IL-21 Receptor Blockade Shifts the Follicular T Cell Balance and Reduces De Novo Donor-Specific Antibody Generation.

Donor-specific antibodies (DSAs) play a key role in chronic kidney allograft injury. Follicular T helper (Tfh) cells trigger the humoral alloimmune response and promote DSA generation, while T-follicular regulatory (Tfr) cells inhibit antibody production by suppressing Tfh and B cells. Interleukin (IL)-21 exerts a distinct effect on Tfh and Tfr. Here, we studied whether blocking IL-21R with anti-IL-21R monoclonal antibody (αIL-21R) changes the Tfh/Tfr balance and inhibits DSA generation. First, we investigated the impact of αIL-21R on CD4+ T cell proliferation and apoptosis. The results showed that αIL-21R did not have cytotoxic effects on CD4+ T cells. Next, we examined Tfh and regulatory T cells (Tregs) in an in vitro conditioned culture model. Naïve CD4+ T cells were isolated from 3-month-old C57BL/6 mice and cultured in Tfh differentiation inducing conditions in presence of αIL-21R or isotype IgG and differentiation was evaluated by CXCR5 expression, a key Tfh marker. αIL-21R significantly inhibited Tfh differentiation. In contrast, under Treg differentiation conditions, FOXP3 expression was inhibited by IL-21. Notably, αIL-21R rescued IL-21-inhibited Treg differentiation. For in vivo investigation, a fully mismatched skin transplantation model was utilized to trigger the humoral alloimmune response. Consistently, flow cytometry revealed a reduced Tfh/Tfr ratio in recipients treated with αIL-21R. Germinal center response was evaluated by flow cytometry and lectin histochemistry. We observed that αIL-21R significantly inhibited germinal center reaction. Most importantly, DSA levels after transplantation were significantly inhibited by αIL-21R at different time points. In summary, our results demonstrate that αIL-21R shifts the Tfh/Tfr balance toward DSA inhibition. Therefore, αIL-21R may be a useful therapeutic agent to prevent chronic antibody mediated rejection after organ transplantation.

Regulation of the germinal center and humoral immunity by interleukin-21.

Cytokines play critical roles in regulating the development, survival, differentiation, and function of immune cells. Cytokines exert their function by binding specific receptor complexes on the surface of immune cells and activating intracellular signaling pathways, thereby resulting in induction of specific transcription factors and regulated expression of target genes. While the function of cytokines is often fundamental for the generation of robust and effective immunity following infection or vaccination, aberrant production or function of cytokines can underpin immunopathology. IL-21 is a pleiotropic cytokine produced predominantly by CD4+ T cells. Gene-targeting studies in mice, in vitro analyses of human and murine lymphocytes, and the recent discoveries and analyses of humans with germline loss-of-function mutations in IL21 or IL21R have revealed diverse roles of IL-21 in immune regulation and effector function. This review will focus on recent advances in IL-21 biology that have highlighted its critical role in T cell-dependent B cell activation, germinal center reactions, and humoral immunity and how impaired responses to, or production of, IL-21 can lead to immune dysregulation.

An IL-4/21 Inverted Cytokine Receptor Improving CAR-T Cell Potency in Immunosuppressive Solid-Tumor Microenvironment.

Incorporation of inverted cytokine receptor (ICR) such as interleukin (IL)-4 vs. IL-7 (4/7) ICR is one strategy to improve the antitumor activities of chimeric antigen receptor (CAR) modified T (CAR-T) cells facing immunosuppressive cytokines. Here we report a novel interleukin (IL)-4 vs. IL-21 ICR (4/21 ICR) that enhanced CAR-T cell potency in IL-4+ tumor milieu via a different working-mechanism from 4/7 ICR. Upon IL-4 stimulation, 4/21 ICR activated the STAT3 pathway and promoted Th17-like polarization and tumor-targeted cytotoxicity in CAR-T cells in vitro. Furthermore, 4/21 ICR-CAR T cells persisted and eradicated established IL-4+ tumors in vivo. Thus, 4/21 ICR is a promising clinical CAR-T cell therapeutics for solid tumors rich in IL-4.

Dynamic Immune Phenotypes of B and T Helper Cells Mark Distinct Stages of T1D Progression.

Multiple studies of B- and T-cell compartments and their response to stimuli demonstrate alterations in established type 1 diabetes (T1D). Yet it is not known whether these alterations reflect immune mechanisms that initiate islet autoimmunity, promote disease progression, or are secondary to disease. To address these questions, we used samples from the TrialNet Pathway to Prevention study to investigate T-cell responses to interleukin (IL)-2 and regulatory T cell-mediated suppression, the composition of the B-cell compartment, and B-cell responses to B-cell receptor and IL-21 receptor engagement. These studies revealed stage-dependent T- and B-cell functional and immune phenotypes; namely, early features that differentiate autoantibody-positive at-risk first-degree relatives (FDRs) from autoantibody-negative FDRs and persisted through clinical diagnosis; late features that arose at or near T1D diagnosis; and dynamic features that were enhanced early and blunted at later disease stages, indicating evolving responses along the continuum of T1D. We further explored how these specific phenotypes are influenced by therapeutic interventions. Our integrated studies provide unique insights into stable and dynamic stage-specific immune states and define novel immune phenotypes of potential clinical relevance.

Interleukin 21 collaborates with interferon-γ for the optimal expression of interferon-stimulated genes and enhances protection against enteric microbial infection.

The mucosal surface of the intestinal tract represents a major entry route for many microbes. Despite recent progress in the understanding of the IL-21/IL-21R signaling axis in the generation of germinal center B cells, the roles played by this signaling pathway in the context of enteric microbial infections is not well-understood. Here, we demonstrate that Il21r-/- mice are more susceptible to colonic microbial infection, and in the process discovered that the IL-21/IL-21R signaling axis surprisingly collaborates with the IFN-γ/IFN-γR signaling pathway to enhance the expression of interferon-stimulated genes (ISGs) required for protection, via amplifying activation of STAT1 in mucosal CD4+ T cells in a murine model of Citrobacter rodentium colitis. As expected, conditional deletion of STAT3 in CD4+ T cells indicated that STAT3 also contributed importantly to host defense against C. rodentium infection in the colon. However, the collaboration between IL-21 and IFN-γ to enhance the phosphorylation of STAT1 and upregulate ISGs was independent of STAT3. Unveiling this previously unreported crosstalk between these two cytokine networks and their downstream genes induced will provide insight into the development of novel therapeutic targets for colonic infections, inflammatory bowel disease, and promotion of mucosal vaccine efficacy.

IL-21 dependent Granzyme B production of B-cells is decreased in patients with lupus nephritis.

OBJECTIVES: B-cells play a crucial role in the pathogenesis of lupus nephritis. Recently, a separate subset has been discovered characterized by expression of Granzyme B. The aim of this study is to investigate this subset in patients with systemic lupus erythematosus (SLE). METHODS: Isolated PBMCs of SLE-patients (n=30) and healthy controls (n=21) were in vitro stimulated with CPG, IgG+IgM and IL-21. Patients were sub-grouped in patients with and without biopsy proven lupus nephritis. B-cells were analyzed for intracellular Granzyme B expression by flow cytometry. RESULTS: The strongest stimulus for Granzyme B secretion of B-cells was IgG+IgM in presence of IL-21. SLE-patients had a significant decreased percentage of Granzyme B+ B-cells in particular SLE-patients with active disease and with lupus nephritis. CONCLUSIONS: The frequency of GrB+ producing B-cells is reduced in SLE patients. This may contribute to an imbalanced B-cell regulation towards effector B-cells which might promote the development of lupus nephritis.

Absence of Grail promotes CD8+ T cell anti-tumour activity.

T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity.Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.

Anti-CD20-interleukin-21 fusokine targets malignant B cells via direct apoptosis and NK-cell-dependent cytotoxicity.

In spite of newly emerging therapies and the improved survival of patients with non-Hodgkin lymphoma (NHL), relapses or primary refractory disease are commonly observed and associated with dismal prognosis. Although discovery of the anti-CD20 antibody rituximab has markedly improved outcomes in B-cell NHL, rituximab resistance remains an important obstacle to successful treatment of these tumors. To improve the efficacy of CD20-targeted therapy, we fused interleukin 21 (IL-21), which induces direct lymphoma cytotoxicity and activates immune effector cells, to the anti-CD20 antibody (αCD20-IL-21 fusokine). We observed substantially enhanced IL-21R-mediated signaling by the fusokine compared with native IL-21 at equimolar concentrations. Fusokine treatment led to direct apoptosis of lymphoma cell lines and primary tumors that otherwise were resistant to native IL-21 treatment. In addition to direct cytotoxicity, the fusokine enhanced NK cell activation, effector functions, and interferon γ production, resulting in greater antibody-dependent cell-mediated cytotoxicity compared with IL-21 and/or anti-CD20 antibody treatments. Further, the αCD20-IL-21 fusokine stabilizes IL-21 and prolongs its half-life. In vivo αCD20-IL-21 therapy resulted in a significant tumor control in the rituximab-resistant A20-huCD20 tumors. Collectively, the dual functional ability of the αCD20-IL-21 fusokine to induce direct apoptosis and activate immune effector cells may provide benefit over existing treatments for NHL.

IL-21 and IL-21 receptor in the immunopathogenesis of multiple sclerosis.

Cytokines are considered important factors in the modulation of various immune responses. Among them, interleukin (IL)-21 is one of the major immune modulators, adjusting various immune responses by affecting various immune cells. It has been suggested that IL-21 may enhance autoimmunity through different mechanisms, such as development and activation of helper T (TH)-17 and follicular helper T (TFH) cells, activation of natural killer (NK) cells, enhancing B-cell differentiation and antibody secretion and suppression of regulatory T (Treg) cells. Moreover, IL-21 has also been suggested to be an inducer of autoimmunity when following treatment of MS patients with some therapeutics such as alemtuzumab. This review will seek to clarify the precise role of IL-21/IL-21R in the pathogenesis of MS and, in its animal model, experimental autoimmune encephalomyelitis (EAE).

Direct and immune-mediated cytotoxicity of interleukin-21 contributes to antitumor effects in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin D1 in 95% of patients. MCL patients experience frequent relapses resulting in median survival of 3 to 5 years, requiring more efficient therapeutic regimens. Interleukin (IL)-21, a member of the IL-2 cytokine family, possesses potent antitumor activity against a variety of cancers not expressing the IL-21 receptor (IL-21R) through immune activation. Previously, we established that IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B-cell lymphoma cells. Herein, we demonstrate that IL-21 possesses potent cytotoxicity against MCL cell lines and primary tumors. We identify that IL-21-induced direct cytotoxicity is mediated through signal transducer and activator of transcription 3-dependent cMyc upregulation, resulting in activation of Bax and inhibition of Bcl-2 and Bcl-XL. IL-21-mediated cMyc upregulation is only observed in IL-21-sensitive cells. Further, we demonstrate that IL-21 leads to natural killer (NK)-cell-dependent lysis of MCL cell lines that were resistant to direct cytotoxicity. In vivo treatment with IL-21 results in complete FC-muMCL1 tumor regression in syngeneic mice via NK- and T-cell-dependent mechanisms. Together, these data indicate that IL-21 has potent antitumor activity against MCL cells via direct cytotoxic and indirect, immune-mediated effects.

Interleukin-21 receptor blockade inhibits secondary humoral responses and halts the progression of preestablished disease in the (NZB × NZW)F1 systemic lupus erythematosus model.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. METHODS: Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens. RESULTS: IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. CONCLUSION: Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.

Treatment of IL-21R-Fc control autoimmune arthritis via suppression of STAT3 signal pathway mediated regulation of the Th17/Treg balance and plasma B cells.

Interleukin-21 (IL-21) is a T cell-derived cytokine modulating T cell, B cell, and natural killer cell responses. To determine whether IL-21 contributes to pathologic processes, recombinant IL-21 receptor (R) fusion protein (rhIL-21R-Fc) was examined in mice models of autoimmune arthritis (collagen-induced arthritis). DBA/1J mice were immunized with chicken type II collagen and then treated intraperitoneally with rhIL-21R-Fc, which was initiated after the onset of arthritis symptoms in 20% of the cohort. The mice were assessed 3 times per week for signs of arthritis and histologic features as well as serum immunoglobulin. Cytokine messenger RNA levels in the spleen were also examined. STAT3 phosphorylation is dose dependently activated by IL-21 and inhibited by rhIL-21R-Fc in vitro using T cells. Treatment of DBA/1J mice with rhIL-21R-Fc reduced the clinical and histologic signs of CIA. The IL-17 and STAT3-expressing CD4(+) splenocytes dramatically decreased in the rhIL-21R-Fc treated mice. IL-21R-Fc treated mice also decreased the production of IgG, STAT3 phosphorylation, and plasma cell transcription factor (Blimp1). These findings demonstrate a pathogenic role of IL-21 in animal models of RA, suggesting IL-21 as a promising therapeutic target among human RA.

Inflammation and lymphopenia trigger autoimmunity by suppression of IL-2-controlled regulatory T cell and increase of IL-21-mediated effector T cell expansion.

The dynamic interplay between regulatory T cells (T(regs)) and effector T cells (T(effs)) governs the balance between tolerance and effector immune responses. Perturbations of T(reg) frequency and function or imbalances in T(reg)/T(eff) levels are associated with the development of autoimmunity. The factors that mediate these changes remain poorly understood and were investigated in this study in murine autoimmune arthritis. T(regs) displayed a stable phenotype in arthritic mice and were fully functional in in vitro suppression assays. However, their expansion was delayed relative to T(effs) (T follicular helper cells and Th17 cells) during the early stages of autoimmune reactivity. This imbalance is likely to have led to insufficient T(reg) control of T(effs) and induced autoimmunity. Moreover, a counterregulatory and probably IL-7-driven increase in thymic T(reg) production and recruitment to inflamed tissues was too slow for disease prevention. Increased T(eff) over T(reg) expansion was further aggravated by inflammation and lymphopenia. Both these conditions contribute to autoimmune pathogenesis and were accompanied by decreases in the availability of IL-2 and increases in levels of IL-21. IL-2 neutralization or supplementation was used to show that T(reg) expansion mainly depended on this cytokine. IL-21R(-/-) cells were used to demonstrate that IL-21 promoted the maintenance of T(effs). Thus, at inflammatory sites in experimental arthritis, a deficit in IL-2 hampers T(reg) proliferation, whereas exaggerated IL-21 levels overwhelm T(reg) control by supporting T(eff) expansion. This identifies IL-2 and IL-21 as targets for manipulation in therapies for autoimmunity.

Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans.

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.

The cellular source and target of IL-21 in K/BxN autoimmune arthritis.

IL-21 is a pluripotent cytokine that regulates B cell and plasma cell differentiation and is thought be an autocrine factor for follicular helper T cell (T(FH)) and Th17 differentiation. Although IL-21 has been implicated in autoimmune diseases, its relevant cellular source and target cells have not been well characterized. We investigated this issue in the K/BxN mouse model of autoimmune arthritis. Adoptive transfer of KRN-transgenic CD4⁺ T cells into appropriate hosts drives germinal center (GC) formation and autoantibody production against glucose-6-phosphate isomerase, leading to joint inflammation and destruction. By comparing transfer of T or B cells deficient in IL-21 or IL-21R, we were able to dissect the contribution of each cell type. T cells deficient in IL-21 did not induce GC formation or autoantibody production, but they went through normal T(FH) differentiation. However, T cells lacking IL-21R induced Ab titers, GC B cell frequency, and arthritis development similar to wild-type T cells, suggesting that IL-21 is not required for T(FH) differentiation and function. IL-21 acts on B cells, because IL-21R expression on B cells was required to induce disease. In contrast, Th17 cells, a T cell subset that also produces IL-21 and can provide help to B cells, are not required for the GC response and arthritis. These data have implications in developing effective therapies for rheumatoid arthritis and other Ab-mediated autoimmune diseases.

Premature ageing of the immune system relates to increased anti-lymphocyte antibodies (ALA) after an immunization in HIV-1-infected and kidney-transplanted patients.

Low-affinity immunoglobulin (Ig)G with potential autoreactivity to lymphocytes and hypergammaglobulinaemia have been described previously in HIV-1-infected patients. Whether such antibodies increase after challenging the immune system, for example with an immunization, is not known. In the present study, the modulation of antibodies with low affinity and potential autoreactivity was evaluated after 2012-13 seasonal flu vaccination with a simple empirical laboratory test measuring the titres of anti-lymphocyte antibodies (ALA) in two different models of secondary immunodeficiency: HIV-1 vertically infected patients (HIV) and patients treated with immunosuppressive therapies after kidney transplantation (KT) compared to healthy individuals (HC). In parallel, the activation status of B cells and their degree of immune senescence was evaluated by measuring the B cell interleukin (IL)-21R expression/plasma IL-21 levels and the frequencies of mature-activated (MA) and double-negative (DN) B cells. A significant increase of ALA titres was observed after vaccination in HIV and KT but not in HC, and this correlated directly with the frequencies of both MA and DN and inversely with the B cell IL-21R expression. This suggests that the quality of an immune response triggered by flu vaccination in HIV and KT may depend upon the activation status of B cells and on their degree of immune senescence. Further investigations are needed to verify whether high frequencies of MA and DN may also relate to increase autoimmunity after immunization in high-risk populations.

Signal transducer and activator of transcription 3 (STAT3) mutations underlying autosomal dominant hyper-IgE syndrome impair human CD8(+) T-cell memory formation and function.

BACKGROUND: The capacity of CD8(+) T cells to control infections and mediate antitumor immunity requires the development and survival of effector and memory cells. IL-21 has emerged as a potent inducer of CD8(+) T-cell effector function and memory development in mouse models of infectious disease. However, the role of IL-21 and associated signaling pathways in protective CD8(+) T-cell immunity in human subjects is unknown. OBJECTIVE: We sought to determine which signaling pathways mediate the effects of IL-21 on human CD8(+) T cells and whether defects in these pathways contribute to disease pathogenesis in patients with primary immunodeficiencies caused by mutations in components of the IL-21 signaling cascade. METHODS: Human primary immunodeficiencies resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Lymphocytes from patients with loss-of-function mutations in signal transducer and activator of transcription 1 (STAT1), STAT3, or IL-21 receptor (IL21R) were used to assess the respective roles of these genes in human CD8(+) T-cell differentiation in vivo and in vitro. RESULTS: Mutations in STAT3 and IL21R, but not STAT1, led to a decrease in multiple memory CD8(+) T-cell subsets in vivo, indicating that STAT3 signaling, possibly downstream of IL-21R, regulates the memory cell pool. Furthermore, STAT3 was important for inducing the lytic machinery in IL-21-stimulated naive CD8(+) T cells. However, this defect was overcome by T-cell receptor engagement. CONCLUSION: The IL-21R/STAT3 pathway is required for many aspects of human CD8(+) T-cell behavior but in some cases can be compensated by other signals. This helps explain the relatively mild susceptibility to viral disease observed in STAT3- and IL-21R-deficient subjects.

IL-6 activated integrated BATF/IRF4 functions in lymphocytes are T-bet-independent and reversed by subcutaneous immunotherapy.

IL-6 plays a central role in supporting pathological T(H2) and T(H17) cell development and inhibiting the protective T regulatory cells in allergic asthma. T(H17) cells have been demonstrated to regulate allergic asthma in general and T-bet-deficiency-induced asthma in particular. Here we found an inverse correlation between T-bet and Il-6 mRNA expression in asthmatic children. Moreover, experimental subcutaneous immunotherapy (SIT) in T-bet((-/-)) mice inhibited IL-6, IL-21R and lung T(H17) cells in a setting of asthma. Finally, local delivery of an anti-IL-6R antibody in T-bet((-/-)) mice resulted in the resolution of this allergic trait. Noteworthy, BATF, crucial for the immunoglobulin-class-switch and T(H2),T(H17) development, was found down-regulated in the lungs of T-bet((-/-)) mice after SIT and after treatment with anti-IL-6R antibody, indicating a critical role of IL-6 in controlling BATF/IRF4 integrated functions in T(H2), T(H17) cells and B cells also in a T-bet independent fashion in allergic asthma.

The cytokines IL-21 and GM-CSF have opposing regulatory roles in the apoptosis of conventional dendritic cells.

Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

pSTAT3: a target biomarker to study the pharmacology of the anti-IL-21R antibody ATR-107 in human whole blood.

BACKGROUND: IL-21 has been shown to play an important role in autoimmune diseases. ATR-107 is an antibody which directly targets the IL-21 receptor (IL-21R). To aid the clinical development of ATR-107, there is a need for understanding the mechanism of action (MOA) of this antibody when assessing target engagement in human subjects. METHODS: To determine ATR-107 biological activity and potency in human blood, its inhibitory function against IL-21 induced STAT3 phosphorylation in human peripheral T and B cells was measured. RESULTS: The data show that IL-21 induces STAT3 phosphorylation in a concentration-dependent manner, consistent with its migration to the nuclear. Using a flow cytometry based functional whole blood assay, ATR-107 is demonstrated to be a potent IL-21 pathway inhibitor. It competes with IL-21 for receptor binding in a competitive manner, but once it binds to the receptor it behaves like a non-competitive inhibitor, most probably due to the long observed k(off). The concentration-dependent inhibition observed with ATR-107 correlates inversely with the levels of receptor occupancy, both in ex vivo whole blood assays and directly in human blood when ATR-107 was given to healthy volunteers. CONCLUSIONS: IL-21 induced phosphorylation of STAT3 in T and B cells can be used as a biomarker to evaluate the target engagement of ATR-107 in human whole blood. The antibody behaves like a potent non-competitive inhibitor blocking IL-21 induced STAT3 phosphorylation for a long period of time. These results may help with the translation of preclinical information and dose selection towards ATR-107 clinical efficacy.