Pilot study to evaluate the safety and effectiveness of etidronate treatment for arterial calcification due to deficiency of CD73 (ACDC).

BACKGROUND: Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle-brachial index (ABI). METHODS: Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams. RESULTS: Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort. CONCLUSIONS: Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort.(ClinicalTrials.gov Identifier NCT01585402).

Multiomics integration of 22 immune-mediated monogenic diseases reveals an emergent axis of human immune health.

Monogenic diseases are often studied in isolation due to their rarity. Here we utilize multiomics to assess 22 monogenic immune-mediated conditions with age- and sex-matched healthy controls. Despite clearly detectable disease-specific and "pan-disease" signatures, individuals possess stable personal immune states over time. Temporally stable differences among subjects tend to dominate over differences attributable to disease conditions or medication use. Unsupervised principal variation analysis of personal immune states and machine learning classification distinguishing between healthy controls and patients converge to a metric of immune health (IHM). The IHM discriminates healthy from multiple polygenic autoimmune and inflammatory disease states in independent cohorts, marks healthy aging, and is a pre-vaccination predictor of antibody responses to influenza vaccination in the elderly. We identified easy-to-measure circulating protein biomarker surrogates of the IHM that capture immune health variations beyond age. Our work provides a conceptual framework and biomarkers for defining and measuring human immune health.

RGC-32' dual role in smooth muscle cells and atherogenesis.

Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-ß-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-ß-induced ECM production.

TNF inhibition in vasculitis management in adenosine deaminase 2 deficiency (DADA2).

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations. OBJECTIVE: We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients. METHODS: We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment. RESULTS: We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2. CONCLUSIONS: Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment.

RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis.

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-ß signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32's ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-ß. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-ß-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.

Multifocal calcific periarthritis with distinctive clinical and radiological features in patients with CD73 deficiency.

OBJECTIVES: Arterial calcification due to deficiency of CD73 (ACDC) is a hereditary autosomal recessive ectopic mineralization syndrome caused by loss-of-function mutations in the ecto-5'-nucleotidase gene. Periarticular calcification has been reported but the clinical characterization of arthritis as well as the microstructure and chemical composition of periarticular calcifications and SF crystals has not been systematically investigated. METHODS: Eight ACDC patients underwent extensive rheumatological and radiological evaluation over a period of 11 years. Periarticular and synovial biopsies were obtained from four patients. Characterization of crystal composition was evaluated by compensated polarized light microscopy, Alizarin Red staining for synovial fluid along with X-ray diffraction and X-ray micro tomosynthesis scanner for periarticular calcification. RESULTS: Arthritis in ACDC patients has a clinical presentation of mixed erosive-degenerative joint changes with a median onset of articular symptoms at 17 years of age and progresses over time to the development of fixed deformities and functional limitations of small peripheral joints with, eventually, larger joint and distinct axial involvement later in life. We have identified calcium pyrophosphate and calcium hydroxyapatite (CHA) crystals in SF specimens and determined that CHA crystals are the principal component of periarticular calcifications. CONCLUSION: This is the largest study in ACDC patients to describe erosive peripheral arthropathy and axial enthesopathic calcifications over a period of 11 years and the first to identify the composition of periarticular calcifications and SF crystals. ACDC should be considered among the genetic causes of early-onset OA, as musculoskeletal disease signs may often precede vascular symptoms.

Characterization of autoantibodies, immunophenotype and autoimmune disease in a prospective cohort of patients with idiopathic CD4 lymphocytopenia.

OBJECTIVE: Characterize autoantibodies and autoimmune diseases in a prospective cohort of patients with Idiopathic CD4 Lymphocytopenia (ICL) a rare immunodeficiency characterized by an absolute CD4+ T count of <300 cells/µl in the absence of HIV or HTLV infection. METHODS: Single-Center prospective study of 67 patients conducted over an 11-year period. Rheumatologic evaluation and measurement of autoantibodies were systematically conducted, and flow cytometry of immune cell subsets was performed in a subset of patients. RESULTS: 54% of referred patients had clinical evidence of autoimmunity, with 34% having at least one autoimmune disease, most commonly autoimmune thyroid disease. 19%, had autoantibodies or incomplete features of autoimmune disease. Patients with autoimmune disease had more elevated serum immunoglobulins, and more effector memory T cells than those without autoimmunity. CONCLUSIONS: Evidence of autoimmunity, including autoimmune diseases, is more prevalent in ICL than the general population, and should be considered part of this syndrome.

The Spectrum of the Deficiency of Adenosine Deaminase 2: An Observational Analysis of a 60 Patient Cohort.

The deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessively inherited disease that has undergone extensive phenotypic expansion since being first described in patients with fevers, recurrent strokes, livedo racemosa, and polyarteritis nodosa in 2014. It is now recognized that patients may develop multisystem disease that spans multiple medical subspecialties. Here, we describe the findings from a large single center longitudinal cohort of 60 patients, the broad phenotypic presentation, as well as highlight the cohort's experience with hematopoietic cell transplantation and COVID-19. Disease manifestations could be separated into three major phenotypes: inflammatory/vascular, immune dysregulatory, and hematologic, however, most patients presented with significant overlap between these three phenotype groups. The cardinal features of the inflammatory/vascular group included cutaneous manifestations and stroke. Evidence of immune dysregulation was commonly observed, including hypogammaglobulinemia, absent to low class-switched memory B cells, and inadequate response to vaccination. Despite these findings, infectious complications were exceedingly rare in this cohort. Hematologic findings including pure red cell aplasia (PRCA), immune-mediated neutropenia, and pancytopenia were observed in half of patients. We significantly extended our experience using anti-TNF agents, with no strokes observed in 2026 patient months on TNF inhibitors. Meanwhile, hematologic and immune features had a more varied response to anti-TNF therapy. Six patients received a total of 10 allogeneic hematopoietic cell transplant (HCT) procedures, with secondary graft failure necessitating repeat HCTs in three patients, as well as unplanned donor cell infusions to avoid graft rejection. All transplanted patients had been on anti-TNF agents prior to HCT and received varying degrees of reduced-intensity or non-myeloablative conditioning. All transplanted patients are still alive and have discontinued anti-TNF therapy. The long-term follow up afforded by this large single-center study underscores the clinical heterogeneity of DADA2 and the potential for phenotypes to evolve in any individual patient.

Morphology and chemical identity of periarticular and vascular calcification in a patient with the rare genetic disease of arterial calcification due to deficiency of CD73 (ACDC).

A 54-year old female patient with the genetic disease of arterial calcification due to deficiency of CD73 was studied under the Undiagnosed Disease Program of the National Institutes of Health. She presented with symptoms of claudication in her 40s and later developed arthritic symptoms, ectopic calcification in her left hand and severe arterial calcifications of the lower extremities. Since little was known about the composition of the calcifications in arterial calcification due to deficiency of CD73, we investigated their chemical identity and microscopic morphology in this patient with imaging and x-ray diffraction analysis. We found that, microscopically, the bulk calcifications consisted of fragments of either solid or porous internal structure. Both periarticular and arterial calcifications were primarily hydroxyapatite crystals of the same crystalline anisotropy, but different crystalline grain sizes. This was consistent with the presence of hydroxyapatite crystals along with birefringent calcium pyrophosphate dihydrate crystals in the synovial fluid of the patients by polarized light microscopy. The result suggests that tissue calcification in both locations follow a similar biochemical mechanism caused by an increase in extracellular tissue-nonspecific alkaline phosphatase activity.

Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia.

BACKGROUNDIdiopathic CD4 lymphopenia (ICL) is defined by persistently low CD4+ cell counts (<300 cells/µL) in the absence of a causal infection or immune deficiency and can manifest with opportunistic infections. Approximately 30% of ICL patients develop autoimmune disease. The prevalence and breadth of their autoantibodies, however, and their potential contribution to pathogenesis of ICL remain unclear.METHODSWe hybridized 34 and 51 ICL patients' sera to a 9,000-human-proteome array and to a 128-known-autoantigen array, respectively. Using a flow-based method, we characterized the presence of anti-lymphocyte Abs in the whole cohort of 72 patients, as well as the Ab functional capability of inducing Ab-dependent cell-mediated cytotoxicity (ADCC), complement deposition, and complement-dependent cytotoxicity (CDC). We tested ex vivo the activation of the classical complement pathway on ICL CD4+ T cells.RESULTSAll ICL patients had a multitude of autoantibodies mostly directed against private (not shared) targets and unrelated quantitatively or qualitatively to the patients' autoimmune disease status. The targets included lymphocyte intracellular and membrane antigens, confirmed by the detection by flow of IgM and IgG (mostly IgG1 and IgG4) anti-CD4+ cell Abs in 50% of the patients, with half of these cases triggering lysis of CD4+ T cells. We also detected in vivo classical complement activation on CD4+ T cells in 14% of the whole cohort.CONCLUSIONOur data demonstrate that a high prevalence of autoantibodies in ICL, some of which are specific for CD4+ T cells, may contribute to pathogenesis, and may represent a potentially novel therapeutic target.TRIAL REGISTRATIONClinicalTrials.gov NCT00867269.FUNDINGNIAID and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH.

Revisiting TNF Receptor-Associated Periodic Syndrome (TRAPS): Current Perspectives.

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory syndrome characterized by prolonged and recurrent episodes of fever, abdominal and/or chest pain, arthralgia, myalgia, and erythematous rash. TRAPS is associated with heterozygous variants in the TNFRSF1A gene, which encodes the TNFR1 (tumor necrosis factor receptor 1) receptor. Disease-causing variants are found exclusively in the extracellular domain of TNFR1 and affect receptor structure and binding to the TNF ligand. The precise mechanism of the disease is still unclear, but it is thought that intracellular accumulation of misfolded mutant protein leads to endoplasmic reticulum stress and enhanced inflammatory responses through constitutive activation of various immune pathways. Other possible mechanisms contributing to the disease pathogenesis include defective receptor shedding, TNF-induced cell death, production of reactive oxygen species, and autophagy impairment. Patients' leucocytes are hyperresponsive to stimulation and produce elevated levels of proinflammatory cytokines. Systemic autoimmune (AA) amyloidosis is an important cause of morbidity and mortality in TRAPS. Over the last two decades, new therapies have changed the progression and outcome of the disease. In this review, we summarize clinical data from 209 patients with validated pathogenic variants reported in the literature and discuss TRAPS diagnosis, pathogenesis, and treatment options.

Generation of human induced pluripotent stem cells (NIHTVBi004-A, NIHTVBi005-A, NIHTVBi006-A, NIHTVBi007-A, NIHTVBi008-A) from 5 CADASIL patients with NOTCH3 mutation.

We have successfully generated induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells of five patients with Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These cells carry the genetic NOTCH3 mutation present in their parental cells. These iPSC cells exhibited normal karyotype and phenotype, which were sustained through propagation. Furthermore, these iPSCs displayed the capacity of differentiating toward the three germ layers in vitro.

RGC-32 Regulates Generation of Reactive Astrocytes in Experimental Autoimmune Encephalomyelitis.

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.

Generation of human induced pluripotent stem cell lines (NIHTVBi011-A, NIHTVBi012-A, NIHTVBi013-A) from autosomal dominant Hyper IgE syndrome (AD-HIES) patients carrying STAT3 mutation.

Autosomal dominant Hyper IgE syndrome (AD-HIES), a rare immune deficiency affecting fewer than one per million people, is caused by heterozygous deleterious mutations in STAT3. STAT3 signaling plays crucial roles in basic cellular functions affecting broad aspects of cellular homeostasis. Accordingly, in addition to immunological deficits, patients experience severe multisystem non-immunological features. Human induced pluripotent stem cells (hiPSC) are well established as in vivo disease models for various human pathologies. We describe the generation of iPSC from three AD-HIES patients. These iPSCs express pluripotency markers, differentiate into three germ layers, have normal karyotype and similar genome identity to parental cells.

Generation of human induced pluripotent stem cells from individuals with a homozygous CCR5Δ32 mutation.

Chemokine receptor 5 (CCR5) is the primary coreceptor for HIV entry into macrophages. Individuals with a homozygous deletion of 32 bp in the CCR5 gene (CCR5Δ32) are highly resistant to HIV infection (Samson et al., 1996). Allogeneic stem cell transplantation from a healthy donor with the homozygous CCR5Δ32 variant to an HIV positive individual has demonstrated efficient long-term control of HIV. We identified three individuals with this homozygous CCR5Δ32 variant, and successfully generated induced pluripotent stem cell (iPSC) lines from their dermal fibroblasts. The iPSCs lines carrying homozygous CCR5Δ32 variant displayed phenotypically normal and the potential to differentiation toward the three germ layers.

Intracerebral matrix metalloproteinase 9 in fatal diabetic ketoacidosis.

There is increasing awareness that in addition to the metabolic crisis of diabetic ketoacidosis (DKA) caused by severe insulin deficiency, the immune inflammatory response is likely an active multicomponent participant in both the acute and chronic insults of this medical crisis, with strong evidence of activation for both the cytokine and complement system. Recent studies report that the matrix metalloproteinase enzymes and their inhibitors are systemically activated in young Type 1 diabetes mellitus (T1D) patients during DKA and speculate on their involvement in blood-brain barrier (BBB) disruption. Based on our previous studies, we address the question if matrix metalloproteinase 9 (MMP9) is expressed in the brain in the fatal brain edema (BE) of DKA. Our data show significant expression of MMP9 on the cells present in brain intravascular areas. The presence of MMP9 in intravascular cells and that of MMP+ cells seen passing the BBB indicates a possible role in tight junction protein disruption of the BBB, possibly leading to neurological complications including BE. We have also shown that MMP9 is expressed on neurons in the hippocampal areas of both BE/DKA cases investigated, while expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was reduced in the same areas. We can speculate that intraneuronal MMP9 can be a sign of neurodegeneration. Further studies are necessary to determine the role of MMP9 in the pathogenesis of the neurologic catastrophe of the brain edema of DKA. Inhibition of MMP9 expression might be helpful in preserving neuronal function and BBB integrity during DKA.

CADASIL: new advances in basic science and clinical perspectives.

PURPOSE OF REVIEW: Recent advances in genetic evaluation improved the identification of several variants in the NOTCH3 gene causing Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). Despite improved diagnosis, the disease mechanism remains an elusive target and an increasing number of scientific/clinical groups are investigating CADASIL to better understand it. The purpose of this review is to summarize the current knowledge in CADASIL. RECENT FINDINGS: CADASIL is a genotypically and phenotypically diverse condition involving multiple molecular systems affecting small blood vessels. Cerebral white matter changes observed by MRI are a key CADASIL characteristic in young adult patients often before severe symptoms and trigger NOTCH3 genetic testing. NOTCH3 mutation locations are highly variable, correlate to disease severity and consistently affect the cysteine balance within extracellular Notch3. Granular osmiophilic material deposits around blood vessels are also a unique CADASIL feature and appear to have a role in sequestering proteins that are essential for blood vessel homeostasis. As potential biomarkers and therapeutic targets are being actively investigated, neurofilament light chain can be detected in patient serum and may be a promising circulating biomarker. SUMMARY: CADASIL is a complex, devastating disease with unknown mechanism and no treatment options. As we increase our understanding of CADASIL, translational research bridging basic science and clinical findings needs to drive biomarker and therapeutic target discovery.

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis.

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-ß downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-ß-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-ß stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-ß-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-ß-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-ß-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.

RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis.

Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-ß that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.