AUTOIMMUNITY

Research ArticleAutoimmunityImmunology Free access | 10.1172/JCI127949 Splicing factor SRSF1 controls T cell hyperactivity and systemic autoimmunity Takayuki Katsuyama,1 Hao Li,1 Denis Comte,1,2 George C. Tsokos,1 and Vaishali R. Moulton1 First published September 5, 2019 - More info Abstract Systemic lupus erythematosus (SLE) is a devastating autoimmune disease in which hyperactive T cells play a critical role. Understanding molecular mechanisms underlying the T cell hyperactivity will lead to identification of specific therapeutic targets. Serine/arginine-rich splicing factor 1 (SRSF1) is an essential RNA-binding protein that controls posttranscriptional gene expression. We have demonstrated that SRSF1 levels are aberrantly decreased in T cells from patients with SLE and that they correlate with severe disease, yet the role of SRSF1 in T cell physiology and autoimmune disease is largely unknown. Here we show that T cell–restricted Srsf1-deficient mice develop systemic autoimmunity and lupus-nephritis. Mice exhibit increased frequencies of activated/effector T cells producing proinflammatory cytokines, and an elevated T cell activation gene signature. Mechanistically, we noted increased activity of the mechanistic target of rapamycin (mTOR) pathway and reduced expression of its repressor PTEN. The mTOR complex 1 (mTORC1) inhibitor rapamycin suppressed proinflammatory cytokine production by T cells and alleviated autoimmunity in Srsf1-deficient mice. Of direct clinical relevance, PTEN levels correlated with SRSF1 in T cells from patients with SLE, and SRSF1 overexpression rescued PTEN and suppressed mTORC1 activation and proinflammatory cytokine production. Our studies reveal the role of a previously unrecognized molecule, SRSF1, in restraining T cell activation, averting the development of autoimmune disease, and acting as a potential therapeutic target for lupus. Graphical Abstract Introduction Systemic lupus erythematosus (SLE) is a chronic debilitating multi-system autoimmune disease that disproportionately afflicts women, causing significant morbidity and mortality (1, 2). Failure of the immune system to recognize self-antigens leads to inappropriate immune responses, systemic autoimmunity, and organ damage. The identification of novel molecules and mechanisms underlying the pathogenic immune response in SLE will guide the development of appropriate therapeutic targets. While autoreactive B cells, autoantibodies, and immune complexes are important in disease pathogenesis (3), and elevated type I interferons (IFNs) have been implicated in disease (4, 5), therapeutic strategies targeting B cells and type I IFN signaling in SLE have shown limited success. In this regard, the role of T cells in disease pathogenesis is beginning to be understood, and therapeutic strategies targeting T cells are emerging (6). Aberrantly activated effector T (Teff) cells and cytokines that orchestrate not only germinal center B cell responses for autoantibody production (7–9) but also promote inflammatory infiltration in target organs (10, 11), are critical players in disease pathogenesis. These Teff cells and cytokines are found both in lupus-prone mice and in patients with SLE (12–15), and therefore are potential promising therapeutic targets. Yet the molecules and mechanisms which control the activated T cell phenotype in SLE are poorly understood. A key pathway involved in the activation of T cells is the phosphoinositide-3 kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway. The mTOR pathway determines cell fate decisions of T cell activation, differentiation, proliferation, and cell death (16). Resting quiescent T cells have low levels of mTOR activity, while mTOR complex 1 (mTORC1) activation leads to increased proportions of activated/effector cells and a decreased pool of naive cells (17). Recently, mTOR activation has emerged as a central regulator of T cell dysfunction in systemic autoimmune diseases and as a promising therapeutic target for SLE (15, 18). Increased activity of the mTORC1 pathway in T cells is known to promote the aberrant production of cytokines IFN-γ, IL-17, and IL-4 (19, 20), and contribute to systemic autoimmunity (9). Furthermore, activation of mTORC1 is associated with altered aberrantly activated T cell responses in patients with SLE (21, 22). The phosphatase and tensin homolog (PTEN), a lipid phosphatase, negatively regulates the PI3K/Akt signaling pathway (23) and inhibits the activity of the mTOR pathway (24). PTEN-deficient T cells are hyperresponsive to TCR stimulation (25), and Pten deletion in T cells in mice leads to autoimmune lymphoproliferative disease and aberrant production of cytokines (26, 27). PTEN expression is decreased in B cells from patients with SLE and contributes to B cell hyperactivity (28). However, it is still unknown whether PTEN is altered in T cells from patients with SLE and whether it contributes to T cell hyperactivity. Serine arginine-rich splicing factor 1 (SRSF1), previously known as splicing factor 2/alternative splicing factor (SF2/ASF), is the prototype member of the highly conserved serine arginine (SR) family of RNA-binding proteins. SRSF1 controls posttranscriptional gene expression via pre-mRNA alternative splicing, mRNA stability, and translation (29). While SRSF1 is an essential ubiquitous molecule that controls genes involved in cell survival and proliferation (30), little is known of its role in the immune system. T cells from patients with SLE display hyperactive T cell receptor (TCR)/CD3+ signaling and proinflammatory cytokine production (1, 31). This overactive lupus T cell phenotype is associated with a reduced expression of the CD3 zeta (ζ) chain (32), attributed in part to defective alternative splicing of its 3′ UTR (33). Using discovery approaches, we found that SRSF1 binds to the 3′ UTR of the CD3ζ mRNA in human T cells (34, 35). We showed that SRSF1 promotes normal expression of CD3ζ by limiting expression of an unstable isoform of its 3′ UTR (35) and that SRSF1 levels decrease upon T cell activation (36). We demonstrated that SRSF1 expression is aberrantly decreased in T cells from patients with SLE and correlates with severe disease (37)(38). In addition, decreased SRSF1 expression levels have been linked to the defective expression of multiple autoantigen transcripts in the organ-specific autoimmune inflammatory myositis (39). Thus, studies in humans suggest that the reduced expression of SRSF1 correlates with an activated T cell phenotype and the expression of autoimmune disease. However, evidence of a causal in vivo relationship among SRSF1, T cell dysfunction, and autoimmune disease is still lacking. Here we show that mice that lack Srsf1 in mature T cells develop systemic autoimmunity and lupus nephritis with increased proportions of activated/effector T cell populations and proinflammatory cytokine-producing T cells. Mechanistically, we show that T cells from Srsf1-deficient mice exhibit increased mTOR activity and reduced expression of its negative regulator PTEN, whereas rapamycin and overexpression of PTEN normalize the inflammatory cytokine production. Importantly, PTEN levels are reduced in patients with SLE with lower SRSF1 expression and SRSF1 rescues PTEN levels, suppresses mTORC1 activity, and reduces proinflammatory cytokine production by T cells from patients with SLE. Our studies demonstrate that SRSF1 is a previously unrecognized molecule that restrains T cell activation and controls the development of autoimmune disease. Results Srsf1-cKO mice developed autoimmunity and lupus nephritis. Because germline Srsf1 deletion is embryonic lethal (40), we generated conditional distal.Lckcre.Srsf1fl/fl (Srsf1-cKO hereafter) mice to study the role of SRSF1 in T cells (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI127949DS1). At birth, Srsf1-cKO mice were viable with normal external features and body weight (Supplemental Figure 1C). As expected (the distal Lck promoter is expressed late in thymic development and mainly in mature T cells), T cell development was normal in Srsf1-cKO mice (Supplemental Figure 1, C–F). We asked whether the Srsf1-cKO mice develop signs of autoimmunity. We measured autoantibodies in the sera of Srsf1-cKO mice using microarray analyses of serum IgG reactivity against a 128-autoantigen panel, which includes the lupus-associated autoantigens dsDNA, histones, and a number of nuclear, membrane, phospholipid, and cytoplasmic protein autoantigens (Figure 1A). Serum from a lupus-prone MRL/lpr mouse, which spontaneously develops an autoimmune lupus-like disease, was included as positive control. We found that Srsf1-cKO mice develop a wide array of lupus-associated autoantibodies at levels comparable to those seen in MRL/lpr mice (Figure 1A). We confirmed by ELISA the presence of antinuclear (ANA) antibodies in sera from Srsf1-cKO mice (Supplemental Figure 2A). In aged mice (12–18 months), we observed a lymphoproliferative phenotype (Figure 1B and Supplemental Figure 2B). We also found that T cells from Srsf1-cKO mice exhibit increased proliferative capacity as assessed by Ki67 staining (Figure 1C). The kidneys of aged Srsf1-cKO mice showed histopathologic evidence of lupus nephritis with glomerulonephritis, mesangial and endocapillary proliferation, and interstitial inflammatory cell infiltration (Figure 1D) with significantly higher histopathology scores compared with control mice (Figure 1E). Immunohistochemistry staining showed T cell infiltration in kidneys of Srsf1-cKO mice (Supplemental Figure 2C). Srsf1-cKO mice developed significant proteinuria compared with WT mice (Figure 1F). These results demonstrate that the loss of SRSF1 in T cells leads to systemic autoimmunity and lupus-like disease. Figure 1 Srsf1-cKO mice develop systemic autoimmunity and lupus nephritis. (A) Heatmaps show autoantibody microarray data from sera of 12-week-old WT and Srsf1-cKO mice (n = 3 each) and 1 MRL/lpr mouse, analyzed for IgG reactivity against a 128-autoantigen panel. (B) Spleen images from aged (12- to 18-month-old) WT and Srsf1-cKO mice. Data are representative of at least 4 independent experiments. (C) Flow cytometry plots and graphs show Ki67 staining on live-gated CD4+ and CD8+ T cells from spleen of WT and Srsf1-cKO mice (2- to 18-month-old, n = 11 [WT], n = 8 [KO]). (D) Representative light microscopy images of H&E-stained paraffin sections of kidneys from aged (9- to 18-month-old) mice. Original magnification ×40. Arrows represent mesangial and endocapillary proliferation (upper) and interstitial inflammatory cell infiltration (lower). (E) Graph shows semiquantitative scores for kidney histopathology (n = 12 [WT], n = 9 [KO]). (F) Graph shows proteinuria from WT and Srsf1-cKO mice (n = 7 each). Mann-Whitney U test (E); unpaired t test (C and F); *P < 0.05, ***P < 0.005. T cells from Srsf1-cKO mice exhibited a hyperactive phenotype and produced proinflammatory cytokines. Examination of peripheral lymphoid T cells from spleen revealed significantly increased proportions of activated CD69hi cells (Figure 2, A and B), and effector/memory CD44hiCD62Llo and CD44hiCD62Lhi populations in the CD4 and CD8 compartments (Figure 2, C and D). In parallel, we recorded a significant reduction in the naive T cell pool in both CD4 and CD8 compartments (Figure 2E). Further, we found increased proportions of IL-17–, IFN-γ–, and IL-4–producing CD4+ T cells, and IFN-γ–producing CD8+ T cells from Srsf1-cKO mice (Figure 2F). In addition, we found that the frequencies of CD4+ CXCR5+PD1+ T follicular helper (Tfh) cells were increased in Srsf1-cKO mice (Figure 2G). We also detected the increased presence of spontaneous germinal centers in the spleens from Srsf1-cKO mice (Supplemental Figure 2D). We found that the frequencies of GL7+Fas+ germinal center B cells were increased in Srsf1-cKO mice (Supplemental Figure 2E). In addition, IgD– memory/plasma B cells were also increased in Srsf1-cKO mice (Supplemental Figure 2F). There was no difference in the frequencies and absolute numbers of B cells, CD11b+ cells, and CD11c+ cells in spleen of control WT and Srsf1-cKO mice (Supplemental Figure 3, A and B). The frequencies of CD4+CD25+FoxP3+ Treg cells in Srsf1-cKO mice were comparable with those of control mice (Supplemental Figure 4, A and B) and suppressed the proliferation of conventional T (Tconv) cells normally in in vitro Treg suppression assays for 3 days (Supplemental Figure 4, C and D). These results show that loss of SRSF1 induces the development of activated, inflammatory cytokine-producing T cells. Figure 2 T cells from Srsf1-cKO mice have a hyperactive phenotype and produce proinflammatory cytokines. Spleen cells were isolated from WT and Srsf1-cKO mice and analyzed by flow cytometry. (A) Plots show CD69 expression gated on live CD4+ T cells. (B) Graph shows percent of CD69+CD4+ T cells (n = 13 [WT], n = 14 [KO]; < 20-week-old mice). (C) Plots show CD62L and CD44 staining gated on live CD4+ and CD8+ T cells in spleen (D) Graphs show percent of activated effector/ memory subsets (CD44hiCD62Lhi and CD44hiCD62Llo) of CD4+ and CD8+ T cells (CD4+: n = 11 [WT], n = 13 [KO]; CD8+: n = 18 [WT], n = 16 [KO]; < 20-week-old mice). (E) Graphs show percentage of naive (CD44loCD62Lhi) CD4+ and CD8+ T cells (CD4+: n = 11 [WT]; n = 13 [KO]; CD8+: n = 18 [WT], n = 16 [KO]; < 20-week-old mice). (F) Spleen cells were stimulated for 4 hours with PMA, ionomycin with monensin followed by surface and intracellular cytokine staining for flow cytometry. Plots show IL-17, IL-4, and IFN-γ staining gated on live CD4+ and CD8+ T cells. Graphs below show percentage of cytokine-producing CD4+ and CD8+ T cells from spleen (IL-17: n = 8 each; IL-4: n = 6–7; CD4+ IFN-γ: n = 13 each; CD8+ IFN-γ: n = 3–4; CD4+: < 20-week-old mice; CD8+: 9- to 40-week-old mice). (G) Splenocytes from WT or Srsf1-cKO mice were stained for PD1 and CXCR5 by flow cytometry. Plots show cells gated on live CD4+ T cells. Graph below shows percentage of Tfh cells (n = 20 [WT]; n = 21 [KO]; 10- to 28-week-old mice). Unpaired t test, *P < 0.05, **P < 0.005. Transcriptomics analysis of CD4+ Teff cells from Srsf1-cKO mice revealed an elevated T cell activation gene signature. Because the Srsf1-cKO mice develop autoimmune inflammatory disease, and exhibit increased proportions of proinflammatory Teff cells, we asked whether SRSF1 controls the genes and pathways involved in T cell activation and differentiation. We performed transcriptomics analysis by RNA-sequencing of CD4+ Teff cells derived by stimulation of naive CD4+ T cells with anti-CD3 and anti-CD28 for 72 hours, from 7-week-old control WT or Srsf1-cKO mice with 3 mice per group (Figure 3 and Table 1). At the fold change greater than 1, and a P value less than 0.05, 890 genes were upregulated and 1021 genes downregulated in Srsf1-cKO mice compared with control mice, whereas at the fold change greater than 2 cutoff, and a P value less than 0.05, 312 genes were upregulated and 300 genes were downregulated in Srsf1-cKO mice compared with normal mice (Figure 3A). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed the top pathways to include genes associated with T cell activation, regulation of protein secretion, cytokine-cytokine receptor interaction, cell cycle, Th17 differentiation, and Th1 and Th2 differentiation (Table 1). Cluster analysis revealed predominantly genes involved in T cell activation, protein secretion, cytokine production, ribosome assembly, cell cycle, and chromatin modifications (Figure 3B). The expression levels of multiple cytokines including Ifng, Il17, Il4, and Il21 were found to be increased (Figure 3C). Because IL-21 is known to play important roles in Tfh differentiation, the increased Il21 expression may contribute to the observed increase in Tfh cells in Srsf1-cKO mice. These data indicate that SRSF1 controls genes involved in T cell activation, differentiation, and cytokine production, and the loss of SRSF1 leads to unrestrained activation and proinflammatory phenotype of T cells. Figure 3 Transcriptomics analysis of CD4+ Teff cells from Srsf1-cKO mice reveals an elevated T cell activation gene signature. (A) CD4+ Teff cells were generated as follows. Naive CD4+ T cells were sorted from spleens of WT and Srsf1-cKO mice (n = 3 each), and stimulated with anti-CD3 (0.5 μg/mL) and anti-CD28 (1.0 μg/mL) for 72 hours. RNA-sequencing data analysis shows differentially expressed (DE) genes with fold change (FC) differences at P < 0.05. (B) GO terms enrichment map of DE genes. The size of the red circles indicates the number of genes within a given pathway, and the color represents the P values relative to the other displayed terms. Outlines (added manually) indicate groups of similar GO terms. (C) Heatmap showing average expression of DE cytokine genes in WT and Srsf1-cKO mice. Table 1 Pathway analysis of RNA-sequencing of CD4+ Teff cells from Srsf1-cKO mice T cells from Srsf1-cKO mice displayed increased activation of the mTORC1 pathway and reduced expression of PTEN. It is known that Pten-deficient mice exhibit features of T cell hyperactivity with increased proinflammatory cytokines and autoimmunity (27). Further, activation of the mTOR pathway is associated with hyperactive T cell responses with inflammatory cytokine production in patients with SLE (15, 21, 22). In our transcriptomics analysis from Srsf1-cKO mice, a number of genes of the PI3K/AKT/mTOR pathway were differentially expressed, and the expression of its repressor PTEN was found to be downregulated (Figure 4A). Given these findings, we examined the activity of the mTOR pathway in the Srsf1-cKO mice. We found increased expression of phosphorylated S6 (pS6) protein levels in CD4+ Teff cells derived by stimulation of naive CD4+ T cells with anti-CD3 and anti-CD28 for 72 hours, indicating an increased activity of the mTORC1 pathway (Figure 4B). It was previously reported that SRSF1 regulates the splicing of S6K1, an mTORC1 downstream signaling molecule, and modulates the mTOR pathway in cell lines (41). We found that deletion of Srsf1 in T cells results in the decrease of the short isoform of S6K1 (Supplemental Figure 5), consistent with previous reports. Although this short isoform of S6K1 associates with activation of the mTORC1 pathway (42), in the present study we found that serine-threonine kinase AKT, upstream of S6K1, is activated in CD4+ Teff cells from Srsf1-cKO mice (Figure 4C). Therefore, we hypothesized that decreased PTEN levels result in the activation of mTOR pathway. We confirmed the decreased mRNA (Figure 4D) and protein (Figure 4E) expression levels of PTEN in T cells from Srsf1-cKO mice. Because SRSF1 controls gene expression at the posttranscriptional level, which is frequently mediated through the 3′ UTR, we investigated the effect of SRSF1 on the 3′ UTR activity of PTEN. Cotransfection of a PTEN 3′ UTR–luciferase construct along with an Srsf1-expression vector in HEK293T cells revealed an increase in PTEN 3′ UTR activity (Figure 4F). Transfection of the PTEN 3′ UTR into freshly isolated total T cells from Srsf1-cKO mice led to decreased 3′ UTR activity, which was rescued by the overexpression of SRSF1 (Figure 4G). These results show that SRSF1 controls the expression of PTEN and loss of SRSF1 leads to reduced PTEN expression and increased activity of the mTOR pathway. Figure 4 T cells from Srsf1-cKO mice exhibit increased activation of the mTORC1 pathway and reduced expression of PTEN. (A) Fold expression of the mTOR pathway genes from RNA-sequencing data. (B–D) Naive CD4+ T cells were isolated from spleens of mice, and stimulated with anti-CD3 (0.5 μg/mL) and anti-CD28 (1.0 μg/mL) for 72 hours. (B) Total protein was immunoblotted for pS6 and total S6. Data are from 1 representative of 3 independent experiments. Graph shows relative densitometry quantitation of pS6 and S6 (n = 3 each). (C) After 72 hours, additional stimulation with anti-CD3 (10 μg/mL) and anti-CD28 (10 μg/mL) for 5 minutes was performed. Total protein was immunoblotted for p-AKT and total AKT. Data are from 1 representative of 4 independent experiments. Graph shows relative densitometry quantitation of p-AKT and AKT (n = 4 each). (D) Total RNA was isolated and reverse transcribed. Pten expression was measured by real-time qPCR and normalized to housekeeping gene cyclophilin A (n = 5 each). (E) Total protein was isolated from total T cells and immunoblotted for PTEN and β-actin. Data are from 1 representative of 4 independent experiments. Graph shows densitometric quantitation of PTEN normalized to β-actin (n = 4 each). (F) HEK293T cells were transfected with a pGL3–PTEN 3′ UTR luciferase plasmid and cotransfected with either a pcDNA3.1 empty vector (EV) or an Srsf1 (pSrsf1) plasmid, and included an internal control pRL-TK luciferase plasmid. Cells were lysed and dual luciferase assays performed. Graph shows relative luciferase units normalized to the internal luciferase control from n = 4 in 4 independent experiments. (G) T cells from spleens of mice were transfected with a PTEN-3′ UTR luciferase plasmid and either EV or pSrsf1 plasmid. Twenty-four hours later, cells were lysed and luciferase activity measured as in F (n = 7). Unpaired t test (B–E); 1-way ANOVA with Bonferroni’s correction (F and G); *P < 0.05, **P < 0.005, ***P < 0.0005; mean ± SEM. Rapamycin treatment reduced proinflammatory cytokine production by T cells and alleviated autoimmunity in Srsf1-cKO mice. Recently, hyperactivity of the mTOR pathway has been associated with aberrant T cell responses in SLE (15, 43), and targeted inhibition of this pathway with the lipophilic macrolide antibiotic rapamycin has led to improved T cell function and shown efficacy in a recent clinical trial in patients with active SLE (44). To assess whether the increased mTORC1 activation of T cells from Srsf1-cKO mice contributes to their proinflammatory phenotype and autoimmune phenotype, we examined the effect of rapamycin. Treatment of spleen cells from Srsf1-cKO mice ex vivo with rapamycin reduced the production of proinflammatory cytokines IL-17 (Figure 5, A and B) and IFN-γ (Figure 5, C and D) from CD4+ T cells. Furthermore, to assess the effects of rapamycin on disease in vivo, we treated Srsf1-cKO mice with rapamycin and evaluated the effect on features of autoimmunity. We observed reduced levels of IL-17 cytokine production ex vivo (Figure 5E), and decreased proportions of CD4+ PD1+CXCR5+ Tfh cells in the spleens from cKO mice (Figure 5F). Importantly, we observed a reduction in the levels of serum dsDNA and histone autoantibodies (Figure 5G). In addition, treatment with rapamycin reduced T cell infiltration into the kidneys (Figure 5H). These data indicate that deficiency of SRSF1 leads to increased activation of the mTOR pathway, the ensuing proinflammatory function of T cells, and systemic autoimmunity, which can be corrected by rapamycin. Figure 5 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 < 0.05, **P < 0.005; mean ± SEM. PTEN overexpression reduced proinflammatory cytokine production by T cells from Srsf1-cKO mice. To confirm that SRSF1 modulates T cell activation via regulation of PTEN expression, PTEN was overexpressed by transient transfections in T cells from WT and Srsf1-cKO mice, followed by stimulation with PMA and ionomycin for 4 hours. We confirmed the overexpression of PTEN at the mRNA and protein levels (Supplemental Figure 6, A and B). We found that cytokine production is decreased by PTEN overexpression in T cells from Srsf1-cKO mice but not in WT T cells (Figure 6, A and B). Of note, transfection by electroporation is toxic to cells and accounts for the overall reduced frequencies of IFN-γ–producing cells (Figure 6) compared with untransfected cells (Figure 5C and Supplemental Figure 6C). These data indicate that deficiency of SRSF1 leads to decreased PTEN expression, increased activation of the mTOR pathway and the ensuing proinflammatory function of T cells (Supplemental Figure 7) which contribute to systemic autoimmune disease. Figure 6 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 < 0.05, mean ± SEM. PTEN is decreased in T cells from patients with SLE with low SRSF1 levels. Although PTEN expression is decreased and contributes to the hyperactivity of B cells in patients with SLE (28), its role in T cells in SLE has not been studied. Given our findings of increased mTOR activity and the decreased expression and regulatory role of PTEN in T cells from the Srsf1-cKO mice (Figure 4, Figure 5, and Figure 6), we asked whether PTEN expression levels are altered in T cells from patients with SLE. Therefore, we assessed PTEN and SRSF1 protein expression levels in T cells from patients with SLE and compared with age-, race-, and sex-matched healthy control individuals. We found that the expression of PTEN was decreased in T cells from patients with SLE who had decreased expression levels of SRSF1, compared with those from healthy individuals (Figure 7A). Importantly, we observed a linear correlation between PTEN and SRSF1 expression levels in T cells from patients with SLE (R2 = 0.226, P = 0.0163, Figure 7B). These results suggest that the low SRSF1 levels may contribute to the reduced expression of PTEN in T cells from patients with SLE. Figure 7 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 < 0.05, mean ± SEM. The mTOR pathway via the regulation of PTEN expression. In T cells from KO mice and patients with SLE, reduced SRSF1 levels contribute to reduced PTEN levels and increased activity of the mTOR pathway. Rapamycin blocks the mTORC1 pathway and reduces proinflammatory cytokines. SRSF1 overexpression increased PTEN levels, suppressed mTORC1 activity, and reduced proinflammatory cytokine production in T cells from patients with SLE. To assess whether SRSF1 overexpression would increase PTEN expression and reduce mTORC1 activity and the aberrant cytokine production by T cells from patients with SLE, we transiently transfected T cells from patients with SLE with an Srsf1-expression vector followed by stimulation with anti-CD3 and anti-CD28. We found that SRSF1 overexpression increased PTEN protein levels (Figure 7C), reduced activation of the mTORC1 pathway as evidenced by a reduction in pS6 expression levels (Figure 7D), and led to reduced frequencies of IL-17– and IFN-γ–producing CD4+ T cells (Figure 7E). These findings show that decreased SRSF1 expression contributes to mTORC1 activation and proinflammatory cytokine production in T cells from patients with SLE through the control of PTEN. Discussion In this study, we demonstrate that selective deletion of SRSF1 in T cells in mice leads to aberrantly activated proinflammatory cytokine-producing Teff cells, leading to systemic autoimmunity and lupus nephritis (Figure 1 and Figure 2). We report that SRSF1 controls the expression of genes involved in T cell activation, differentiation, and cytokine signaling (Figure 3). At the mechanistic level, we demonstrate that SRSF1 regulates the 3′ UTR of the mRNA of the mTOR inhibitor PTEN and limits its expression, causing increased mTOR activity (Figure 4). Rapamycin treatment corrects proinflammatory cytokine production in T cells and alleviates autoimmunity in Srsf1-deficient mice (Figure 5). Of clinical relevance is our finding that T cells from patients with SLE with low expression levels of SRSF1 had decreased levels of PTEN, which correlate with decreased expression levels of SRSF1 (Figure 7). Further, restitution of SRSF1 levels corrected the proinflammatory cytokine production by hyperactive SLE T cells (Figure 7). Thus, we uncover SRSF1 as what we believe is a novel molecular brake in the control of T cell hyperactivity and the prevention of autoimmune disease. Our findings highlight an important role for SRSF1 in the immune system and in the control of immune-mediated disease. SRSF1 deficiency unrestrains T cell activation, unleashes the production of proinflammatory cytokines IL-17 and IFN-γ by CD4+ Teff cells, and leads to activation of B cells and inflammatory infiltration in target organs, leading to systemic autoimmunity and organ inflammation. Increased activity of the mTOR pathway contributes to the proinflammatory phenotype, which is rescued by rapamycin administration. The mTOR pathway is a recently emerging crucial therapeutic target in autoimmune diseases, including SLE (15, 43). Increased mTORC1 activity has been demonstrated in T cells from patients with SLE and correlates with expansion of proinflammatory cytokine-producing T cells (21, 22). Treatment of lupus-prone MRL/lpr mice with rapamycin, an inhibitor of mTORC1 signaling, improved disease parameters (45). Importantly, a recent clinical trial of rapamycin in patients with SLE has shown efficacy in patients with active SLE (44). Here, we find that SRSF1 deficiency in mice results in increased activation of mTORC1 pathway, which parallels the observed increased mTORC1 activity (15) and the decreased SRSF1 expression in patients with SLE (37). Although PTEN is known to suppress the mTOR pathway and the expression of PTEN is decreased in B cells from patients with SLE (28), the role of PTEN in T cells from patients with SLE remains unknown. We previously showed that the expression levels of SRSF1 are decreased in SLE T cells and inversely correlate with disease activity, suggesting the important role of SRSF1 in SLE T cells (37). Our current study extends this concept with a number of crucial new findings. Here we show a linear correlation between PTEN and SRSF1 in T cells from patients with SLE, and that patients with lupus with low SRSF1 expression levels exhibit lower expression levels of PTEN. Furthermore, the overexpression of SRSF1 rescues PTEN expression in lupus T cells (Figure 7). This is the first evidence showing the reduction of PTEN in T cells in a subset of patients with SLE. Moreover, the overexpression of SRSF1 in lupus T cells reduces mTOR activity and the production of proinflammatory cytokines, suggesting that the SRSF1-PTEN-mTOR pathway may be a novel therapeutic target in patients with SLE. T cell quiescence is enforced by molecules able to restrain T cell activation, and the expression of such molecules generally subsides upon TCR stimulation to enable effector differentiation programs. These genes include, among others, the Kruppel-like factor (KLF) genes, Schlafen-2, Tsc2, and the Forkhead box (Foxo) family (46). T cells from the Srsf1-cKO mice preferentially expressed genes involved in the Foxo1 signaling pathway, cell cycle, T cell activation, cytokine-cytokine receptor interaction, and Th differentiation programs. Therefore, it appears that SRSF1 may be involved in the maintenance of resting T cell quiescence. In agreement with these findings is our previous finding that high SRSF1 protein levels in resting human T cells decreased following TCR stimulation due to degradation by the ubiquitin-proteasome pathway (36). In contrast, deficiency or decreased expression of SRSF1 as recorded in our Srsf1-cKO mice and in patients with SLE, respectively, contributes to T cell activation, loss of tolerance, autoimmunity, and organ inflammation. Of direct clinical relevance is the observation that T cells from patients with SLE exhibit a hyperactive phenotype and functional defects (13), which are linked mechanistically to decreased SRSF1 expression (37). A limitation of our study is that the T cell–restricted deletion of Srsf1 is under the distal Lck promoter, which is expressed late during thymic development, as opposed to the proximal Lck promoter, which is expressed early in the double-negative thymocytes. Therefore, the role of SRSF1 in early thymic development is bypassed, which may differentially influence T cell physiology and function. Also, Srsf1 is absent from birth and does not address the possibility of altering T cell function should its levels decrease later in life. Deletion of Srsf1 in adult mice may result in differential physiological and pathophysiological outcomes. SRSF1 levels are decreased in T cells from patients with SLE but are not completely absent. Therefore, while the homozygous Srsf1-cKO mice are used in proof-of-concept studies here, the heterozygous (distal.Lckcre.Srsf1flox/wt mice) Srsf1-cKO mice, which also develop some features of T cell activation and signs of autoimmune disease (unpublished observations), may serve as a better model for human disease in future studies. Because SRSF1 regulates target genes via various mechanisms, it is possible that other pathways also contribute to T cell activation and autoimmunity. In addition, although the frequency of Tregs and functional results from short-term in vitro suppression assays are comparable between WT and Srsf1-cKO mice, further studies (including generating Treg conditional Srsf1-cKO mice) are required to assess precisely the role of SRSF1 in Tregs. Furthermore, the differences observed in the distribution of activated/memory T cell populations within the CD4 and CD8 compartments imply a potentially differential role of SRSF1 in CD4+ and CD8+ T cells (Figure 2, C–E). Besides these limitations, our patient numbers in the human studies are small, and a larger cohort of patients with SLE is needed to systematically define the clinical characteristics of patients stratified based on molecular levels of SRSF1 and PTEN. In addition, whether T cells from patients with SLE with and without low SRSF1 expression exhibit differential response to rapamycin needs to be examined. Increased activation of the mTOR pathway is associated with altered metabolism in T cells in mice and in patients with SLE (47, 48). It would be interesting to assess whether Srsf1-deficient T cells exhibit such defects. In conclusion, we have uncovered what we believe is a previously unrecognized role of SRSF1 in T cell physiology and demonstrated the impact of its absence on the expression of autoimmune disease. Loss of SRSF1 limits the expression of the mTOR repressor PTEN, leading to aberrant T cell activation and the expression of systemic autoimmunity. Our results suggest that prevention of the loss of SRSF1 should be of therapeutic value to autoimmune diseases. Methods Human subjects. Patients with SLE, all fulfilling the American College of Rheumatology classification criteria (49), were recruited at the rheumatology clinic at Beth Israel Deaconess Medical Center (BIDMC), Boston, MA (Supplemental Table 1). Age-, race-, and sex-matched healthy individuals were recruited as controls. Peripheral blood was drawn from patients by venipuncture. Mice. C57BL/6J (stock 000664), B6.129S4-Srsf1-flox (stock 018020), and B6.dLck.Cre (stock 012837) mice were purchased from The Jackson Laboratory. In most experiments, sex-matched littermates were used as WT controls. All mice were maintained in the specific pathogen–free animal facility at BIDMC. Antibodies and reagents. The following flow cytometry antibodies were from Biolegend: anti–mouse-CD4 (clone GK1.5), CD8a (clone 53-6.7), CD11b (clone M1/70), CD11c (clone N418), CD19 (clone 6D5), CD25 (clone PC61), CD44 (clone IM7), B220 (clone RA3-6B2), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD90.2 (clone 53-2.1), TCR-β (clone H57-597), IgD (clone 11-26c.2a), IL-4 (clone 11B11), IL-17A (clone JC11-18F10.1), IFN-γ (clone XMG1.2), CXCR5 (clone L138D7), Ki67 (clone 16A8), CD127 (clone A019D5), PD-1 (clone 29F.1A12), GL7 (clone GL7), purified anti–mouse-CD3 (clone 145-2C11), CD28 (clone 37.51), CD16/32 (Fc block), anti–human CD3 (clone OKT3), CD4 (clone OKT4), CD8 (clone SK1), and IFN-γ (clone 4S.B1). Brilliant Violet 421–conjugated streptavidin, Zombie Aqua Fixable Viability kit mouse IL-17A ELISA kit, and carboxyfluorescein succinimidyl ester (CFSE) were also from Biolegend. Anti-mouse Foxp3 (clone FJK-16s) antibody, mouse regulatory T cell staining kit, and anti–human IL-17A antibody (clone eBIO64DEC17) were from eBioscience. Anti–mouse Fas (clone Jo2) antibody, Cytofix/Cytoperm, Perm/Wash buffer, and monensin were from BD Biosciences. ACK lysing buffer was from Thermo Fisher Scientific. SRSF1 rabbit polyclonal antibody for flow cytometry was from LifeSpan Biosciences and monoclonal antibody for Western blot (clone 96) was from Thermo Fisher Scientific. Anti-S6 ribosomal protein phospho-Ser 235 and Ser 236 (clone D57.2 2E), anti-S6 ribosomal protein (clone 5G10), anti-PTEN (clone 138G6), anti–phospho-AKT (Ser473; clone D9E), anti–phospho-AKT (Thr308; clone 244F9), and anti-AKT (clone C67E7) antibodies were from Cell Signaling Technology. Phorbol myristic acid (PMA), ionomycin, mitomycin C, and β-actin antibody (clone AC-74) were from Sigma-Aldrich. Goat anti–rabbit IgG horseradish peroxidase (HRP) and goat anti–mouse IgG HRP antibodies were from Thermo Fisher Scientific. Rapamycin and goat anti–mouse IgG were from Millipore. HEK293T cells were purchased from American Type Culture Collection (ATCC). The pcDNA3.1-SRSF1 expression plasmid was a gift from James Manley (Columbia University, NY), and the pGL3-PTEN 3′ UTR plasmid was a gift from Jeffrey Rosen (Addgene plasmid 28104). The pcDNA3-FLAG PTEN plasmid was a gift from Jaewhan Song (Addgene plasmid 78777). Cell isolation and tissue processing. Spleens and lymph nodes were homogenized using a syringe plunger and mesh cell strainer. After perfusion, kidneys were homogenized and digested in HBSS medium with 1.5 mg/mL collagenase B (Boehringer Mannheim) at 37°C for 30 minutes. RBC lysis was performed with ACK lysing buffer. All cell cultures were in RPMI complete medium (RPMI plus 10% FBS plus penicillin and streptomycin antibiotics). Blood samples from mice were collected by tail vein incision and capillary tube collection, and serum was separated by centrifugation. Urine samples were collected from mice in individual metabolic cages and urine protein was measured by Multistix (Siemens). Human T cells from peripheral blood were isolated using the Rosette Sep human T cell enrichment cocktail (STEMCELL Technologies). For histopathology, tissues were immediately fixed in 10% formalin overnight, processed in an automated tissue processor, embedded into paraffin blocks, and sent to the BIDMC histopathology core for sectioning and slide staining with H&E. Slides were evaluated blind for histopathology scoring, utilizing semiquantitative scoring criteria for lupus nephritis (50). Flow cytometry. Zombie aqua viability dye was used for live/dead cell staining. Surface staining was performed in FACS staining buffer (PBS plus 2% FBS) on ice for 20 minutes with Fc block. For cytokine production, cells from mice were stimulated for 4 hours in culture medium with PMA (100 ng/mL), ionomycin (1 μM), and monensin (1 μL/mL). Human T cells were stimulated with PMA (10 ng/mL), ionomycin (400 ng/mL), and monensin (1 μL/mL) for 4 hours. Cells were surface stained followed by fixation and permeabilization. Appropriate antibodies were used for intracellular staining for cytokines or Foxp3 and Ki67. Flow cytometry data were acquired on a BD LSRII or CytoFLEX LX and analyzed with FlowJo software. All procedures were performed according to the manufacturer’s instructions. Autoantibody detection. Sera from 12-week-old WT and Srsf1-cKO mice and one 16-week-old MRL/lpr mouse (as positive control) were analyzed for the presence of autoantibodies using the Autoantigen Microarray Super Panel (128-antigen panel) at the Genomics and Microarray Core facility at the University of Texas, Southwestern Medical Center (Dallas, TX). Heatmaps were prepared using R software. Antinuclear antibody (ANA) in serum was measured using an ELISA kit (Alpha Diagnostic International) following the manufacturer’s instructions. Briefly, the serum was diluted and assayed for antigen reactivity on precoated plates followed by incubation for 60 minutes. Bound IgG was detected with anti–mouse IgG HRP antibody and developed with a tetramethylbenzidine (TMB) substrate. Plates were read at 450 nm on a spectrophotometer. To detect anti–double stranded (ds)DNA and anti–histone antibodies, Immulon II plates (Dynatech) precoated with BSA were coated individually with 50 μg/mL calf thymus DNA (Sigma-Aldrich) or 50 μg/mL calf thymus histone (Sigma-Aldrich). The serum was diluted (1:500) and assayed for autoantigen reactivity against the antigens described above by incubation overnight at 4°C. Bound IgG was detected with a goat polyclonal HRP–anti-mouse IgG detection antibody (SouthernBiotech) and visualized at 450 nm using a TMB substrate (Sigma-Aldrich). mRNA expression and RT-PCR. Total RNA was isolated using the RNeasy mini kit (Qiagen) and reverse-transcribed into cDNA using the RNA to cDNA premix (Clontech). Real-time PCR amplification was carried out with SYBR Green I using a LightCycler 480 (Roche) and the following program: initial denaturation at 95°C for 5 minutes; 40 cycles of amplification (denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 30 seconds); one cycle of melting curves (95°C for 15 seconds, 65°C for 2 minutes, and 97°C continuous), and a final cooling at 37°C. Threshold cycle values were used to calculate relative mRNA expression by the ΔCt relative quantification method. Primer sequences are listed in Supplemental Table 2. RNA sequencing. Naive CD4+ T cells were isolated from spleens of 7-week-old WT and Srsf1-cKO mice (n = 3 each) and stimulated with anti-CD3 (0.5 μg/mL) and anti-CD28 (1 μg/mL) for 72 hours. Cells were restimulated with PMA (100 ng/mL) and ionomycin (1 μM) for 4 hours. Total RNA was extracted using the RNeasy mini kit (Qiagen) and submitted for sequencing to the Molecular Biology Core Facility (MBCF) at the Dana-Farber Cancer Institute (DFCI). Libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits according to the manufacturer’s protocols. Samples were sequenced on an Illumina NextSeq500 run with single-end 75-bp reads. Data analyses were performed by the MBCF at DFCI and the Harvard T.H. Chan School of Public Health Biostatistics Core. The data were deposited in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE 136286. Western blot. To detect pS6, naive CD4+ T cells were isolated from spleens of 9- to 10-week-old WT and Srsf1-cKO mice and stimulated with anti-CD3 (0.5 μg/mL) and anti-CD28 (1 μg/mL) for 72 hours. Human T cells were stimulated with soluble anti-CD3 (5 μg/mL), anti-CD28 (2.5 μg/mL) and cross linker goat anti–mouse IgG (10 μg/mL) for 5 minutes. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Boston Bioproducts). Lysates were electrophoresed on NuPAGE 4%–12% Bis-Tris gels (Life Technologies) and transferred to PVDF membrane. Membranes were blocked with 5% (wt/vol) nonfat milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1 hour, incubated with primary antibody (1:1000, or 1:10,000 for β-actin antibody) in 5% milk in TBS-T or Hikari solution A (Nacalai Tesque) at 4°C overnight or at room temperature for 2 hours for β-actin antibody. Membranes were washed 3 times with TBS-T, incubated with HRP-conjugated secondary antibody (1:2000 for ECL detection reagents or 1:4000 for ECL prime detection reagents; GE Healthcare) for 1 hour, washed 3 times with TBS-T, developed with ECL reagents, and visualized by a Fujifilm LAS-4000 imager or Bio-Rad ChemiDoc imager. Densitometry was performed using Quantity One software (Bio-Rad). Transfections. HEK293T cells were cultured in DMEM complete medium. One day prior to transfection, 0.3 × 106 cells were seeded in a 12-well plate. Cells were cotransfected with the pGL3-PTEN 3′ UTR luciferase plasmid and either pcDNA3.1 empty vector (EV) or pcDNA3.1-Srsf1 vector using Lipofectamine (Invitrogen). All transfections included a pRL-TK Renilla luciferase plasmid as internal control. Human T cells and mouse T cells were transfected using the Amaxa human T cell nucleofector kit and the mouse T cell nucleofector kit, respectively, following the manufacturer’s instructions. Briefly, 3 × 106 to 6 × 106 cells were resuspended in 100 μL nucleofector solution, plasmid DNA was added, and cells were transferred into a cuvette and electroporated using the U-014 program for human cells or the X-001 program for mouse cells in the nucleofector device. Cells were immediately rescued into prewarmed medium and cultured overnight. Luciferase assays. Cells were transfected as described above. After 24 hours in culture, cells were collected, lysed in passive lysis buffer (Promega), and luciferase activity was quantified on a luminometer using the dual-luciferase assay system (Promega) according to the manufacturer’s instructions. Immunofluorescence and immunohistochemistry staining. For immunofluorescence staining, spleens were embedded in OCT tissue media (Tissue-Tek) and frozen on dry ice. Frozen sections (7-μm thickness) were fixed to slides in ice-cold acetone for 15 minutes and air dried for 30 seconds. The sections were blocked with 10% horse serum for 30 minutes at room temperature, then stained for 30 minutes at room temperature in a humidified chamber with fluorescence-conjugated anti-IgD (catalog 405717; Biolegend), anti-CD4 (catalog 100533; Biolegend), anti-B220 (catalog 103207; Biolegend), and biotinylated PNA (catalog B-1075; Vector Laboratories). Biotinylated PNA was detected using by streptavidin–Alexa Fluor 350 (catalog S-11249; Life Technologies). For immunohistochemistry staining, slides of formalin-fixed, paraffin-embedded kidneys were incubated with purified anti–mouse CD3 antibody (clone 100201; Biolegend) overnight at 4°C after antigen retrieval. The primary antibody was detected with rabbit anti–rat HRP secondary antibody (clone ab6734, Abcam). In vitro suppression assays. Total T cells were isolated from spleens by magnetic assisted cell sorting (MACS), using the Pan T cell isolation kit (Miltenyi Biotech). Conventional CD4+ T cells (Tconv) (CD4+CD25–CD127hi) and Treg cells (CD4+CD25+CD127lo) were sorted by flow cytometry (FACSAria II, BD Bioscience). Tconv cells were labeled with CFSE and cocultured with Treg cells at increasing ratios for 3 days in the presence of mitomycin C–treated splenocytes and anti-CD3 and anti-CD28, and proliferation of Tconv was analyzed by flow cytometry. Rapamycin treatment. Rapamycin was dissolved in DMSO and diluted in PBS. For in vitro culture treatment experiments, a final concentration of 1 nM or 10 nM rapamycin was added for the duration of cell cultures. For in vivo treatment experiments, rapamycin (2 mg/kg dissolved in DMSO, diluted in 150 μL PBS) or PBS (equal volume DMSO in 150 μL PBS) was administered to mice by intraperitoneal injection once every 2 days for 4 weeks. After 4 weeks of treatment, mice were euthanized and spleen cells were analyzed by flow cytometry, and sera was collected to measure autoantibodies by ELISA. In some experiments, mice were treated with rapamycin every day for 1 week. Kidneys were collected to analyze T cell infiltration by flow cytometry. Spleen cells were cultured with anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) for 24 hours and IL-17A was measured in supernatants by ELISA (Biolegend). Statistics. Student’s 2-tailed t test, Mann-Whitney U test, and 1-way ANOVA were used to calculate statistical significance among groups. A P value of less than 0.05 was considered significant. In all graphs, data represent mean ± SEM. Study approval. For human studies, written informed consent was obtained from all participants and all studies were approved by the institutional review board (Committee on Clinical Investigations) at BIDMC. All animal studies were approved by the Institutional Animal Care and Use Committee at BIDMC. Author contributions VRM conceptualized the study. TK, HL, DC, and VRM designed and performed the experiments, and analyzed and interpreted data. TK, HL, GCT, and VRM prepared the manuscript. Supplemental material View Supplemental data Acknowledgments We thank Andrew Gillooly, Michael Mosho, and Melissa Carr-Reynolds for excellent technical assistance; Suzanne Krishfield for recruiting patients with SLE and healthy individuals; Donna Farber and Martin Flajnik for critical reading of the manuscript; and Amir Sharabi and Abel Suarez-Fueyo for helpful discussions. This work was funded by NIH grants K01AR060781 and R01AR068974 to VRM, R01AI42269 to GCT, and an SICPA Foundation grant to DC. Footnotes Conflict of interest: The authors have declared that no conflict of interest exists. Copyright: © 2019, American Society for Clinical Investigation. Reference information: J Clin Invest. https://doi.org/10.1172/JCI127949. References Tsokos GC. Systemic lupus erythematosus. N Engl J Med. 2011;365(22):2110–2121. View this article via: PubMed CrossRef Google Scholar Yen EY, Singh RR. Brief report: lupus-an unrecognized leading cause of death in young females: a population-based study using nationwide death certificates, 2000-2015. Arthritis Rheumatol. 2018;70(8):1251–1255. View this article via: PubMed CrossRef Google Scholar Malkiel S, Barlev AN, Atisha-Fregoso Y, Suurmond J, Diamond B. Plasma cell differentiation pathways in systemic lupus erythematosus. Front Immunol. 2018;9:427. View this article via: PubMed Google Scholar Banchereau J, Pascual V. 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One of the more perplexing questions in biomedical research is—why does the body's protective shield against infections, the immune system, attack its own vital cells, organs, and tissues? The answer to this question is central to understanding an array of autoimmune diseases, such as rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus, and Sjogren's syndrome. When some of the body's cellular proteins are recognized as "foreign" by immune cells called T lymphocytes, a destructive cascade of inflammation is set in place. Current therapies to combat these cases of cellular mistaken identity dampen the body's immune response and leave patients vulnerable to life-threatening infections. Research on stem cells is now providing new approaches to strategically remove the misguided immune cells and restore normal immune cells to the body. Presented here are some of the basic research investigations that are being guided by adult and embryonic stem cell discoveries. Introduction The body's main line of defense against invasion by infectious organisms is the immune system. To succeed, an immune system must distinguish the many cellular components of its own body (self) from the cells or components of invading organisms (nonself). "Nonself" should be attacked while "self" should not. Therefore, two general types of errors can be made by the immune system. If the immune system fails to quickly detect and destroy an invading organism, an infection will result. However, if the immune system fails to recognize self cells or components and mistakenly attacks them, the result is known as an autoimmune disease. Common autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosis (lupus), type 1 diabetes, multiple sclerosis, Sjogren's syndrome and inflammatory bowel disease. Although each of these diseases has different symptoms, they share the unfortunate reality that, for some reason, the body's immune system has turned against itself (see Box 6.1. Immune System Components: Common Terms and Definitions). How Does the Immune System Normally Keep Us Healthy? The "soldiers" of the immune system are white blood cells, including T and B lymphocytes, which originate in the bone marrow from hematopoietic stem cells. Every day the body comes into contact with many organisms such as bacteria, viruses, and parasites. Unopposed, these organisms have the potential to cause serious infections, such as pneumonia or AIDS. When a healthy individual is infected, the body responds by activating a variety of immune cells. Initially, invading bacteria or viruses are engulfed by an antigen presenting cell (APC), and their component proteins (antigens) are cut into pieces and displayed on the cell's surface. Pieces of the foreign protein (antigen) bind to the major histocompatibility complex (MHC) proteins, also known as human leukocyte antigen (HLA) molecules, on the surface of the APCs (see Figure 6.1 Immune Response to Self or Foreign Antigens). This complex, formed by a foreign protein and an MHC protein, then binds to a T cell receptor on the surface of another type of immune cell, the CD4 helper T cell. They are so named because they "help" immune responses proceed and have a protein called CD4 on their surface. This complex enables these T cells to focus the immune response to a specific invading organism. The antigen-specific CD4 helper T cells divide and multiply while secreting substances called cytokines, which cause inflammation and help activate other immune cells. The particular cytokines secreted by the CD4 helper T cells act on cells known as the CD8 "cytotoxic" T cells (because they can kill the cells that are infected by the invading organism and have the CD8 protein on their surface). The helper T cells can also activate antigen-specific B cells to produce antibodies, which can neutralize and help eliminate bacteria and viruses from the body. Some of the antigen-specific T and B cells that are activated to rid the body of infectious organisms become long-lived "memory" cells. Memory cells have the capacity to act quickly when confronted with the same infectious organism at later times. It is the memory cells that cause us to become "immune" from later reinfections with the same organism. Figure 6.1. Immune Response to Self or Foreign Antigens. (© 2001 Terese Winslow) How Do the Immune Cells of the Body Know What to Attack and What Not To? All immune and blood cells develop from multipotent hematopoietic stem cells that originate in the bone marrow. Upon their departure from the bone marrow, immature T cells undergo a final maturation process in the thymus, a small organ located in the upper chest, before being dispersed to the body with the rest of the immune cells (e.g., B cells). Within the thymus, T cells undergo an important process that "educates" them to distinguish between self (the proteins of their own body) and nonself (the invading organism's) antigens. Here, the T cells are selected for their ability to bind to the particular MHC proteins expressed by the individual. The particular array of MHCs varies slightly between individuals, and this variation is the basis of the immune response when a transplanted organ is rejected. MHCs and other less easily characterized molecules called minor histocompatibility antigens are genetically determined and this is the reason why donor organs from relatives of the recipient are preferred over unrelated donors. In the bone marrow, a highly diverse and random array of T cells is produced. Collectively, these T cells are capable of recognizing an almost unlimited number of antigens. Because the process of generating a T cell's antigen specificity is a random one, many immature T cells have the potential to react with the body's own (self) proteins. To avoid this potential disaster, the thymus provides an environment where T cells that recognize self-antigens (autoreactive or self-reactive T cells) are deleted or inactivated in a process called tolerance induction. Tolerance usually ensures that T cells do not attack the "autoantigens" (self-proteins) of the body. Given the importance of this task, it is not surprising that there are multiple checkpoints for destroying or inactivating T cells that might react to auto-antigens. Autoimmune diseases arise when this intricate system for the induction and maintenance of immune tolerance fails. These diseases result in cell and tissue destruction by antigen-specific CD8 cytotoxic T cells or autoantibodies (antibodies to self-proteins) and the accompanying inflammatory process. These mechanisms can lead to the destruction of the joints in rheumatoid arthritis, the destruction of the insulinproducing beta cells of the pancreas in type 1 diabetes, or damage to the kidneys in lupus. The reasons for the failure to induce or maintain tolerance are enigmatic. However, genetic factors, along with environmental and hormonal influences and certain infections, may contribute to tolerance and the development of autoimmune disease [4, 7]. Hematopoietic Stem Cell Therapy for Autoimmune Diseases The current treatments for many autoimmune diseases include the systemic use of anti-inflammatory drugs and potent immunosuppressive and immunomodulatory agents (i.e., steroids and inhibitor proteins that block the action of inflammatory cytokines). However, despite their profound effect on immune responses, these therapies are unable to induce clinically significant remissions in certain patients. In recent years, researchers have contemplated the use of stem cells to treat autoimmune disorders. Discussed here is some of the rationale for this approach, with a focus on experimental stem cell therapies for lupus, rheumatoid arthritis, and type 1 diabetes. The immune-mediated injury in autoimmune diseases can be organ-specific, such as type 1 diabetes which is the consequence of the destruction of the pancreatic beta islet cells or multiple sclerosis which results from the breakdown of the myelin covering of nerves. These autoimmune diseases are amenable to treatments involving the repair or replacement of damaged or destroyed cells or tissue (see Chapter 7. Stem Cells and Diabetes and Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). In contrast, non-organ-specific autoimmune diseases, such as lupus, are characterized by widespread injury due to immune reactions against many different organs and tissues. One approach is being evaluated in early clinical trials of patients with poorly responsive, life-threatening lupus. This is a severe disease affecting multiple organs in the body including muscles, skin, joints, and kidneys as well as the brain and nerves. Over 239,000 Americans, of which more than 90 percent are women, suffer from lupus. In addition, lupus disproportionately afflicts African-American and Hispanic women [11]. A major obstacle in the treatment of non-organ-specific autoimmune diseases such as lupus is the lack of a single specific target for the application of therapy. The objective of hematopoietic stem cell therapy for lupus is to destroy the mature, long-lived, and auto-reactive immune cells and to generate a new, properly functioning immune system. In most of these trials, the patient's own stem cells have been used in a procedure known as autologous (from "one's self") hematopoietic stem cell transplantation. First, patients receive injections of a growth factor, which coaxes large numbers of hematopoietic stem cells to be released from the bone marrow into the blood stream. These cells are harvested from the blood, purified away from mature immune cells, and stored. After sufficient quantities of these cells are obtained, the patient undergoes a regimen of cytotoxic (cell-killing) drug and/or radiation therapy, which eliminates the mature immune cells. Then, the hematopoietic stem cells are returned to the patient via a blood transfusion into the circulation where they migrate to the bone marrow and begin to differentiate to become mature immune cells. The body's immune system is then restored. Nonetheless, the recovery phase, until the immune system is reconstituted represents a period of dramatically increased susceptibility to bacterial, fungal, and viral infection, making this a high-risk therapy. Recent reports suggest that this replacement therapy may fundamentally alter the patient's immune system. Richard Burt and his colleagues [18] conducted a long-term follow-up (one to three years) of seven lupus patients who underwent this procedure and found that they remained free from active lupus and improved continuously after transplantation, without the need for immunosuppressive medications. One of the hallmarks of lupus is that during the natural progression of disease, the normally diverse repertoire of T cells become limited in the number of different antigens they recognize, suggesting that an increasing proportion of the patient's T cells are autoreactive. Burt and colleagues found that following hematopoietic stem cell transplantation, levels of T cell diversity were restored to those of healthy individuals. This finding provides evidence that stem cell replacement may be beneficial in reestablishing tolerance in T cells, thereby decreasing the likelihood of disease reoccurrence. Development of Hematopoietic Stem Cell Lines for Transplantation The ability to generate and propagate unlimited numbers of hematopoietic stem cells outside the body—whether from adult, umbilical cord blood, fetal, or embryonic sources—would have a major impact on the safety, cost, and availability of stem cells for transplantation. The current approach of isolating hematopoietic stem cells from a patient's own peripheral blood places the patient at risk for a flare-up of their autoimmune disease. This is a potential consequence of repeated administration of the stem cell growth factors needed to mobilize hematopoietic stem cells from the bone marrow to the blood stream in numbers sufficient for transplantation. In addition, contamination of the purified hematopoietic stem cells with the patient's mature autoreactive T and B cells could affect the success of the treatment in some patients. Propagation of pure cell lines in the laboratory would avoid these potential drawbacks and increase the numbers of stem cells available to each patient, thus shortening the at-risk interval before full immune reconstitution. Whether embryonic stem cells will provide advantages over stem cells derived from cord blood or adult bone marrow hematopoietic stem cells remains to be determined. However, hematopoietic stem cells, whether from umbilical cord blood or bone marrow, have a more limited potential for self-renewal than do pluripotent embryonic stem cells. Although new information will be needed to direct the differentiation of embryonic stem cells into hematopoietic stem cells, hematopoietic cells are present in differentiated cultures from human embryonic stem cells [9] and from human fetal-derived embryonic germ stem cells [17]. One potential advantage of using hematopoietic stem cell lines for transplantation in patients with autoimmune diseases is that these cells could be generated from unaffected individuals or, as predisposing genetic factors are defined, from embryonic stem cells lacking these genetic influences. In addition, use of genetically selected or genetically engineered cell types may further limit the possibility of disease progression or reemergence. One risk of using nonself hematopoietic stem cells is of immune rejection of the transplanted cells. Immune rejection is caused by MHC protein differences between the donor and the patient (recipient). In this scenario, the transplanted hematopoietic stem cells and their progeny are rejected by the patient's own T cells, which are originating from the patient's surviving bone marrow hematopoietic stem cells. In this regard, embryonic stem cell-derived hematopoietic stem cells may offer distinct advantages over cord blood and bone marrow hematopoietic stem cell lines in avoiding rejection of the transplant. Theoretically, banks of embryonic stem cells expressing various combinations of the three most critical MHC proteins could be generated to allow close matching to the recipient's MHC composition. Additionally, there is evidence that embryonic stem cells are considerably more receptive to genetic manipulation than are hematopoietic stem cells (see Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). This characteristic means that embryonic stem cells could be useful in strategies that could prevent their recognition by the patient's surviving immune cells. For example, it may be possible to introduce the recipient's MHC proteins into embryonic stem cells through targeted gene transfer. Alternatively, it is theoretically possible to generate a universal donor embryonic stem cell line by genetic alteration or removal of the MHC proteins. Researchers have accomplished this by genetically altering a mouse so that it has little or no surface expression of MHC molecules on any of the cells or tissues. There is no rejection of pancreatic beta islet cells from these genetically altered mice when the cells are transplanted into completely MHC-mismatched mice [13]. Additional research will be needed to determine the feasibility of these alternative strategies for prevention of graft rejection in humans [6]. Jon Odorico and colleagues have shown that expression of MHC proteins on mouse embryonic stem cells and differentiated embryonic stem cell progeny is either absent or greatly decreased compared with MHC expression on adult cells [8]. These preliminary findings raise the intriguing possibility that lines derived from embryonic stem cells may be inherently less susceptible to rejection by the recipient's immune system than lines derived from adult cells. This could have important implications for the transplantation of cells other than hematopoietic stem cells. Another potential advantage of using pure populations of donor hematopoietic stem cells achieved through stem cell technologies would be a lower incidence and severity of graft-versus-host disease, a potentially fatal complication of bone marrow transplantation. Graft-versus-host disease results from the immune-mediated injury to recipient tissues that occurs when mature organ-donor T cells remain within the organ at the time of transplant. Such mature donor alloreactive T cells would be absent from pure populations of multipotent hematopoietic stem cells, and under ideal conditions of immune tolerance induction in the recipient's thymus, the donor-derived mature T cell population would be tolerant to the host. Gene Therapy and Stem Cell Approaches for the Treatment of Autoimmune Diseases Gene therapy is the genetic modification of cells to produce a therapeutic effect (see Chapter 11. Use of Genetically Modified Stem Cells in Experimental Gene Therapies). In most investigational protocols, DNA containing the therapeutic gene is transferred into cultured cells, and these cells are subsequently administered to the animal or patient. DNA can also be injected directly, entering cells at the site of the injection or in the circulation. Under ideal conditions, cells take up the DNA and produce the therapeutic protein encoded by the gene. Currently, there is an extensive amount of gene therapy research being conducted in animal models of autoimmune disease. The goal is to modify the aberrant, inflammatory immune response that is characteristic of autoimmune diseases [15, 19]. Researchers most often use one of two general strategies to modulate the immune system. The first strategy is to block the actions of an inflammatory cytokine (secreted by certain activated immune cells and inflamed tissues) by transferring a gene into cells that encodes a "decoy" receptor for that cytokine. Alternatively, a gene is transferred that encodes an anti-inflammatory cytokine, redirecting the auto-inflammatory immune response to a more "tolerant" state. In many animal studies, promising results have been achieved by using these approaches, and the studies have advanced understanding of the disease processes and the particular inflammatory cytokines involved in disease progression [15, 19]. Serious obstacles to the development of effective gene therapies for humans remain, however. Foremost among these are the difficulty of reliably transferring genetic material into adult and slowly dividing cells (including hematopoietic stem cells) and of producing long-lasting expression of the intended protein at levels that can be tightly controlled in response to disease activity. Importantly, embryonic stem cells are substantially more permissive to gene transfer compared with adult cells, and embryonic cells sustain protein expression during extensive self-renewal. Whether adult-derived stem cells, other than hematopoietic stem cells, are similarly amenable to gene transfer has not yet been determined. Ultimately, stem cell gene therapy should allow the development of novel methods for immune modulation in autoimmune diseases. One example is the genetic modification of hematopoietic stem cells or differentiated tissue cells with a "decoy" receptor for the inflammatory cytokine interferon gamma to treat lupus. For example, in a lupus mouse model, gene transfer of the decoy receptor, via DNA injection, arrested disease progression [12]. Other investigators have used a related but distinct approach in a mouse model of type 1 diabetes. Interleukin-12 (IL-12), an inflammatory cytokine, plays a prominent role in the development of diabetes in these mice. The investigators transferred the gene for a modified form of IL-12, which blocks the activity of the natural IL-12, into pancreatic beta islet cells (the target of autoimmune injury in type 1 diabetes). The islet cell gene therapy prevented the onset of diabetes in these mice [20]. Theoretically, embryonic stem cells or adult stem cells could be genetically modified before or during differentiation into pancreatic beta islet cells to be used for transplantation. The resulting immune-modulating islet cells might diminish the occurrence of ongoing autoimmunity, increase the likelihood of long-term function of the transplanted cells, and eliminate the need for immunosuppressive therapy following transplantation. Researchers are exploring similar genetic approaches to prevent progressive joint destruction and loss of cartilage and to repair damaged joints in animal models of rheumatoid arthritis. Rheumatoid arthritis is a debilitating autoimmune disease characterized by acute and chronic inflammation, in which the immune system primarily attacks the joints of the body. In a recent study, investigators genetically transferred an anti-inflammatory cytokine, interleukin-4 (IL-4), into a specialized, highly efficient antigen-presenting cell called a dendritic cell, and then injected these IL-4-secreting cells into mice that can be induced to develop a form of arthritis similar to rheumatoid arthritis in humans. These IL-4-secreting dendritic cells are presumed to act on the CD4 helper T cells to reintroduce tolerance to self-proteins. Treated mice showed complete suppression of their disease and, in addition to its immune-modulatory properties, IL-4 blocked bone resorption (a serious complication of rheumatoid arthritis), making it a particularly attractive cytokine for this therapy [10]. However, one obstacle to this approach is that human dendritic cells are difficult to isolate in large numbers. Investigators have also directed the differentiation of dendritic cells from mouse embryonic stem cells, indicating that a stem cell-based approach might work in patients with rheumatoid arthritis [5]. Longer-term follow-up and further characterization will be needed in animal models before researchers proceed with the development of such an approach in humans. In similar studies, using other inhibitors of inflammatory cytokines such as a decoy receptor for tumor necrosis factor-α (a prominent inflammatory cytokine in inflamed joints), an inhibitor of nuclear factor-κB (a protein within cells that turns on the production of many inflammatory cytokines), and interleukin-13 (an anti-inflammatory cytokine), researchers have shown promising results in animal models of rheumatoid arthritis [19]. Because of the complexity and redundancy of immune system signaling networks, it is likely that a multifaceted approach involving inhibitors of several different inflammatory cytokines will be successful, whereas approaches targeting single cytokines might fail or produce only short-lived responses. In addition, other cell types may prove to be even better vehicles for the delivery of gene therapy in this disease. Chondrocytes, cells that build cartilage in joints, may provide another avenue for stem cell-based treatment of rheumatoid arthritis. These cells have been derived from human bone marrow stromal stem cells derived from human bone marrow [14]. Little is known about the intermediate cells that ultimately differentiate into chondrocytes. In addition to adult bone marrow as a source for stromal stem cells, human embryonic stem cells can differentiate into precursor cells believed to lead ultimately to the stromal stem cells [16]. However, extensive research is needed to reliably achieve the directed derivation of the stromal stem cells from embryonic stem cells and, subsequently, the differentiation of chondrocytes from these stromal stem cells. The ideal cell for optimum cartilage repair may be a more primitive cell than the chondrocyte, such as the stromal cell, or an intermediate cell in the pathway (e.g., a connective tissue precursor) leading to the chondrocyte. Stromal stem cells can generate new chondrocytes and facilitate cartilage repair in a rabbit model [3]. Such cells may also prove to be ideal targets for the delivery of immune-modulatory gene therapy. Like hematopoietic stem cells, stromal stem cells have been used in animal models for delivery of gene therapy [1]. For example, a recent study demonstrated that genetically engineered chondrocytes, expressing a growth factor, can enhance the function of transplanted chondrocytes [2]. Two obstacles to the use of adult stromal stem cells or chondrocytes are the limited numbers of these cells that can be harvested and the difficulties in propagating them in the laboratory. Embryonic stem cells, genetically modified and expanded before directed differentiation to a connective tissue stem cell, may be an attractive alternative. Collectively, these results illustrate the tremendous potential these cells may offer for the treatment of rheumatoid arthritis and other autoimmune diseases. Conclusion Stem cell-based therapies offer many exciting possibilities for the development of novel treatments, and perhaps even cures, for autoimmune diseases. A challenging research effort remains to fully realize this potential and to address the many remaining questions, which include how best to direct the differentiation of specific cell types and determine which particular type of stem cell will be optimum for each therapeutic approach. Gene therapy with cytokines or their inhibitors is still in its infancy, but stem cells or their progeny may provide one of the better avenues for future delivery of immune-based therapies. Ultimately, the potential to alleviate these devastating chronic diseases with the use of stem cell-based technologies is enormous. References Allay, J.A., Dennis, J.E., Haynesworth, S.E., Majumdar, M.K., Clapp, D.W., Shultz, L.D., Caplan, A.I., and Gerson, S.L. (1997). LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors. Hum. Gene Ther. 8, 1417–1427. Brower-Toland, B.D., Saxer, R.A., Goodrich, L.R., Mi, Z., Robbins, P.D., Evans, C.H., and Nixon, A.J. (2001). Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function. Hum. Gene Ther. 12, 117–129. Caplan, A.I., Elyaderani, M., Mochizuki, Y., Wakitani, S., and Goldberg, V.M. (1997). Principles of cartilage repair and regeneration. Clin. Orthop. 342, 254–269. Cooper, G.S., Dooley, M.A., Treadwell, E.L., St Clair, E.W., Parks, C.G., and Gilkeson, G.S. (1998). Hormonal, environmental, and infectious risk factors for developing systemic lupus erythematosus. Arthritis Rheum. 41, 1714–1724. Fairchild, P.J., Brook, F.A., Gardner, R.L., Graca, L., Strong, V., Tone, Y., Tone, M., Nolan, K.F., and Waldmann, H. (2000). Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr. Biol. 10, 1515–1518. Gearhart, J. (1998). New potential for human embryonic stem cells. Science. 282, 1061–1062. Grossman, J.M. and Tsao, B.P. (2000). Genetics and systemic lupus erythematosus. Curr. Rheumatol. Rep. 2, 13–18. Harley, C.B., Gearhart, J., Jaenisch, R., Rossant, J., and Thomson, J. (2001). Keystone Symposia. Pluripotent stem cells: biology and applications. Durango, CO. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. (2000). Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol. Med. 6, 88–95. Kim, S.H., Kim, S., Evans, C.H., Ghivizzani, S.C., Oligino, T., and Robbins, P.D. (2001). Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4. J. Immunol. 166, 3499–3505. Lawrence, R.C., Helmick, C.G., Arnett, F.C., Deyo, R.A., Felson, D.T., Giannini, E.H., Heyse, S.P., Hirsch, R., Hochberg, M.C., Hunder, G.G., Liang, M.H., Pillemer, S.R., Steen, V.D., and Wolfe, F. (1998). Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum. 41, 778–799. Lawson, B.R., Prud'homme, G.J., Chang, Y., Gardner, H.A., Kuan, J., Kono, D.H., and Theofilopoulos, A.N. (2000). Treatment of murine lupus with cDNA encoding IFN-gammaR/Fc. J. Clin. Invest. 106, 207–215. Osorio, R.W., Ascher, N.L., Jaenisch, R., Freise, C.E., Roberts, J.P., and Stock, P.G. (1993). Major histocompatibility complex class I deficiency prolongs islet allograft survival. Diabetes. 42, 1520–1527. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143–147. Prud'homme, G.J. (2000). Gene therapy of autoimmune diseases with vectors encoding regulatory cytokines or inflammatory cytokine inhibitors. J. Gene. Med. 2, 222–232. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D., and Benvenisty, N. (2000). Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 97, 11307–11312. Shamblott, M.J., Axelman, J., Littlefield, J.W., Blumenthal, P.D., Huggins, G.R., Cui, Y., Cheng, L., and Gearhart, J.D. (2000). Human embryonic germ cell derivatives express a broad range of develpmentally distinct markers and proliferate extensively in vitro. Proc. Natl. Acad. Sci. U. S. A. 98, 113–118. Traynor, A.E., Schroeder, J., Rosa, R.M., Cheng, D., Stefka, J., Mujais, S., Baker, S., and Burt, R.K. (2000). Treatment of severe systemic lupus erythematosus with high-dose chemotherapy and haemopoietic stem-cell transplantation: a phase I study. Lancet. 356, 701–707. Tsokos, G.C. and Nepom, G.T. (2000). Gene therapy in the treatment of autoimmune diseases. J. Clin. Invest. 106, 181–183. Yasuda, H., Nagata, M., Arisawa, K., Yoshida, R., Fujihira, K., Okamoto, N., Moriyama, H., Miki, M., Saito, I., Hamada, H., Yokono, K., and Kasuga, M. (1998). Local expression of immunoregulatory IL-12p40 gene prolonged syngeneic islet graft survival in diabetic NOD mice. J. Clin. Invest. 102, 1807–1814.  Chapter 5 | Table of Contents | Chapter 7  Historical content: June 17, 2001

Toll-like receptors play an important role in host defense. TLR-7 recognizes viral ssRNA, but also plays a role in the development of systemic lupus erythematosus (SLE). Genetic ablation of a similar receptor, TLR-9, results in opposite effects, with severe disease and kidney involvement.

Case Reports in Hematology is a peer-reviewed, Open Access journal that publishes case reports and case series in all areas of hematology, including general hematology, pathology, and oncology, with a specific focus on lymphomas and leukemias.

New research on the autoimmune disease systemic lupus erythematosus provides hints to the origins of the puzzling disorder. The results were published in Nature Immunology.

Epstein&ndash;Barr Virus (EBV) has been associated with development of rheumatic connective tissue diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in genetically susceptible individuals.

Although antimitochondrial antibodies are present in SLE patients, researchers have has difficulty investigating the phenomenon due to barriers in isolating pure mitochondria. The study describes a technique that allows for such an investigation and provides insight into how the adaptive immune...

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Systemic lupus erythematosus (SLE) is an autoimmune disease featuring enhanced expression of type I interferon (IFN) and autoantibody production triggering inflammation of, and damage to, multiple organs.

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Clot analysis after endovascular thrombectomy can be a useful diagnostic tool and guide therapy.

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The diagnosis of SLE and antiphospholipid syndrome can be further supported by clot histopathology and histoimmunochemistry.

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A negative TEE may not exclude Libman-Sacks endocarditis.

Systemic lupus erythematosus Fatigue in SLE: diagnostic and pathogenic impact of anti-N-methyl-D-aspartate receptor (NMDAR) autoantibodies Andreas Schwarting1,2, Tamara Möckel1, Freya Lütgendorf2, Konstantinos Triantafyllias2, Sophia Grella1, Simone Boedecker1, Arndt Weinmann1, Myriam Meineck1, Clemens Sommer3, Ingrid Schermuly4, Andreas Fellgiebel4, Felix Luessi5, Julia Weinmann-Menke1 Division of Rheumatology and Clinical Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Acura Rheumatology Center Rhineland Palatinate, Bad Kreuznach, Germany Institute of Neuropathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Department of Psychiatry and Psychotherapy, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Department of Neurology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Correspondence to Andreas Schwarting, Division of Rheumatology and Clinical Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz 55131, Germany; schwarting{at}uni-mainz.de Abstract Objectives We explored the impact of circulating anti-N-methyl-D-aspartate receptor (NMDAR) antibodies on the severity of fatigue in patients with systemic lupus erythematosus (SLE). Methods Serum samples of 426 patients with SLE were analysed for the presence of antibodies to the NR2 subunit of the NMDAR. In parallel, the severity of fatigue was determined according to the Fatigue Scale for Motor and Cognitive functions questionnaire. In a subgroup of patients with SLE, the hippocampal volume was correlated with the levels of anti-NR2 antibodies. Isolated immunoglobulin G from patients with anti-NR2 antibodies were used for murine immunohistochemical experiments and functional assays on neuronal cell lines. Treatment effects were studied in 86 patients with lupus under belimumab therapy. Results We found a close correlation between the titre of anti-NR2 antibodies, the severity of fatigue, the clinical disease activity index (Systemic Lupus Erythematosus Disease Activity Index 2000) and anti-double stranded DNA antibodies—independently of the presence of neuropsychiatric lupus manifestations. Pathogenic effects could be demonstrated by (1) detection of anti-NR2 antibodies in the cerebrospinal fluid, (2) in situ binding of anti-NR2 antibodies to NMDAR of the hippocampus area and (3) distinct functional effects in vitro: downregulating the energy metabolism of neuronal cells without enhanced cytotoxicity. Treatment with belimumab for at least 6 months affected both the severity of fatigue and the levels of anti-NR2 antibodies. Conclusion The presence of anti-NR2 antibodies in patients with SLE with fatigue is a helpful diagnostic tool and may offer a major approach in the therapeutic management of this important disabling symptom in patients with SLE. View Full Text Statistics from Altmetric.com View Full Text Footnotes Handling editor Josef S Smolen Contributors AS and JW conceived and supervised the study. AS, AW, JW, SG and MM maintained the SLE database. TM, MM, CS, AF, IS, SG and FL contributed to the acquisition of data. All authors have made substantial contributions to the analysis or interpretation of data and revised the manuscript critically for important intellectual content and approved the version for publication. Funding The Investigator-initiated-study was supported by GSK (data acquisition) and the Deutsche Forschungsgemeinschaft. Competing interests AS, TM, FL, KT, SG, SB, AW, FL, MM, CS, IS and AF have nothing to declare; AS has received speaker fees (less than US$10 000) and grant/research support by AbbVie, Novartis, Roche and GSK; KT has received speaker fees (less than US$10 000) and research support from Pfizer outside the submitted work. Patient and public involvement statement Indirect Patient and Public Involvement. We did not directly include PPI in this study, but the initiative of the study was driven by lupus patients and their representatives. Patient consent for publication Not required. Ethics approval The study protocol was approved by the local ethics committee (Ethics Commission of the State Chamber of Medicine in Rheinland-Pfalz, Mainz, Germany) in adherence to the Declaration of Helsinki. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement All data relevant to the study are included in the article or uploaded as supplementary information. Request Permissions If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways. Copyright information: © Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Read the full text or download the PDF: Subscribe Log in

Abstract Objectives In a multi‐ethnic/racial, prospective SLE inception cohort, to determine the frequency, clinical characteristics, associations and outcomes in different types of peripheral nerv...

Objective To develop new classification criteria for systemic lupus erythematosus (SLE) jointly supported by the European League Against Rheumatism (EULAR) and the American College of Rheumatology ...

Function-altering variants of immune-related genes cause rare autoimmune syndromes, whereas their contribution to common autoimmune diseases remains uncharacterized. Here the authors show that rare variants of lupus-associated genes are present in the majority of lupus patients and healthy...