Fig 2: Polyfunctional T cells are enriched with stem cell–associated genetic signatures.(A) Heatmap of gene expression of memory CD8+ T cells treated by DMSO or TWS119 for 7 days. Red and blue reflect increased and decreased gene expression, respectively. (B) GSEA demonstrated that TWS119-treated memory T cells were enriched with progenitor and hematopoietic stem cell signatures. (C) Heatmap showing the expression of immunological- and self-renewal–related genes. (D) Fold change of PRMT gene expression following TWS119 treatment. (E) PRMT1 and PRMT5 expression in memory CD8+ T cells was upregulated by Wnt agonist treatment. Numbers indicate MFI value of each protein. (F) Quantitative RT-PCR analysis of PRMT1 and PRMT5 in response to Wnt activation (n = 3). Dunn’s test for multiple comparisons. #P < 0.05 by Dunn’s test.

Fig 3: PRMT1 epigenetically controls CD8+ T cell polyfunctionality.(A) Memory CD8+ T cells were stimulated with CD3/CD28 in the presence or absence of SKL2001 for 1 day and transduced with either nontarget (NT) sequence virus or PRMT1 knockdown virus. On day 7, PRMT1 RNA expression was analyzed (n = 3). Mann-Whitney U test. (B) PRMT1 protein detection by flow cytometry in PRMT1 knockdown or nontarget virus in presence or absence of SKL2001. (C) Polyfunctionality profile of cells transduced with nontarget or PRMT1 knockdown virus in DMSO or SKL2001 treatment. 0, 1, 2, 3 are defined as the number of positive cytokines. (D and E) H4R3 dimethylation by PRMT1 was enhanced by Wnt agonists (n = 4). Dunn’s test for multiple comparisons. (F) ChIP assay of memory CD8+ cells stimulated with DMSO or SKL2001 for 7 days. ChIP assays were performed with antibodies to H3Ac and H4R3me2a. Each ChIP eluate was amplified by qPCR at the indicated regions of the IL-2 locus (n = 3). Mann-Whitney U test. ***P < 0.001. #P < 0.05 by Dunn’s test.

Fig 4: N-terminal amino acid sequences, genomic configuration and expression of the prmt1 gene in chicken.(A) The N-terminal amino acid sequences of chicken PRMT1 v1 and v2. (B) Schematic illustration of the three major prmt1 variants due to alternative splicing at the 5’ end in human, chicken and zebrafish. The gene structure of prmt8 very close to prmt1 v1 is also shown. The nomenclature of the exons according to human PRMT1 by Goulet et al. [26] is shown in the parenthesis. (C) Expression of prmt1 variants in chicken analyzed by RT-PCR. The products amplified by the primers for the constitutive exons (prmt1-1) and the products amplified by the primer set encompassing the alternative exon (prmt1-2) are shown. Mouse prmt1 transcripts were also analyzed in comparison. Gapdh indicates the RT-PCR product of glyceraldehyde 3-phosphate dehydrogenase as the control. NC: negative control. L: liver, M: muscle, B: brain.

Fig 5: PRMT1 highly correlates with CD8+ T cell polyfunctionality.(A) Intracellular staining for cytokines and PRMT1 expression in memory CD8+ T cells in different treatment conditions. PRMT1 staining is illustrated on the x axes versus cytokine production (y axes). Black and red numbers represent percentages of cytokine-positive cells and PRMT1 MFI, respectively. (B) Pie charts showing the polyfunctionality profile of the different treatment conditions. Bar graphs display PRMT1 MFI of T cell subsets of different degrees of polyfunctionality of each treatment condition (n = 4). 0, 1, 2, 3 are defined as the number of positive cytokines. (C) PRMT1 MFI in IL-2+, IFN-γ+, or TNF-α+ CD8+ cells undergoing 7 days of CD3/CD28 stimulation from multiple healthy donors (n = 10). Mann-Whitney U test. (D) Combined PRMT1 protein and RNA detection with polyfunctionality profile by flow cytometry. Green and red boxes represent top and bottom 30% of PRMT1-expressing cells, respectively. Black lines in the plots indicate the MFI of PRMT1 protein and RNA in control group. Pie charts show the polyfunctionality profile of PRMT1hi/lo (green/red) cells in the presence or absence of SKL2001. (E) MFI of PRMT1 protein and mRNA were plotted by different polyfunctionality combinations. Squares and circles represent PRMT1 protein and mRNA levels, respectively. (F) Percentages of IFN-γ+ (black), TNF-α+ (blue), and IL-2+ (red) of all CD8+ cells in PRMT1 hi versus lo cells (filled or grid) following treatment with DMSO or SKL2001 (n = 3). Mann-Whitney U test. (G) Memory CD8+ cells treated with DMSO or SKL2001 were stratified into approximately 20–30 tiers according to MFI of PRMT1. Each tier contained at least 500 cells, and polyfunctionality was computed from individual tiers before being plotted against PRMT1 MFI in linear regression analysis.*P < 0.05; **P < 0.01; ***P < 0.001.