Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells.

BACKGROUND: Dysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC. METHODS: The clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells. RESULTS: HCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells. CONCLUSIONS: IL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

IL-21/IL-21R Regulates the Neutrophil-Mediated Pathologic Immune Response during Chlamydial Respiratory Infection.

IL-21/IL-21R was documented to participate in the regulation of multiple infection and inflammation. During Chlamydia muridarum (C. muridarum) respiratory infection, our previous study had revealed that the absence of this signal induced enhanced resistance to infection with higher protective Th1/Th17 immune responses. Here, we use the murine model of C. muridarum respiratory infection and IL-21R deficient mice to further identify a novel role of IL-21/IL-21R in neutrophilic inflammation. Resistant IL-21R-/- mice showed impaired neutrophil recruitment to the site of infection. In the absence of IL-21/IL-21R, pulmonary neutrophils also exhibited reduced activation status, including lower CD64 expression, MPO activity, and neutrophil-produced protein production. These results correlated well with the decrease of neutrophil-related chemokines (KC and MIP-2), inflammatory cytokines (IL-6, IL-1ß, and TNF-α), and TLR/MyD88 pathway mediators (TLR2, TLR4, and MyD88) in infected lungs of IL-21R-/- mice than normal mice. Complementarily, decreased pulmonary neutrophil infiltration, activity, and levels of neutrophilic chemotactic factors and TLR/MyD88 signal in infected lungs can be corrected by rIL-21 administration. These results revealed that IL-21/IL-21R may aggravate the neutrophil inflammation through regulating TLR/MyD88 signal pathway during chlamydial respiratory infection.

Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence.

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.

Combining random mutagenesis, structure-guided design and next-generation sequencing to mitigate polyreactivity of an anti-IL-21R antibody.

Despite substantial technological advances in antibody library and display platform development, the number of approved biotherapeutics from displayed libraries remains limited. In vivo, 20-50% of peripheral B cells undergo a process of receptor editing, which modifies the variable and junctional regions of light chains to delete auto-reactive clones. However, in vitro antibody evolution relies primarily on interaction with antigen, with no in-built checkpoints to ensure that the selected antibodies have not acquired additional specificities or biophysical liabilities during the optimization process. We had previously observed an enrichment of positive charge in the complementarity-determining regions of an anti-IL-21 R antibody during affinity optimization, which correlated with more potent IL-21 neutralization, but poor in vivo pharmacokinetics (PK). There is an emerging body of data that has correlated antibody nonspecificity with poor PK in vivo, and established a series of screening assays that are predictive of this behavior. In this study we revisit the challenge of developing an anti-IL-21 R antibody that can effectively compete with IL-21 for its highly negatively charged paratope while maintaining favorable biophysical properties. In vitro deselection methods that included an excess of negatively charged membrane preparations, or deoxyribonucleic acid, during phage selection of optimization libraries were unsuccessful in avoiding enrichment of highly charged, nonspecific antibody variants. However, a combination of structure-guided rational library design, next-generation sequencing of library outputs and application of linear regression models resulted in the identification of an antibody that maintained high affinity for IL-21 R and exhibited a desirable stability and biophysical profile.

IL-21 promotes osteoblastic differentiation of human valvular interstitial cells through the JAK3/STAT3 pathway.

Objectives: This study amied to whether IL-21 promotes osteoblast transdifferentiation of cultured human Valvular interstitial cells (VICs). Methods: We first confirmed that IL-21 alters gene expression between CAVD aortic valve tissue and normal samples by immunohistochemistry, qPCR, and western blotting. VICs were cultured and treated with IL-21. Gene and protein expression levels of the osteoblastic markers ALP and Runx2, which can be blocked by specific JAK3 inhibitors and/or siRNA of STAT3, were measured. Results: IL-21 expression was upregulated in calcified aortic valves and promotes osteogenic differentiation of human VICs. IL-21 accelerated VIC calcification through the JAK3/STAT3 pathway. Conclusion: Our data suggest that IL-21 is a key factor in valve calcification and a promising candidate for targeted therapeutics for CAVD.

Nobiletin suppresses IL-21/IL-21 receptor-mediated inflammatory response in MH7A fibroblast-like synoviocytes (FLS): An implication in rheumatoid arthritis.

The mechanisms driving the development and progression of Rheumatoid arthritis (RA) are complex, novel targeted therapies are gaining traction as potential methods to prevent or slow the progression of RA. Nobiletin is a derivative of citrus fruit that has been shown to attenuate the development of osteoarthritis and inhibit the expression of proinflammatory cytokines. However, the exact mechanisms by which nobiletin exerts these chondroprotective effects remain poorly understood. In the present study, we investigated the impact of nobiletin in mediating the effects of interleukin-21 (IL-21) in MH7A fibroblast-like synoviocytes (FLS), the main cell type found in the articular synovium. Firstly, we demonstrate that nobiletin (25 µM and 50 µM) reduced the expression of the IL-21 receptor by 29% and 51%, respectively, in FLS. Additionally, our findings demonstrate that nobiletin potently ameliorated IL-21-induced increased production of reactive oxygen species and 4-hydroxynonenal, increased expression of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and high-mobility group box 1 (HMGB1), and decreased mitochondrial membrane potential. We also demonstrate the ability of nobiletin to attenuate IL-21-induced expression of matrix metalloproteinases 3 and 13 (MMP-3, MMP-13), key degradative enzymes involved in RA-associated cartilage destruction. Finally, we show that the effects of nobiletin are mediated through the JAK1/STAT3 pathway, as nobiletin significantly reduced the phosphorylation of both JAK1 and STAT3. Taken together, our findings indicate that nobiletin may offer a safe and effective treatment against the development and progression of RA induced by the expression of IL-21 and its receptor.

Inhibition of tyrosine kinase signaling by tyrphostin AG126 downregulates the IL-21/IL-21R and JAK/STAT pathway in the BTBR mouse model of autism.

Autism spectrum disorder (ASD) comprises a broad range of neurodevelopmental disorders that are associated with deficits in social interaction and communication. The tyrosine kinase inhibitor tyrphostin AG126 represents a promising therapeutic agent for several neuroinflammatory disorders. There are currently no treatments available that can improve ASD and we previously showed that AG126 treatment exerts beneficial effects on BTBR T+ Itpr3tf/J (BTBR) mice, a model for autism that shows the core features of ASD; however, the immunological mechanisms and molecular targets associated with this effect were previously unclear. This study was undertaken to delineate the neuroprotective effect of AG126 on BTBR mice. Here, using this mouse model, we investigated the effects of AG126 administration on IL-21R, IL-21, IL-22, TNF-α, NOS2, STAT3, IL-27, and Foxp3 production by CD8+ T cells in the spleen by flow cytometry. We further explored the mRNA and protein expression of IL-21, IL-22, IL-1ß, TNF-α, NOS2, JAK1, STAT3, IL-27, and Foxp3 in brain tissue by RT-PCR, and western blotting. We found that BTBR mice treated with AG126 exhibited significant decreases in IL-21R-, IL-21-, IL-22-, TNF-α-, NOS2-, STAT3-producing, and increases in IL-27- and Foxp3-producing, CD8+ T cells. Our results further demonstrated that AG126 treatment effectively decreased IL-21, IL-22, IL-1ß, TNF-α, NOS2, JAK1, and STAT3, and increased IL-27 and Foxp3 mRNA and protein expression in brain tissues. Our findings suggest that AG126 elicits a neuroprotective response through downregulation of the IL-21/IL-21R and JAK/STAT pathway in BTBR mice, which could represent a promising novel therapeutic target for ASD treatment.

Interleukin (IL)-21 in Inflammation and Immunity During Parasitic Diseases.

Parasitic diseases cause significant morbidity and mortality in the developing and underdeveloped countries. No efficacious vaccines are available against most parasitic diseases and there is a critical need for developing novel vaccine strategies for care. IL-21 is a pleiotropic cytokine whose functions in protection and immunopathology during parasitic diseases have been explored in limited ways. IL-21 and its cognate receptor, IL-21R, are highly expressed in parasitized organs of infected humans as well in murine models of the human parasitic diseases. Prior studies have indicated the ability of the IL-21/IL-21R signaling axis to regulate the effector functions (e.g., cytokine production) of T cell subsets by enhancing the expression of T-bet and STAT4 in human T cells, resulting in an augmented production of IFN-γ. Mice deficient for either IL-21 (Il21-/-) or IL-21R (Il21r-/-) showed significantly reduced inflammatory responses following parasitic infections as compared with their WT counterparts. Targeting the IL-21/IL-21R signaling axis may provide a novel approach for the development of new therapeutic agents for the prevention of parasite-induced immunopathology and tissue destruction.

Short Communication: Nonprogressive HIV-1 Infection Is Associated with Expansion of IL-21R Expressing Class-Switched Memory B Cells.

HIV perturbs the functionality of B cells resulting in defective humoral responses. As efficient humoral immune responses are important in controlling HIV-disease progression, we characterized the memory B cell population for its subsets and their activation (CD38 expression) and functional [interleukin (IL)-21R expression] profile in individuals with nonprogressive [long-term nonprogressors (LTNPs), N = 16] and progressive HIV disease (progressors, N = 16) along with 10 HIV uninfected healthy controls (HCs). The frequencies of total memory B cells were similar in HCs and HIV-infected individuals, whereas the frequencies of unswitched memory B (UMB) cells and CD38+ UMB cells were significantly higher in progressors than LTNPs and HCs (p < .03). LTNPs showed higher frequencies of class-switched memory B (SMB) cells and IL-21R expressing SMB cells than seen in progressors (p = .019), which were similar to that seen in HCs. The %UMB cells correlated inversely (p = .0002, r = -0.6053) and %SMB cells correlated positively (p = .0005, r = 0.5804) with CD4 count. IL-21/IL-21R interaction is required for class switching of B cells and differentiation into antibody-secreting plasma cells. The higher expression of IL-21R on class SMB cells from LTNPs might be resulting in efficient plasma cell differentiation and the functional humoral immune response that might be responsible for mounting efficient antibody response against the encountered infections. The more insights in this area might be required to further understand the role of IL-21R expressing class SMB cells in HIV infection.

IL-21R functions as an oncogenic factor and is regulated by the lncRNA MALAT1/miR-125a-3p axis in gastric cancer.

Interleukin-21 receptor (IL-21R) is involved in the immunological regulation of immune cells and tumor progression in multiple malignancies. However, the potential molecular mechanisms through which non-coding RNAs (ncRNAs) modulate IL-21R signaling in gastric cancer (GC) remain elusive. In this study, the expression of IL-21R was detected by RT-qPCR and western blot analysis in GC cell lines. The association between IL-21R expression and clinicopathological characteristics and the prognosis of patients with GC was analyzed by immunohistochemistry and Kaplan-Meier plotter analysis. The biological functions of IL-21R were analyzed by a series of in vitro and in vivo experiments, and its regulation by ncRNAs was predicted by bioinformatics analysis and confirmed by luciferase assays and rescue experiments. As a result, the expression of IL-21R was found to be significantly increased in GC cell lines and tissues as compared with normal tissues, and was associated with tumor size and lymphatic metastasis, acting as an independent prognostic factor of poor survival and recurrence in patients with GC. The knockdown of IL-21R markedly suppressed GC cell proliferation and invasion, and IL-21R expression was further validated to be negatively regulated by miR-125a-3p (miR-125a). The overexpression of IL-21R reversed the tumor suppressive effects of miR-125a in vitro and in vivo. Moreover, lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acted as a sponge of miR-125a to modulate the IL-21R signaling pathway in GC cells and represented a risk factor for survival and recurrence in patients with GC. Taken together, the findings of this study reveal an oncogenic role for IL-21R in gastric tumorigenesis and verify that its activation is partly due to the dysregulation of the lncRNA MALAT1/miR-125a axis. These findings may provide a potential prognostic marker for patients with GC.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

IL-6 Impairs Vaccine Responses in Neonatal Mice.

The inability of infants to mount proper follicular helper T (TFH) cell response renders this age group susceptible to infectious diseases. Initial instruction of T cells by antigen presenting cells and subsequent differentiation into TFH cells are controlled by T cell receptor signal strength, co-stimulatory molecules and cytokines such as IL-6 and IL-21. In immunized adults, IL-6 promotes TFH development by increasing the expression of CXCR5 and the TFH master transcription factor, B cell lymphoma 6. Underscoring the importance of IL-6 in TFH generation, we found improved antibody responses accompanied by increased TFH cells and decreased follicular regulatory helper T (TFR) cells, a Foxp3 expressing inhibitory CD4+ T cell occupying the germinal center (GC), when a tetanus toxoid conjugated pneumococcal polysaccharide type 14 vaccine was injected in adult mice together with IL-6. Paradoxically, in neonates IL-6 containing PPS14-TT vaccine suppressed the already impaired TFH development and antibody responses in addition to increasing TFR cell population. Supporting the diminished TFH development, we detected lower frequency of phospho-STAT-3+ TFH in immunized neonatal T cells after IL-6 stimulation than adult cells. Moreover, IL-6 induced more phospho-STAT-3+ TFR in neonatal cells than adult cells. We also measured lower expression of IL-6R on TFH cells and higher expression on TFR cells in neonatal cells than adult cells, a possible explanation for the difference in IL-6 induced signaling in different age groups. Supporting the flow cytometry findings, microscopic examination revealed the localization of Treg cells in the splenic interfollicular niches of immunized adult mice compared to splenic follicles in neonatal mice. In addition to the limitations in the formation of IL-21 producing TFH cells, neonatal mice GC B cells also expressed lower levels of IL-21R in comparison to the adult mice cells. These findings point to diminished IL-6 activity on neonatal TFH cells as an underlying mechanism of the increased TFR: TFH ratio in immunized neonatal mice.

Interleukin-21 receptor signalling is not critically required for imiquimod-induced psoriasiform dermatitis in mice.

Psoriasis is largely mediated by interleukin (IL)-23/T helper (Th) 17 axis, and IL-21 is a pleiotropic cytokine expressed by Th17 cells. Despite previously reported possible pathogenic roles of IL-21 in human psoriasis, we found that IL-21 receptor (IL-21R) signalling was not crucial for imiquimod-induced psoriatic inflammation, using IL-21R-/- mice. The severity of imiquimod-induced psoriatic manifestation and pro-inflammatory Th17 cytokine levels, IL-17A-producing γδ T cells and CD4+ T cells, and in vitro IL-17A production by γδ T cells after IL-23 stimulation was comparable between wild-type and IL-21R-/- mice. Collectively, IL-21R signalling was not critically involved in IMQ-induced psoriatic inflammation despite an increased IL-21 expression in the IMQ-treated mouse skin. Our data may represent the significant differences between human psoriasis and murine psoriasis model, and further studies using other models will be required to elucidate the role of IL-21 in psoriasis pathogenesis.

PI3K-Akt pathway enhances the differentiation of interleukin-27-induced type 1 regulatory T cells.

Interleukin 27 (IL-27) has been identified as a potent cytokine in the differentiation of type 1 regulatory T (Tr1) cells through interactions with several key elements, including transcription factors such as aryl hydrocarbon receptor and IL-21. Autocrine production of IL-21 is known to be important for maintaining IL-10 expression by Tr1 cells. Although previous studies have shown that the phosphoinositide 3-kinase (PI3K) -Akt axis contributes to the differentiation of helper T-cell subsets, the role of the PI3K pathway on Tr1 cell differentiation remains to be elucidated. Here, we demonstrate that suppression of the PI3K-Akt pathway results in impairment of IL-27-induced Tr1 (IL-27-Tr1) cell differentiation in vitro and in vivo. Furthermore, this suppression down-regulates IL-21 receptor expression by Tr1 cells, followed by suppression of IL-10 expression by IL-27-Tr1 cells. These results suggest that the PI3K pathway enhances IL-10 expression by IL-27-Tr1 cells through up-regulation of IL-21 receptors.

Natural allelic variation of the IL-21 receptor modulates ischemic stroke infarct volume.

Risk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.

MiR-30a inhibits Th17 differentiation and demyelination of EAE mice by targeting the IL-21R.

T helper cells 17 (Th17) are recognized as key participants in the pathogenesis of chronic autoimmune diseases such as multiple sclerosis (MS). Regulation of Th17 differentiation is a valuable strategy for diagnosis and treatment of these complicated immune disorders. Here, by genome-wide expression profiling of microRNAs (miRs), we screened miR-30a, whose level was greatly decreased during Th17 differentiation and the process of demyelination disease, both in MS patients and experimental autoimmune encephalomyelitis (EAE) mice. Enforced constitutive expression of miR-30a in naïve T cells inhibited their differentiation into Th17, and in vivo overexpression of miR-30a resulted in fewer Th17 and alleviative EAE. Moreover, target prediction analysis and dual luciferase report assay revealed that interleukin-21 receptor (IL-21R) was a direct target of miR-30a, a finding consistent with the results that miR-30a downregulated the expression of IL-21R, while overexpression of IL-21R alleviated the inhibitory effect of miR-30a on Th17 differentiation. Taken together, our findings imply that miR-30a inhibits Th17 differentiation and the pathogenesis of MS by targeting IL-21R.

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.

Elevated frequencies of CD4(+) IL-21(+) T, CD4(+) IL-21R(+) T and IL-21(+) Th17 cells, and increased levels of IL-21 in bleomycin-induced mice may be associated with dermal and pulmonary inflammation and fibrosis.

AIM: Systemic sclerosis (SSc) is characterized by immune abnormalities, progressive fibrosis of the skin and internal organs, and microvascular injury and damage. Interleukin-21 receptor (IL-21R) is expressed in the epidermis from patients with SSc. However, information describing the role of IL-21 in SSc is limited. METHODS: We established a mouse model of bleomycin (BLM)-induced fibrosis. The frequency of CD4(+) IL-21(+) T, CD4(+) IL-21R(+) T and IL-21(+) Th17 cells in peripheral blood, skin and lungs of BLM-induced mice were detected by flow cytometry; IL-21 levels in the peripheral blood were evaluated by enzyme-linked immunosorbent assay (ELISA). CD4(+) T cells were isolated from the spleen of BLM-induced and control mice and cultured in vitro alone or in the presence of mrIL-21 or mrIL-21 plus transforming growth factor (TGF)-ß1. The frequency of Th17 cells was detected by flow cytometry; levels of IL-17 were evaluated by ELISA, and the expression of IL-17A and retinoic-acid-receptor-related orphan receptors gamma t (RORγt) messenger RNA were analyzed by real-time polymerase chain reaction. RESULTS: Compared to control mice, the frequency of CD4(+) IL-21(+) T, CD4(+) 21R(+) T and IL-21(+) Th17 cells and the levels of IL-21 were significantly increased in BLM-induced mice. The frequency of CD4(+) IL-21(+) T, CD4(+) 21R(+) T and IL-21(+) Th17 cells and the levels of IL-21 were correlated with dermal and pulmonary inflammation and fibrosis. In vitro analyses indicate that IL-21 promoted the differentiation of Th17 cells from CD4(+) cells isolated from the spleen of BLM-induced mice. CONCLUSION: IL-21 may play an important role in the pathogenesis of SSc as a Th17 effector cytokine, and IL-21 may induce the differentiation of Th17 cells in the BLM-induced SSc mouse model.

IL-21-driven neoplasms in SJL mice mimic some key features of human angioimmunoblastic T-cell lymphoma.

SJL/J mice exhibit a high incidence of mature B-cell lymphomas that require CD4(+) T cells for their development. We found that their spleens and lymph nodes contained increased numbers of germinal centers and T follicular helper (TFH) cells. Microarray analyses revealed high levels of transcripts encoding IL-21 associated with high levels of serum IL-21. We developed IL-21 receptor (IL21R)-deficient Swiss Jim Lambart (SJL) mice to determine the role of IL-21 in disease. These mice had reduced numbers of TFH cells, lower serum levels of IL-21, and few germinal center B cells, and they did not develop B-cell tumors, suggesting IL-21-dependent B-cell lymphomagenesis. We also noted a series of features common to SJL disease and human angioimmunoblastic T-cell lymphoma (AITL), a malignancy of TFH cells. Gene expression analyses of AITL showed that essentially all cases expressed elevated levels of transcripts for IL21, IL21R, and a series of genes associated with TFH cell development and function. These results identify a mouse model with features of AITL and suggest that patients with the disease might benefit from therapeutic interventions that interrupt IL-21 signaling.

CpG oligodeoxynucleotide CpG-685 upregulates functional interleukin-21 receptor on chronic lymphocytic leukemia B cells through an NF-κB mediated pathway.

CpG oligodeoxynucleotides (ODNs) upregulate the interleukin-21 receptor (IL21R) and enhance IL-21-mediated cytotoxicity in chronic lymphocytic leukemia (CLL) B cells. We demonstrate that treatment of CLL B cells with the ODN CpG-685 leads to increased IL21R expression, and that this increased expression enhances the effects of IL-21 treatment as evidenced by increased phosphorylation of JAK1, STAT1, and STAT3, as compared to IL-21 treatment without prior CpG stimulation. Induction of IL21R by CpG-685 also enhanced IL-21-mediated cytotoxicity. The mechanism by which CpG ODNs upregulate IL21R has not been elucidated, although IL21R regulation in T cells has been shown to be linked to T cell receptor-induced Sp1 binding to the IL21R promoter. Here, we demonstrate that luciferase reporter constructs containing the Sp1 binding site have increased basal luciferase activity compared to constructs lacking the Sp1 binding site, but fail to increase luciferase activity with CpG-685 stimulation in CLL B cells. By treating CLL cells with an NF-κB inhibitor, we inhibit the CpG ODN-mediated induction of IL21R, thus demonstrating that CpG-685 upregulates IL21R through an NF-κB mediated pathway. These findings suggest an alternative mechanism for induction of IL-21 receptor in CLL B cells and provide a basis for creation of future combination therapies.