Genomic Spectrum and Phenotypic Heterogeneity of Human IL-21 Receptor Deficiency.

Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.

CXCL13-mediated recruitment of intrahepatic CXCR5+CD8+ T cells favors viral control in chronic HBV infection.

BACKGROUND & AIMS: Although CD8+T cell exhaustion hampers viral control during chronic HBV infection, the pool of CD8+T cells is phenotypically and functionally heterogeneous. Therefore, a specific subpopulation of CD8+T cells should be further investigated. This study aims to dissect a subset of CD8+T cells expressing C-X-C motif chemokine receptor 5 (CXCR5) in chronic HBV infection. METHODS: The frequency of CXCR5+CD8+T cells and the levels of C-X-C motif chemokine ligand 13 (CXCL13), a chemokine of CXCR5, were measured in patients with chronic HBV infection. C57BL/6, interleukin (IL)-21 receptor- or B cell-deficient mice were hydrodynamically injected with pAAV-HBV1.2 plasmids. Phenotype and functions of peripheral and intrahepatic CXCR5+ and CXCR5-CD8+T cells were assessed. RESULTS: CXCR5+CD8+T cells were partially exhausted but possessed a stronger antiviral ability than the CXCR5- subset in patients with chronic HBV infection; moreover, CXCR5+CD8+T cells were associated with a favorable treatment response in patients with chronic hepatitis B (CHB). High levels of CXCL13 from patients with CHB facilitated the recruitment of intrahepatic CXCR5+CD8+T cells, and this subpopulation produced high levels of HBV-specific interferon (IFN)-γ and IL-21. Notably, PD1 (programmed death 1) blockade and exogenous IL-21 enhanced the production of IFN-γ. More strikingly, mice injected with CXCR5+CD8+T cells showed remarkably decreased expression of HBsAg. Additionally, an impaired production of HBV-specific IFN-γ from intrahepatic CXCR5+CD8+T cells was observed in IL-21 receptor- or B cell-deficient mice. CONCLUSION: CXCL13 promotes the recruitment of CXCR5+CD8+T cells to the liver, and this subpopulation improves viral control in chronic HBV infection. The identification of this unique subpopulation may contribute to a better understanding of CD8+T cell functions and provide a potential immunotherapeutic target in chronic HBV infection. LAY SUMMARY: Exhaustion of CD8+ T cells is an important factor in the development of chronic hepatitis B virus (HBV) infection. CD8+ T cells expressing the receptor CXCR5 are partially exhausted, but have potent antiviral activity, as they produce high levels of HBV-specific cytokines in chronic HBV infection. Increased expression of CXCL13 within the liver facilitates the recruitment of CXCR5+CD8+T cells and establishes effective immune control of HBV infection.

Alternative Splicing Rescues Loss of Common Gamma Chain Function and Results in IL-21R-like Deficiency.

Inborn errors in interleukin 2 receptor, gamma (IL2RG) perturb signaling of the common gamma chain family cytokines and cause severe combined immunodeficiency (SCID). Here, we report two brothers suffering from chronic cryptosporidiosis, severe diarrhea, and cholangitis. Pan T, B, and NK cell numbers were normal, but immunophenotyping revealed defective B cell differentiation. Using whole exome sequencing, we identified a base pair deletion in the first exon of IL2RG predicted to cause a frameshift and premature stop. However, flow cytometry revealed normal surface expression of the IL-2Rγ chain. While IL-2, IL-7, and IL-15 signaling showed only mild defects of STAT5 phosphorylation in response to the respective cytokines, IL-4- and IL-21-induced phosphorylation of STAT3 and STAT6 was markedly reduced. Examination of RNA isoforms detected alternative splicing downstream of IL2RG exon 1 in both patients resulting in resolution of the predicted frameshift and 16 mutated amino acids. In silico modeling suggested that the IL-2Rγ mutation reduces the stabilization of IL-4 and IL-21 cytokine binding by affecting the N-terminal domain of the IL-2Rγ. Thus, our study shows that IL2RG deficiency can be associated with differential signaling defects. Confounding effects of alternative splicing may partially rescue genetic defects and should be considered in patients with inborn errors of immunity.

CTL-Promoting Effects of IL-21 Counteract Murine Lupus in the Parent→F1 Graft-versus-Host Disease Model.

IL-21 promotes B cell and CTL responses in vivo, conferring IL-21 with a role in both humoral and cellular responses. Because CTL can target and eliminate autoreactive B cells, we investigated whether IL-21R signaling in CD8 T cells would alter the expansion of autoreactive B cells in an autoimmune setting. We addressed this question using the parent→F1 murine model of acute and chronic (lupus-like) graft-versus-host disease (GVHD) as models of a CTL-mediated or T-dependent B cell-mediated response, respectively. Induction of acute GVHD using IL-21R-deficient donor T cells resulted in decreased peak donor CD8 T cell numbers and decreased CTL effector function due to impaired granzyme B/perforin and Fas/Fas ligand pathways and a phenotype of low-intensity chronic GVHD with persistent host B cells, autoantibody production, and mild lupus-like renal disease. CTL effector maturation was critically dependent on IL-21R signaling in Ag-specific donor CD8, but not CD4, T cells. Conversely, treatment of DBA/2J→F1 chronic GVHD mice with IL-21 strongly promoted donor CD8 T cell expansion and rescued defective donor anti-host CTLs, resulting in host B cell elimination, decreased autoantibody levels, and attenuated renal disease, despite evidence of concurrently enhanced CD4 help for B cells and heightened B cell activation. These results demonstrate that, in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, IL-21 signaling on Ag-specific donor CD8 T cells is critical for CTL effector maturation, whereas a lack of IL-21R downregulates CTL responses that would otherwise limit B cell hyperactivity and autoantibody production.

IL-21R signaling is critical for induction of spontaneous experimental autoimmune encephalomyelitis.

IL-17-producing CD4+ T cells (Th17 cells) have well-described pathogenic roles in tissue inflammation and autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE); however, the involvement of IL-21 in these processes has remained controversial. While IL-21 is an essential autocrine amplification factor for differentiation of Th17 cells, the loss of IL-21 or IL-21 receptor (IL-21R) does not protect mice from actively induced EAE. Here, we utilized a transgenic EAE mouse model, in which T and B cells overexpress receptors for myelin oligodendrocyte glycoprotein (MOG) (referred to as 2D2xTH mice), and demonstrated that IL-21 is critical for the development of a variant form of spontaneous EAE in these animals. Il21r deletion in 2D2xTH mice reduced the incidence and severity of spontaneous EAE, which was associated with a defect in Th17 cell generation. Moreover, IL-21R deficiency limited IL-23R expression on Th17 cells and inhibited expression of key molecules involved in the generation of pathogenic Th17 cells. Conversely, loss of IL-23R in 2D2xTH mice resulted in complete resistance to the development of spontaneous EAE. Our data identify a previously unappreciated role for IL-21 in EAE and reveal that IL-21-mediated signaling supports generation and stabilization of pathogenic Th17 cells and development of spontaneous autoimmunity.

IL-21 promotes late activator APC-mediated T follicular helper cell differentiation in experimental pulmonary virus infection.

IL-21 is a type-I cytokine that has pleiotropic immuno-modulatory effects. Primarily produced by activated T cells including NKT and TFH cells, IL-21 plays a pivotal role in promoting TFH differentiation through poorly understood cellular and molecular mechanisms. Here, employing a mouse model of influenza A virus (IAV) infection, we demonstrate that IL-21, initially produced by NKT cells, promotes TFH differentiation by promoting the migration of late activator antigen presenting cell (LAPC), a recently identified TFH inducer, from the infected lungs into the draining lymph nodes (dLN). LAPC migration from IAV-infected lung into the dLN is CXCR3-CXCL9 dependent. IL-21-induced TNF-α production by conventional T cells is critical to stimulate CXCL9 expression by DCs in the dLN, which supports LAPC migration into the dLN and ultimately facilitates TFH differentiation. Our results reveal a previously unappreciated mechanism for IL-21 modulation of TFH responses during respiratory virus infection.

Potential role of myeloid cell/eosinophil-derived IL-17 in LPS-induced endotoxin shock.

IL-17RA is a shared receptor subunit for several cytokines of the IL-17 family, including IL-17A, IL-17C, IL-17E (also called IL-25) and IL-17F. It has been shown that mice deficient in IL-17RA are more susceptible to sepsis than wild-type mice, suggesting that IL-17RA is important for host defense against sepsis. However, it is unclear which ligands for IL-17RA, such as IL-17A, IL-17C, IL-17E/IL-25 and/or IL-17F, are involved in the pathogenesis of sepsis. Therefore, we examined IL-17A, IL-17E/IL-25 and IL-17F for possible involvement in LPS-induced endotoxin shock. IL-17A-deficient mice, but not IL-25- or IL-17F-deficient mice, were resistant to LPS-induced endotoxin shock, as compared with wild-type mice. Nevertheless, studies using IL-6-deficient, IL-21Rα-deficient and Rag-2-deficient mice, revealed that neither IL-6 and IL-21, both of which are important for Th17 cell differentiation, nor Th17 cells were essential for the development of LPS-induced endotoxin shock, suggesting that IL-17A-producing cells other than Th17 cells were important in the setting. In this connection, IL-17A was produced by macrophages, DCs and eosinophils after LPS injection. Taken together, these findings indicate that IL-17A, but not IL-17F or IL-25, is crucial for LPS-induced endotoxin shock. In addition, macrophages, DCs and eosinophils, but not Th17 cells or γδ T cells, may be sources of IL-17A during LPS-induced endotoxin shock.

Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells.

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell-dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10- and IL-21-mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21-induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.

IL-21 promotes lupus-like disease in chronic graft-versus-host disease through both CD4 T cell- and B cell-intrinsic mechanisms.

T cell-driven B cell hyperactivity plays an essential role in driving autoimmune disease development in systemic lupus erythematosus. IL-21 is a member of the type I cytokine family with pleiotropic activities. It regulates B cell differentiation and function, promotes T follicular helper (T(FH)) cell and Th17 cell differentiation, and downregulates the induction of T regulatory cells. Although IL-21 has been implicated in systemic lupus erythematosus, the relative importance of IL-21R signaling in CD4(+) T cells versus B cells is not clear. To address this question, we took advantage of two induced models of lupus-like chronic graft-versus-host disease by using wild-type or IL-21R(-/-) mice as donors in the parent-into-F1 model and as hosts in the Bm12→B6 model. We show that IL-21R expression on donor CD4(+) T cells is essential for sustaining T(FH) cell number and subsequent help for B cells, resulting in autoantibody production and more severe lupus-like renal disease, but it does not alter the balance of Th17 cells and regulatory T cells. In contrast, IL-21R signaling on B cells is critical for the induction and maintenance of germinal centers, plasma cell differentiation, autoantibody production, and the development of renal disease. These results demonstrate that IL-21 promotes autoimmunity in chronic graft-versus-host disease through both CD4(+) T cell- and B cell-intrinsic mechanisms and suggest that IL-21 blockade may attenuate B cell hyperactivity, as well as the aberrant T(FH) cell pathway that contributes to lupus pathogenesis.

Interleukin-21 receptor-mediated signals control autoreactive T cell infiltration in pancreatic islets.

It remains unclear how interleukin-21 receptor (IL-21R) contributes to type 1 diabetes. Here we have shown that dendritic cells (DCs) in the pancreas required IL-21R not for antigen uptake, but to acquire the chemokine receptor CCR7 and migrate into the draining lymph node. Consequently, less antigen, major histocompatibility complex (MHC) class II, and CD86 was provided to autoreactive effector cells in Il21r(-/-) mice, impairing CD4(+) T cell activation, CD40:CD40L interactions, and pancreatic infiltration by autoreactive T cells. CD40 crosslinking restored defective CD4(+) cell expansion and CD4 independently expanded autoreactive CD8(+) cells, but CD8(+) cells still required CD4(+) cells to reach the pancreas and induce diabetes. Diabetes induction by transferred T cells required IL-21R-sufficient host antigen-presenting cells. Transferring IL-21R-sufficient DCs broke diabetes resistance in Il21r(-/-) mice. We conclude that IL-21R controls both antigen transport by DCs and the crucial beacon function of CD4(+) cells for autoreactive CD8(+) cells to reach the islets.

Differential IL-21 signaling in APCs leads to disparate Th17 differentiation in diabetes-susceptible NOD and diabetes-resistant NOD.Idd3 mice.

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.

Altered effector CD4+ T cell function in IL-21R-/- CD4+ T cell-mediated graft-versus-host disease.

We previously showed that transplantation with IL-21R gene-deficient splenocytes resulted in less severe graft-versus-host disease (GVHD) than was observed with wild type splenocytes. In this study, we sought to find mechanism(s) explaining this observation. Recipients of donor CD4(+) T cells lacking IL-21R exhibited diminished GVHD symptoms, with reduced inflammatory cell infiltration into the liver and intestine, leading to prolonged survival. After transplantation, CD4(+) T cell numbers in the spleen were reduced, and MLR and cytokine production by CD4(+) T cells were impaired. These results suggest that IL-21 might promote GVHD through enhanced production of effector CD4(+) T cells. Moreover, we found that CD25 depletion altered neither the impaired MLR in vitro nor the ameliorated GVHD symptoms in vivo. Thus, the attenuated GVHD might be caused by an impairment of effector T cell differentiation itself, rather than by an increase in regulatory T cells and suppression of effector T cells.