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. 2020 Apr 23;5(8):e131099. doi: 10.1172/jci.insight.131099

Figure 1. PRMT5 expression and activity is upregulated in activated mouse and human T cells in vitro and in vivo.

Figure 1

Murine B6 T cells were stimulated with either CD3/CD28 Dynabeads or allogeneic BALB/c DCs for 4 days. (A and B) Prmt5 mRNA expression in T cells quantified by real-time PCR (n = 5). Expression relative to unstimulated (US) T cells, with β-actin used as a normalizer. Results are represented as mean ± SD of 2–3 independent experiments. Each symbol represents an individual donor. (C) PRMT5 protein and histone H3R8 symmetric dimethyl protein (H3R8me2s) analyzed by Western blot. One representative Western blot of 3 independent experiments is shown. (D) T cells isolated from healthy human donor (HD) PBMCs were stimulated with CD3/CD28 Dynabeads, with Prmt5 mRNA expression (n = 3 donors) quantified by real-time PCR, and (E) immunoblotted for PRMT5 protein expression and function (n = 2 donors). (F) In vitro PRMT5 methyltransferase activity in nuclear extracts derived from US versus CD3/CD28-stimulated human T cells. Results show specific activity measured in nanograms of dimethylated H4-Arg3 produced per 20 μg nuclear extract. Each symbol represents an individual replicate measurement, with mean ± SD. One representative assay of 3 independent experiments is shown. (G) Splenic T cells isolated from lethally irradiated B6D2F1 mice that received TCD-BM or TCD-BM + B6 allogeneic splenocytes around 25 days after transplant. mRNA expression (n = 9) and (H) protein expression. Two or 3 spleens were pooled from recipient mice to make 1 pooled sample, and 3 pooled samples were analyzed for PRMT5 protein expression by Western blot. (I) Healthy human PBMCs were collected from HDs (n = 7) and from patients after allogeneic bone marrow transplantation with or without GVHD at the time of collection (Supplemental Table 2). Patient samples were matched for day of transplant at which GVHD occurred. PBMCs were stained and analyzed for PRMT5 expression on T cells using mass cytometry; the gating strategy is shown in Supplemental Figure 8. Each symbol represents an individual donor. Data represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.