Splicing factor SRSF1 controls T cell homeostasis and its decreased levels are linked to lymphopenia in systemic lupus erythematosus

Abstract

Objective: Lymphopenia is a frequent clinical manifestation and risk factor for infections in SLE, but the underlying mechanisms are not fully understood. We previously identified novel roles for the RNA-binding protein serine arginine-rich splicing factor 1 (SRSF1) in the control of genes involved in signalling and cytokine production in human T cells. SRSF1 is decreased in T cells from patients with SLE and associates with severe disease. Because SRSF1 controls the expression of apoptosis-related genes, we hypothesized that SRSF1 controls T cell homeostasis and, when reduced, leads to lymphopenia.

Methods: We evaluated SRSF1 expression in T cells from SLE patients by immunoblots and analysed its correlation with clinical parameters. T cell conditional Srsf1 knockout mice were used to evaluate lymphoid cells and apoptosis by flow cytometry. Quantitative PCR and immunoblots were used to assess Bcl-xL mRNA and protein expression. SRSF1 overexpression was performed by transient transfections by electroporation.

Results: We found that low SRSF1 levels correlated with lymphopenia in SLE patients. Selective deletion of Srsf1 in T cells in mice led to T cell lymphopenia, with increased apoptosis and decreased expression of the anti-apoptotic Bcl-xL. Lower SRSF1 expression correlated with low Bcl-xL levels in T cells and lower Bcl-xL levels associated with lymphopenia in SLE patients. Importantly, overexpression of SRSF1 rescued survival of T cells from patients with SLE.

Conclusion: Our studies uncovered a previously unrecognized role for SRSF1 in the control of T cell homeostasis and its reduced expression as a molecular defect that contributes to lymphopenia in systemic autoimmunity.

Keywords: Bcl-xL; SRSF1; T cells; homeostasis; lymphopenia; systemic lupus erythematosus.

Fig. 1

Low SRSF1 levels correlate with lymphopenia in patients with SLE Peripheral blood T cells were isolated from patients with SLE (n = 61) and healthy control individuals (n = 44). Total protein was immunoblotted for SRSF1 and β-actin. (A) Data are from one representative of eight independent experiments. (B) Densitometric quantitation of Western blots was performed and SRSF1 normalized to β-actin. Relative SRSF1 expression in SLE patients was normalized to matched healthy controls. (C) Graph shows linear correlation between relative SRSF1 protein levels and peripheral blood lymphocyte counts (n = 61). (D) Graphs show associations of relative SRSF1 with SLEDAI and lymphocyte counts with SLEDAI or WBC counts [B, D (middle and right): unpaired t test; D (left): one-way analysis of variance with Tukey’s correction; C: single linear regression, *P < 0.05].

Fig. 2

T cell conditional deletion of Srsf1 in mice leads to lymphopenia Spleen and MLN cells from WT and Srsf1-ko mice were stained with fluorescent antibodies and analysed by flow cytometry. (A) Plots show Thy1.2⁺ TCRβ⁺ T cells (left panel) and CD4⁺ and CD8⁺ T cells (right panel) gated on live cells. (B) Graphs show the percentage of T cells (spleen: n = 24 each, MLN: n = 13 each, mice <20 weeks old). (C) Graphs show absolute number of T cells (spleen: n = 23 each, MLN: n = 12 each, mice <20 weeks old). (D) Graphs show absolute numbers of CD4 and CD8 T cells (spleen: n = 23 each, MLNs: n = 12 each, mice <20 weeks old) (unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005).

Fig. 3

T cell conditional deletion of Srsf1 in mice leads to increased apoptosis Spleen cells were isolated from WT and Srsf1-ko mice, stained with fluorescent antibodies and analysed by flow cytometry. (A) Plots show 7AAD and Annexin V expression on ex vivo gated T cells. (B) Graph shows the percentage of live cells and early apoptotic cells (7AAD⁻Annexin V⁺) (WT, n = 7; ko, n = 8). (C) Spleen cells were stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) antibodies for 48 h. Flow cytometry plots show 7AAD and Annexin V expression on T cells. (D) Graph shows the percentage of live cells and early apoptotic cells (7AAD⁻Annexin V⁺) (n = 4) (unpaired t test, *P < 0.05, **P < 0.005).

Fig. 4

Bcl-xL is decreased in T cells from Srsf1-ko mice (A) RNA sequencing data analysis of CD4 effector T cells from WT and Srsf1-ko mice shows pathways identified by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO) analyses of differentially expressed genes. (B) Conventional CD4 T cells (CD4⁺CD25⁻) were sorted by flow cytometry and stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) antibodies for 24 h. Total RNA was isolated and reverse transcribed. Expression levels of apoptosis-associated genes were measured by real-time quantitative PCR and normalized to housekeeping gene cyclophilin A (n = 4 each, unpaired t test, **P < 0.005, n.s. not significant).

Fig. 5

Low Bcl-xL levels correlate with SRSF1 expression in T cells and associate with lymphopenia in SLE patients SRSF1 overexpression rescues survival of SLE T cells. Peripheral blood T cells were isolated from patients with SLE and healthy control individuals. Total protein was immunoblotted for SRSF1, Bcl-xL and β-actin. (A) Graph shows the relative quantitation of Bcl-xL/β-actin by densitometry (n = 35). (B) The graph shows the linear correlation between Bcl-xL and SRSF1 (n = 35). (C) The graph shows the relative quantitation of Bcl-xL/β-actin by densitometry (n = 23) in association with lymphocyte counts grouped by lymphopenia. (D and E) PBMCs were isolated from peripheral blood from patients with SLE and transfected with empty vector (EV) or Srsf1 overexpression plasmid (pSrsf1). At 16–18 h after transfection, cells were analysed by flow cytometry. Plots show 7AAD and Annexin V expression on gated T cells, CD4 T cells and CD8 T cells (D). Graphs show the percentage of live (7AAD⁻Annexin V⁻) cells (E, n = 4) (A and C: unpaired t test, B: single linear regression, E: paired t test; *P < 0.05, **P < 0.005, ***P < 0.0005).