PRMT5 Is Required for T Cell Survival and Proliferation by Maintaining Cytokine Signaling

Abstract

Arginine methylation is a post-translational modification that regulates many biological processes. However, the role of arginine methylation in immune cells is not well studied. Here we report an essential role of protein arginine methyltransferase 5 (PRMT5) in T cell homeostasis and activation-induced expansion. Using T cell-specific PRMT5 conditional knockout mice, we found that PRMT5 is required for natural killer T (NKT) cell but not for conventional or regulatory T (Treg) cell development after the double positive (DP) stage in the thymus. In contrast, PRMT5 was required for optimal peripheral T cell maintenance, for the transition of naïve T cells to effector/memory phenotype, and for early T cell development before the DP stage in a cell-intrinsic manner. Accordingly, PRMT5-deleted T cells showed impaired IL-7-mediated survival and TCR-induced proliferation in vitro. The latter was more pronounced and attributed to reduced responsiveness to IL-2. Acute deletion of PRMT5 revealed that not only naïve but also effector/memory T cells were impaired in TCR-induced proliferation in a development-independent manner. Reduced expression of common γ chain (γc), a shared receptor component for several cytokines including IL-7 and IL-2, on PRMT5-deleted T cells may be in part responsible for the defect. We further showed that PRMT5 was partially required for homeostatic T cell survival but absolutely required for lymphopenic T cell expansion in vivo. Thus, we propose that PRMT5 is required for T cell survival and proliferation by maintaining cytokine signaling, especially during proliferation. The inhibition of PRMT5 may provide a novel strategy for the treatment of diseases where uncontrolled T cell activation has a role, such as autoimmunity.

Keywords: PRMT5; T cell development; T cell proliferation; T cell survival; arginine methylation; cytokine signaling.

FIGURE 1

T-cell-specific deletion of PRMT5 leads to peripheral T cell lymphopenia in mice. (A–C) Thymocytes from control and CD4^Cre PRMT5^fl/fl mice were analyzed by flow cytometry. (A) Thymocytes were classified into DN (CD8a^– CD4^–), DP (CD8a⁺ CD4⁺), CD8 SP (CD8a⁺ CD4^–), and CD4 SP (CD8a^– CD4⁺) cells. TCRβ^– DN cells were further divided into DN1 (CD44⁺ CD25^–), DN2 (CD44⁺ CD25⁺), DN3 (CD44^– CD25⁺), and DN4 (CD44^– CD25^–) cells. CD8 SP cells include TCRβ^– ISP cells. CD4 SP cells include Foxp3⁺ Treg cells. NKT cells were defined as NK1.1⁺ TCRβ⁺ cells. (B) Representative histograms show PRMT5 expression by thymocytes from three independent experiments. Peripheral CD44^low T cells from CD4^Cre PRMT5^fl/fl mice were used as a negative control for PRMT5 staining. (C) The absolute numbers of thymocytes in control mice (n = 6) and CD4^Cre PRMT5^fl/fl mice (n = 9) from three independent experiments are plotted as mean ± SEM. (D,E) Splenocytes from control and CD4^Cre PRMT5^fl/fl mice were analyzed by flow cytometry. (D) Representative plots show CD8⁺ T (NK-1.1^– TCRβ⁺ CD8a⁺), CD4⁺ T (NK-1.1^– TCRβ⁺ CD4⁺), Treg (NK-1.1^– TCRβ⁺ CD4⁺ Foxp3⁺), and NKT (NK-1.1⁺ TCRβ⁺) cells. (E) The absolute numbers of splenic T cell subsets in control mice (n = 6) and CD4^Cre PRMT5^fl/fl mice (n = 9) from three independent experiments are plotted as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired two-tailed Student’s t-test. NS, not significant.

FIGURE 2

PRMT5 is cell-intrinsically important for peripheral T cell maintenance and activation. (A–D) Lethally irradiated CD90.2⁺ CD45.1⁺ wildtype mice (n = 4/group) were reconstituted with a 1:1 mixture of bone marrow cells from CD90.1⁺ CD45.2⁺ wildtype mice and CD90.2⁺ CD45.2⁺ control or CD4^Cre PRMT5^fl/fl mice. At 8 wk after reconstitution, the thymus and spleen of mixed bone marrow chimeras were analyzed by flow cytometry. (A) Representative plots of CD90.1 versus CD90.2 gated on donor (CD45.2⁺) T cell subsets in the thymus and spleen are shown. (B) The ratio of CD90.2⁺ cells to CD90.1⁺ cells in each T cell population was normalized to the ratio obtained from DN cells for each recipient. Results are plotted as mean ± SEM. (C) Representative plots show the expression of CD62L and CD44 on donor (CD45.2⁺) CD90.1⁺ and CD90.2⁺ T cells in the spleen of mixed bone marrow chimeras that received CD90.1⁺ CD45.2⁺ wildtype and CD90.2⁺ CD45.2⁺ CD4^Cre PRMT5^fl/fl bone marrow cells. (D) The frequencies of CD44^high cells among donor T cells shown in (C) are plotted as mean ± SEM. The data are representative of two independent experiments. (E) Splenocytes from control and CD4^Cre PRMT5^fl/fl mice were analyzed by flow cytometry for the expression of PRMT5 and CD44 by CD8⁺ T cells (NK-1.1^– CD90.2⁺ CD8a⁺), CD4⁺ T cells (NK-1.1^– CD90.2⁺ CD4⁺), and Treg cells (NK-1.1^– CD90.2⁺ CD4⁺ CD25⁺). Representative plots from three independent experiments are shown. **P < 0.01, ***P < 0.001, by unpaired two-tailed Student’s t-test. NS, not significant; WT, wildtype.

FIGURE 3

PRMT5 plays a role in peripheral T cell maintenance and early T cell development in a cell-intrinsic manner. Lethally irradiated CD90.2⁺ CD45.1⁺ wildtype mice (n = 4/group) were reconstituted with a 1:1 mixture of bone marrow cells from CD90.1⁺ CD45.2⁺ wildtype mice and CD90.2⁺ CD45.2⁺ Cre-ERT2 control or Cre-ERT2 PRMT5^fl/fl mice. The mixed bone marrow chimeras were treated with tamoxifen for five consecutive days and the spleen (A–C) and thymus (D–F) were analyzed by flow cytometry at 10 days after the last treatment. (A) Representative plots show the expression of YFP on donor CD45.2⁺ CD90.2⁺ CD8⁺ T (TCRβ⁺ CD8a⁺), CD4⁺ T (TCRβ⁺ CD4⁺), and iNKT (TCRβ⁺ CD1d tet⁺) cells in the spleen of mixed bone marrow chimeras. YFP⁺ CD8⁺ and CD4⁺ T cells were further analyzed for the expression of CD62L and CD44. (B) The frequencies of YFP⁺ cells among donor cells shown in (A) are plotted as mean ± SEM. (C) The frequencies of CD44^high cells among YFP⁺ CD8⁺ and CD4⁺ T cells are plotted as mean ± SEM. (D) Representative plots show the expression of YFP on donor CD45.2⁺ CD90.2⁺ DN (CD8a^– CD4^– TCRβ^–), DP (CD8a⁺ CD4⁺), CD8 SP (CD8a⁺ CD4^– TCRβ⁺), CD4 SP (CD8a^– CD4⁺) and iNKT (TCRβ⁺ CD1d tet⁺) cells in the thymus of mixed bone marrow chimeras. YFP⁺ DN cells were further divided into DN1 (CD44⁺ CD25^–), DN2 (CD44⁺ CD25⁺), DN3 (CD44^– CD25⁺), and DN4 (CD44^– CD25^–) developmental subsets. (E) The frequencies of YFP⁺ cells among donor cells shown in (D) are plotted as mean ± SEM. (F) The frequencies of DN subsets among YFP⁺ DN are plotted as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired two-tailed Student’s t-test. CD1d tet, CD1d tetramers loaded with PBS-57; NS, not significant; WT, wildtype.

FIGURE 4

PRMT5 is partially required for IL-7-induced survival of naïve T cells in vitro. Naïve CD8⁺ and CD4⁺ T cells were purified from control and CD4^Cre PRMT5^fl/fl mice and cultured for 3 d in the presence or absence of IL-7, and analyzed by flow cytometry. (A) The frequency of live cells (Live/Dead near-IR^–) among CD8⁺ T cells (TCRβ⁺ CD8a⁺) and CD4⁺ T cells (TCRβ⁺ CD4⁺) is plotted as mean ± SEM. (B) The counts of live T cells are normalized to the initial (day 0) counts and plotted as mean ± SEM. The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired two-tailed Student’s t-test. NS, not significant.

FIGURE 5

PRMT5 is required for the proliferation of naïve T cells in response to TCR stimulation in vitro. CTV-labeled naïve CD8⁺ and CD4⁺ T cells from control and CD4^Cre PRMT5^fl/fl mice were stimulated for 3 d with anti-CD3 and anti-CD28-coated beads in the presence or absence of anti-IL-2 blocking antibody or IL-2, and analyzed by flow cytometry. (A) The frequency of live cells (Live/Dead near-IR^–) among CD8⁺ T cells (TCRβ⁺ CD8a⁺) and CD4⁺ T cells (TCRβ⁺ CD4⁺) is plotted as mean ± SEM. (B) The counts of live T cells are normalized to the initial (day 0) counts and plotted as mean ± SEM. (C) Representative plots show the dilution of CTV and expression of PRMT5. The CTV dilution of PRMT5^– cells among CD4^Cre PRMT5^fl/fl T cells were overlayed on the CTV dilution profile of control T cells. The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired two-tailed Student’s t-test. NS, not significant.

FIGURE 6

PRMT5 is required for the proliferation of naïve and effector/memory T cells in a development-independent manner. FACS-sorted CTV-labeled naïve and effector/memory CD8⁺ and CD4⁺ T cells from tamoxifen-treated Cre-ERT2 and Cre-ERT2 PRMT5^fl/fl mice were stimulated for 3 d with anti-CD3 and anti-CD28-coated beads in the presence of IL-2, and analyzed by flow cytometry. (A) The frequency of live cells (Live/Dead near-IR^–) among CD8⁺ T cells (CD90.2⁺ CD8a⁺) and CD4⁺ T cells (CD90.2⁺ CD4⁺) is plotted as mean ± SEM. (B) The counts of live T cells are normalized to the initial (day 0) counts and plotted as mean ± SEM. (C) Representative plots show the dilution of CTV and expression of PRMT5. The data are representative of two independent experiments. **P < 0.01, ***P < 0.001, by unpaired two-tailed Student’s t-test. NS, not significant.

FIGURE 7

PRMT5 is important for maintaining IL-7 and IL-2 signaling in T cells. (A,C) Naïve CD8⁺ and CD4⁺ T cells were purified from control and CD4^Cre PRMT5^fl/fl mice and stimulated for the indicated times with anti-CD3 and anti-CD28-coated beads in the presence of IL-2 or anti-IL-2 blocking antibody, and analyzed by flow cytometry for the expression of Nur77, CD69, CD25, γc, and pSTAT5. Representative histograms and plots are shown from at least two independent experiments. (B) Naïve CD4⁺ T cells were purified from control and CD4^Cre PRMT5^fl/fl mice and stimulated for the indicated times with biotinylated anti-CD3, anti-CD4, and anti-CD28 plus streptavidin. The phosphorylation of ERK1/2 was analyzed by Western blot. Total PLCγ1 was used as a loading control. The data are representative of two independent experiments. (D) Naïve CD8⁺ and CD4⁺ T cells were purified from control and CD4^Cre PRMT5^fl/fl mice and left unstimulated or stimulated for 30 min with IL-7, followed by flow cytometric analysis of pSTAT5 expression. The frequency of pSTAT5⁺ cells among IL-7-stimulated T cells of control and CD4^Cre PRMT5^fl/fl mice (n = 4) from two independent experiments is plotted as mean ± SEM. **P < 0.01 by unpaired two-tailed Student’s t-test. (E,F) T cell subsets from the thymus and spleen of control and CD4^Cre PRMT5^fl/fl mice were analyzed for expression of CD25, CD122, CD127, and γc by flow cytometry. Gating strategy is shown in Figures 1A,D and Supplementary Figure S3C. Representative histograms are shown from three independent experiments.

FIGURE 8

PRMT5 is required for homeostatic T cell survival and proliferation in vivo. (A,B) CD90.2⁺ CD45.1⁺ wildtype mice (n = 4/group) were adoptively transferred with a 1:1 mixture of naïve T cells from CD90.1⁺ CD45.2⁺ wildtype mice and CD90.2⁺ CD45.2⁺ control or CD4^Cre PRMT5^fl/fl mice. The cells before transfer (Input) and splenocytes at 7 days after transfer (Day 7 Spleen) were analyzed by flow cytometry. (A) Representative plots show CD90.1 versus CD90.2 among donor CD8⁺ T cells (CD45.2⁺ CD8a⁺) and CD4⁺ T cells (CD45.2⁺ CD4⁺). CD90.2⁺ cells were analyzed for the dilution of CTV and expression of PRMT5 (far right plots). (B) The ratio of total (left plot) and undivided (right plot) CD90.2⁺ cells to CD90.1⁺ cells in donor CD8⁺ and CD4⁺ T cells was normalized to the ratio of the input. Results are plotted as mean ± SEM. (C,D) RAG2 KO mice (n = 4/group) were adoptively transferred with a 1:1 mixture of naïve T cells from CD90.1⁺ wildtype mice and CD90.2⁺ control or CD4^Cre PRMT5^fl/fl mice. The transferred cells (Input) and splenocyte at 7 d after transfer (Day 7 Spleen) were analyzed by flow cytometry. (C) Representative plots showing CD90.1 versus CD90.2 among donor CD8⁺ T cells (CD8a⁺) and CD4⁺ T cells (CD4⁺). CD90.2⁺ cells were further analyzed for the dilution of CTV and expression of PRMT5 (far right plots). (D) The ratio of CD90.2⁺ cells to CD90.1⁺ cells in donor CD8⁺ and CD4⁺ T cells was normalized to the ratio of the input. Results are plotted as mean ± SEM. The data are representative of two independent experiments. *P < 0.05, ***P < 0.001, by unpaired two-tailed Student’s t-test. NS, not significant; WT, wildtype.