells.(E) UMAP gene-expression patterns and violin plots of Lag3, Foxo1, Ikzf4, Ccr7, and Il-10 in the T cell cluster (cluster 14) (FDR < 0.05).Data are expressed as the mean ± SEM. *p < 0.05.

Fig 4: ß cell deletion of Alox15 enhances expression of the immune checkpoint protein PD-L1Islets were isolated from NOD-Cre+ and NOD-?ß mice at 8 weeks of age, dissociated into single cells, and subjected to single-cell RNA sequencing. N = 2 individual animal replicates.(A) UMAP gene expression patterns and violin plots of Esr1, Lncpint, Nfe2l1, Sod1, and Cd74 in the 5 ß cell clusters (clusters 1–5; FDR < 0.05).(B) UMAP gene-expression patterns and violin plots of Cd274 and Stat1 in the 5 ß cell clusters (clusters 1–5; FDR < 0.05).(C) Gene ontology-gene set enrichment analysis (GO-GSEA) in the 5 ß cell clusters (clusters 1–5).(D) Percentage of cells in ß cell cluster 5.(E) Number of non-immune cells (CD45-) expressing PD-L1 as measured by mass cytometry of the pancreas.(F) Representative images of pancreata from 8-week-old mice immunostained for PD-L1 (magenta), ß cells (insulin, green), and nuclei (DAPI, blue), and quantification of PD-L1 pixel density from islets (from N = 5 mice). Scale bars, 50 µm. Data are expressed as the mean ± SEM. *p < 0.05.

Fig 5: Hypoxia differentially affects PD-L1 expression in melanoma cells. (A) The human melanoma cell lines MelAKR, MelJUSO, Mel88.23, Mel136.2, MelWBO, the mouse melanoma cell line B16.F10 and the human cervical cancer cell line HeLa were cultured for 48 h in normoxia or hypoxia, after which cells were harvested and PD-L1 mRNA expression was analyzed by qPCR. Graphs show PD-L1 and BNIP3L mRNA levels depicted as fold change, as compared to normoxia. White bars present normoxic (N) and black bars hypoxic (H) conditions. The HIF1 target BNIP3L (and Vegfa in the mouse B16.F10 cells) were used as markers of HIF1 activity, and (B) Western Blots showing HIF1a protein levels for the indicated cell lines. ß-ACTIN protein levels serve as loading control. (C) Total PD-L1 protein levels (both intracellular and extracellular) were determined in human cell lines by FACS under the same conditions as in (A). Graphs indicate the fold change in PD-L1 protein levels in hypoxia, as compared to normoxia. (D) Cell viability was analyzed for all cell lines in normoxia and hypoxia. All quantified data are presented as the average ± standard deviation, as compared to the control (N) in at least three independent experiments. Statistical significance of differences between normoxia and hypoxia was tested. *p < 0.05, **p < 0.01, ***p < 0.001