Fig 2: Identification of Novel CD274 and PDCD1LG2 cis-Regulatory Elements. (A) Cell surface expression of PD-L2 in brain tumor cells (grey line: isotype control; black line: PD-L2; dotted line: PD-L2 with IFN-? stimulation). (B) CD274 promoter (Pr1) and enhancer (Pr2 and Pr3) regions cloned and assessed. (C) Normalized luciferase activity in cells with high and low endogenous PD-L1 expression using PD-L1.Pr1,.Pr2 and.Pr3 elements. (D) Schematic of PD-L1 enhancer region (Pr3) and truncated minimal region (Pr3M). (E) Luciferase assay of PD-L1.Pr3 (black) and.Pr3M (white) in PD-L1 high- and low-expressing cell lines. (F) PDCD1LG2 element (.Pr1 + 2) and truncated.Pr1 and.Pr2. (G) Luciferase assay of PD-L2.Pr1 + 2 (left) and PD-L2.Pr1 and.Pr2 (right) in PD-L2 high- and low-expressing cells. (H) PD-L2.Pr1 mutants lacking predicted STAT1 (?STAT1), GATA2/3 (?GATA2/3), or both binding sites (?STAT1 & ?GATA2/3). (I) Luciferase assay of PD-L2.Pr1 truncations lacking STAT1 (?STAT1) or GATA2/3 (?GATA2/3)(top) or also both (?STAT1 & ?GATA2/3)(bottom) in high and low expressing cell lines.

Fig 3: The combination therapy leads to enhanced activation markers in the TME.In one experiment, the i.p. MC38-luc tumour model was treated with VV and/or a-PD-L1 as described. Tumour tissues were collected at day 5 post first treatment and were used for extraction of total RNA. RT–qPCR assays were performed to determine the levels of IFN-? (a), granzyme B (b) and perforin (c) in the TME. Data were analysed using Student's t-test (*P<0.05; **P<0.01).

Fig 4: PD-L1/CTLA4-Ig–overexpressing eMSCs delay allogeneic islet rejection in mice.(A) Representative digital images of kidneys transplanted with islets alone (top) or with either MSC or eMSC spheroids (bottom). (B) Blood glucose curves of diabetic C57BL/6 mice transplanted with BALB/c islets without MSCs (no-MSC group) (n = 9), BALB/c islets with MSC spheroids (MSC group) (n = 9), and BALB/c islets with eMSC spheroids (eMSC group) (n = 9) in the kidney capsule. (C) Graft survival curve of indicated groups shown in (B). (D) Blood glucose measurement in the intraperitoneal glucose tolerance test of different groups (n = 3). (E) Bioluminescent images of diabetic C57BL/6 mice transplanted with GFP/luciferase FVB mouse islets without MSCs (no-MSC group) (n = 6), with MSC spheroids (MSC group) (n = 6), or with eMSC spheroids (eMSC group) (n = 8) in the kidney capsule. (F) Quantitative analysis of bioluminescent signals measured in mice with different grafts (n = 6 to 8). (G) Graft survival curve of indicated groups in (F) (n = 6 to 8). Survival curve was analyzed using a Mantel-Cox test. The level of significance was labeled by *** and ****, denoting P values of <0.001 and <0.0001, respectively.

Fig 5: PD-L1/CTLA4-Ig–overexpressing eMSCs improve syngeneic islet transplantation in mice.(A) Live and dead staining of islets cocultured without MSCs, with MSC spheroids, or with eMSC spheroids in vitro for 24 hours (green, live cells; red, dead cells). (B) Quantitative analysis of fluorescence intensity of images shown in (A) (n = 5). (C) Stimulation index of islets (the ratio of insulin secretion at high glucose to that at low glucose) cocultured without MSCs, with MSC spheroids, or with eMSC spheroids for 24 hours (n = 4 to 5). (D) Blood glucose curves of diabetic C57BL/6 mice transplanted with syngeneic islets with a marginal dosage without MSCs (no-MSC group) (n = 3), with MSC spheroids (MSC group) (n = 4), and with eMSC spheroids (eMSC group) (n = 5) in the kidney capsule. (E) Graft survival curves of indicated groups shown in (D). (F) Blood glucose measurement in the intraperitoneal glucose tolerance test of different groups (n = 3 to 5). (G) Representative H&E staining of syngeneic islets with eMSC engrafted in diabetic C57BL/6 mice in the kidney capsule. (H) Representative immunofluorescent staining of syngeneic islets with eMSCs engrafted in diabetic C57BL/6 mice in the kidney capsule. DAPI (gray), insulin (INS, magenta), and glucagon (GCG, green). (I) Higher magnification of islets in (H). One-way ANOVA followed by Tukey’s test was performed for comparing the multigroup data. Survival curve was analyzed using a Mantel-Cox test. The level of significance was labeled by n.s., *, and **, denoting nonsignificant and P values of <0.05 and <0.01, respectively. Scale bars, 50 µm (A and I) and 100 µm (G and H).