Fig 2: (a) Wharton's jelly stripped from the umbilical cord. (b) P3-generation hWJMSCs morphology under an optical inverted microscope. (c) Osteogenic differentiation of hWJMSCs stained with alizarin red ( × 100). (d) hWJMSCs differentiated into fat and stained with oil red O ( × 200). (e) hWJMSCs differentiated into cartilage and stained with alcian blue ( × 40). (f) Flow cytometry showed that hWJMSCs highly expressed CD73, CD90, and CD105 (both ≥95%) but did not express CD34 and CD45 (both ≤2%). (g) TEM shows that Exos have the shape of a disc with a diameter of approximately 100 nm (Exos shown by the red arrowhead). (h) NTA shows the particle size distribution of Exos. (i) WB results for the Exo markers CD9, TSG101, and CD63 and the negative marker Calnexin. CL is a positive control (cell lysate). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Loss of CD14 on the cell surface in BLaER1 monocytes is the result of a general GPI anchor defect. (a) CD14 mRNA expression of BLaER1 clones, quantified by qRT-PCR. Shown is the mean ± SD of n = 3 independent measurements, normalized to GAPDH expression. Dots represent individual values. (b) Western blot analysis of CD14 expression. One representative blot of two is shown. Full-length blots are presented in Supplementary Figure S9. (c) Quantification of the CD14 protein in (b). CD14 expression was normalized to the housekeeping protein β-actin. The mean ± SD of pooled data from three different monoclonal cell lines from n = 2 independent measurements is shown. Dots represent the individual values. (d–f) Surface expression of the GPI-anchored proteins CD48 and CD55 (d,f) and the transmembrane proteins CD45 and CD64 (e) was measured by flow cytometry in transdifferentiated (d,e) or undifferentiated (f) BLaER1 monoclonal cell lines. Shown is the percentage of cells expressing the protein of interest among all GFP-positive cells from n = 2 independent measurements (mean ± SD). Individual values are depicted as dots. (a,c–f) Monoclonal cell lines with higher responsiveness to LPS (dark blue) were compared to cell lines with lower responsiveness to LPS (light blue) by Mann–Whitney test (ns—not significant, **p ≤ 0.01).

Fig 4: PEM-induced immunogenicity is restrained by CD73. A, Tumor volume of HCC827GR6 cells with Scramble (Scr) or CD73sgRNA using humanized murine model (n = 8 in each group). B, Frequency of CD8+ T cells infiltration among CD45+ cells in mouse tumors (n = 5 in each group). C and D, Representative CD8 IHC images and analysis from Scramble or CD73sgRNA mouse tumors. Red arrows highlight intratumoral localization of CD8+ T cells. Scale bar, 100 µmol/L. E, Schematic of study with PEM in humanized murine HCC827GR6 cells models. F, Tumor volume of HCC827GR6 cells with Scramble or CD73 sgRNA with PEM treatment in humanized murine model (n = 3 in each group). G and H, HLA-A,B,C expression on HCC827, HCC827GR6, and HCC827EPR cells treated ± PEM for 48 hours, followed by 24 hours of drug-free. MFI, mean fluorescence intensity. ?MFI, (Pemetrexed-DMSO (dimethyl sulfoxide))/DMSO. Mean ± SEM of n = 5 biological replicates is shown (unpaired two-tailed Student t test). I, CD73 expression on HCC827GR6 cells treated ± PEM for 48 hours, followed by 24 hours of drug-free. J, Adenosine levels in CM derived from HCC827GR6 cell lines with Scramble or CD73 sgRNA treated ± PEM for 48 hours for 72 hours. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). K, ELISA of human IFN? and IL2 levels in CM derived from HCC827GR6 cell lines with Scramble or CD73 sgRNA pretreated ± PEM for 48 hours with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ±1 µg/mL peptides (SSCSSCPLSK) for 3 hours. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). L, Schematic of treatment strategy on in MET-driven EGFR-TKI–resistant cells. *, P < 0.05; **, P < 0.005; ***, P < 0.001; n.s., nonsignificant.

Fig 5: Impaired T-cell antigen-specific recognition of HCC827-GR6 cells despite elevated STING. A, TCRaß expression on J76 cells transduced with or without LMP2B TCR. B, HLA-A*11:01 Tetramer and CD45 expression on J76 cells transduced with or without LMP2B TCR. C, ELISA of human IFN? levels in CM derived from HCC827 cells with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ±10 µg/mL peptides (SSCSSCPLSK) for 3 hours. Each data represents one experiment. D, ELISA of human IFN? levels in CM derived from HCC827 or HCC827GR6 cells with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ± 0.1, 1, 10µg/mL peptides (SSCSSCPLSK) for 3 hours. Mean ± SEM of n = 2 biological replicates is shown (unpaired two-tailed Student t test; left). ELISA of human IL2 levels in CM derived from HCC827 or HCC827GR6 cells with J76 cells transduced with LMP2B TCR for 72 hours and pretreated ±1 µg/mL peptides (SSCSSCPLSK) for 3 hours. Mean ± SEM of n = 3 biological replicates shown (unpaired two-tailed Student t test; right). *, P < 0.05; **, P < 0.005.