Journal: The Journal of Clinical Investigation

Article Title: Splicing factor SRSF1 controls T cell hyperactivity and systemic autoimmunity

doi: 10.1172/JCI127949

Figure Lengend Snippet: PTEN is decreased in T cells from patients with SLE and correlates with SRSF1 levels. SRSF1 overexpression increases PTEN, suppresses mTORC1 activity, and reduces proinflammatory cytokine production. ( A and B ) Peripheral blood T cells were isolated from patients with SLE and normal (N) healthy control individuals. Total protein was immunoblotted for PTEN, SRSF1, and β-actin. ( A ) Data are from 1 representative of 10 independent experiments. Graph shows relative quantitation by densitometry ( n = 14 SRSF1 low and n = 12 SRSF1 normal patients). ( B ) Graph shows a linear correlation between relative PTEN and SRSF1 expression. ( C ) Peripheral blood T cells were isolated from patients with SLE and transfected with empty vector (EV) or SRSF1 overexpression plasmid (pSrsf1). At 16 to 18 hours after transfections, SLE T cells were stimulated with anti-CD3, anti-CD28, and crosslinker for 5 minutes. Total protein was immunoblotted for PTEN. Data are from 1 representative of 5 independent experiments ( n = 10). Graph shows relative quantitation by densitometry. ( D ) Total protein was immunoblotted for pS6 and total S6 in stimulated SLE T cells after transfection as described in C . Data are from 1 representative of 4 independent experiments ( n = 4). Graph shows relative quantitation by densitometry. ( E ) At 16 to 18 hours after transfection, cells were stimulated with PMA and ionomycin in the presence of monensin. After 4 hours, cytokine production was analyzed by flow cytometry. Graphs show quantitation of cytokine data ( n = 8). Paired t test, * P

Article Snippet: Anti–mouse Fas (clone Jo2) antibody, Cytofix/Cytoperm, Perm/Wash buffer, and monensin were from BD Biosciences.

Techniques: Over Expression, Activity Assay, Isolation, Quantitation Assay, Expressing, Transfection, Plasmid Preparation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Splicing factor SRSF1 controls T cell hyperactivity and systemic autoimmunity

doi: 10.1172/JCI127949

Figure Lengend Snippet: Rapamycin treatment reduces proinflammatory cytokine production by T cells and alleviates autoimmunity in Srsf1 -cKO mice. ( A – D ) Spleen cells from WT or Srsf1 -cKO mice were cultured for 4 hours with PMA plus ionomycin in the presence of monensin. Rapamycin (1 nM or 10 nM) was added for the duration of cultures. Cells were collected, surface stained, fixed, and permeabilized for intracellular cytokine staining. ( A and C ) Plots show IL-17 and IFN-γ intracellular staining gated on live CD4 + T cells. ( B ) Graphs show data from n = 8 mice in 7 independent experiments. ( D ) Graph shows data from n = 15 mice each in 10 independent experiments. ( E – H ) Rapamycin (2 mg/kg) or PBS was administered to WT and Srsf1 -cKO mice by intraperitoneal injection once every 2 days for 4 weeks ( F and G ) or every day for 1 week ( E and H ). ( E ) Spleen cells were cultured with anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) for 24 hours and IL-17A measured in the supernatants by ELISA. Data are shown as fold values normalized to controls ( n = 5 mice in 2 independent experiments). ( F ) Spleen cells were analyzed by flow cytometry, and graph shows the frequency of Tfh cells in spleen ( n = 5–6 mice in 2 independent experiments). ( G ) Serum was collected and autoantibodies measured by ELISA ( n = 5–6 mice in 2 independent experiments). ( H ) Cells from kidneys were analyzed by flow cytometry, and graph shows the frequency of T cells in kidneys ( n = 5 mice in 2 independent experiments). One-way ANOVA with Bonferroni correction ( B , D , E , and F ); 2-tailed unpaired t test ( G and H ); * P

Article Snippet: Anti–mouse Fas (clone Jo2) antibody, Cytofix/Cytoperm, Perm/Wash buffer, and monensin were from BD Biosciences.

Techniques: Mouse Assay, Cell Culture, Staining, Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Splicing factor SRSF1 controls T cell hyperactivity and systemic autoimmunity

doi: 10.1172/JCI127949

Figure Lengend Snippet: PTEN overexpression reduces proinflammatory cytokine production by T cells from Srsf1 -cKO mice. ( A ) T cells were isolated from spleens of WT or Srsf1 -cKO mice and transfected by electroporation with empty vector (EV) or PTEN overexpression plasmid (pPTEN). Sixteen hours later, cells were stimulated with PMA plus ionomycin in the presence of monensin for 4 hours and cells were collected, surface stained, fixed, and permeabilized for intracellular cytokine staining. ( B ) Graphs show average data from n = 9 mice in 5 independent experiments. Two-tailed paired t test, * P

Article Snippet: Anti–mouse Fas (clone Jo2) antibody, Cytofix/Cytoperm, Perm/Wash buffer, and monensin were from BD Biosciences.

Techniques: Over Expression, Mouse Assay, Isolation, Transfection, Electroporation, Plasmid Preparation, Staining, Two Tailed Test