Fig 2: H5 ImmTAAI localizes at the target cell–T cell interface and inhibits proximal TCR signaling.(A) Schematic of the cell assay used to visualize H5 ImmTAAI localization. (B) Phase contrast and confocal fluorescence images of H5 ImmTAAI localization on ECN90 ß cell target cells cocultured with either PD-1+ or PD-1– Jurkat cells. Upper panel: phase contrast. Lower panel: fluorescence images, a white arrow indicates ImmTAAI accumulation (representative images from 3 independent studies). Total original magnification, ×100. (C) The numbers of ECN90 ß cell: Jurkat conjugates with ImmTAAI accumulation (white arrow) were counted and plotted as a percentage of the total number of cell conjugates (n = 100 conjugates for conditions using Jurkat Mel5 PD-1 cells and n = 96 for Jurkat Mel5 cells, plotted as mean data from 3 independent studies). (D) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 or ECN90 PD-L1 cells for the indicated times. Whole cell lysates for each condition and time point were resolved by SDS-PAGE, transferred to PVDF membranes, and probed by Western blotting for phospho–SLP-76 (pSLP-76), total SLP-76 (SLP-76), phospho-PLC? (pPLC?), total PLC? (PLC?), phospho–ZAP-70 (pZAP-70), and total ZAP-70 (representative blots from 3 independent experiments). (E) Jurkat Mel5 PD-1 cells were stimulated with Melan-A–pulsed ECN90 cells, in the presence or absence of H5 ImmTAAI, and Western blots were performed as above (representative blots from 3 independent experiments).

Fig 3: CD274-L2A and CD274v1 expression are decoupled under certain stimuli.(A) CD274 variant 1 and CD274-L2A expression across TCGA tumour and GTEx healthy samples. Average TPM is shown per tissue type, with linear regression performed separately for tumour and healthy samples. (B) CD274 variant 1 and CD274-L2A expression (in TPMs) across the CCLE dataset. Dashed lines denote a 10:1 and 1:1 ratio of CD274v1:CD274-L2A, respectively. (C) Expression of CD274 variant 1 and CD274-L2A, measured by qRT-PCR using variant-specific primers in five leukocyte cell lines. Cells were stimulated with IFN-a or IFN-? for 48 hr or were left untreated. Mean (± SEM) expression normalized to HPRT from three independent experiments is shown. (D) Expression of CD274 variant 1 and CD274-L2A (in TPMs), calculated using RNA-seq data (SRP045500) from B cells, monocytes and neutrophils isolated from peripheral blood of healthy individuals of patients with Sepsis, ALS or T1D or from MS patients before and 24 hr after the first treatment with IFN-ß.Figure 3—source data 1.Expression of CD274 variants in the CCLE collection.

Fig 4: SPHK1 transcriptionally regulates MTA3 and c-Myc in melanoma cells. a Heat map of RNA-seq expression z-scores computed the candidate genes in SK-MEL-28 melanoma cells treated with PF543 (25 µM) and IFN-? (200 ng/mL) for 24 h. See also Supplementary Table 5. b RT-PCR of CD274 and candidate transcription factors mRNA level was performed from the same sample as RNA-seq (n = 9). Data represent mean ± SEM. ns non-significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. P values were calculated using unpaired two-sided Student’s t-test. c SPHK1 was positively correlated with MTA3 by using the calculation of Pearson’s correlation coefficient and subsequent significance test in 52 melanoma cell lines from the GDSC database. d Spearman’s correlation between MTA3 expression and MSigDB hallmark pathways across 33 cancer types. Pie charts in the left panel represent the percentage of cancer types with positive (red, Rs > 0.2, p < 0.05), negative (blue, Rs < -0.2, p < 0.05), and non-significant correlation (gray). e Enrichment plot of MYC target gene sets based on RNA sequencing. f Scatterplot for linear-regression of the association between c-Myc and SPHK1, as determined by Pearson’s correlation coefficient and significance test. The solid line corresponds to the regression estimate, and the corresponding 95% CIs, indicated by grey shading. g Overlaid images of MTA3 ChIP-seq enrichment from K562 cell line for two replicates. ChIP-seq tracks are obtained from published data. h SK-MEL-28 melanoma cells were transfected with MTA3 or negative control (CTL) vector. MTA3 expression was analyzed by western blot. i, j Locations of ChIP-qPCR primers at the c-Myc promoter, transcription start site is designated as nucleotide +1. F/R, Forward/Reverse (i). Input % of c-Myc DNA by IgG or Flag antibody were determined by ChIP-qPCR in melanoma cell lines (n = 3). j Data represent mean ± SEM. ns non-significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. P values were calculated using unpaired two-sided Student’s t-test

Fig 5: STAT1 upregulation in MM cells by panobinostat in the presence of IFN-?(A) STAT1 mRNA expression in MM cells. KMS-11, MM.1S and OPM-2 cells were cultured in triplicate for 24 hours with or without 25 nM of panobinostat in the presence or absence of 100 U/ml of IFN-?. STAT1 mRNA expression was quantified by quantitative RT-PCR. Ratios of STAT1 over PPIE mRNA levels were calculated for a normalized target value (defined as 1). PPIE was used as an internal control. Results were expressed as the mean ± SD. *p < 0.05, **p < 0.01. (B) STAT1 and STAT3 expression in MM cells. KMS-11, MM.1S and OPM-2 cells were cultured for 6 or 24 hours with or without 25 nM of panobinostat in the presence or absence of 100 U/ml of IFN-?. STAT1, STAT3 and PD-L1 mRNA expression was analyzed in the MM cells by RT-PCR. (C) Effects of STAT1 gene silencing on PD-L1 expression. STAT1 shRNA (clones #1 and #2) or control Luciferase shRNA were transfected into KMS-11 cells. The knockdown efficacy was examined by RT-PCR (left). The cells were cultured for 6 hours with or without 25 nM of panobinostat in the presence or absence of 100 U/ml of IFN-?. PD-L1 mRNA expression was analyzed by RT-PCR. PPIE was used as an internal control. Pano, panobinostat.