Blockade Of IL-21 Research Articles

Selective blockade of IL-21 by myricetin impedes T follicular helper cell differentiation by negatively regulating the JAK/STAT/Bcl-6 pathway in a rheumatoid arthritis animal model.

The online version contains supplementary material available at 10.1007/s13205-023-03880-w.

Aging-associated and CD4 T-cell–dependent ectopic CXCL13 activation predisposes to anti–PD-1 therapy-induced adverse events

Clinical success of immune-checkpoint blockade (ICB) cancer immunotherapy is compromised by increased risk of immune-related adverse events (irAEs). However, mechanistic action(s) of immune responses underlying development of irAE remain not fully explored. Here, we found that in tumor-bearing aged, but not young, mice, antiprogrammed death receptor (PD)-1 therapy elicited irAE-like multiorgan dysfunctions with ectopic accumulation of T and B cells in damaged organs. In this preclinical model, the organ toxicities were mediated by immunoglobulin G (IgG) deposition because administration of IG from ICB-treated aged mice induced the pathogenicity specifically in naïve aged hosts. Mechanistically, CD4 T-cell-derived interleukin (IL)-21 upregulated B-cell-homing chemokine, CXCL13, preferentially in irAE organs from aged mice treated with anti-PD-1 therapy. The ICB-induced pathogenicity was alleviated by B-cell depletion or by blockade of IL-21 or CXCL13 activity. These results suggest that age-associated immune regulatory milieu contributes to the formation of tertiary lymphoid structure-like lymphocytic aggregates in irAE organs and irAE-related toxicity employing IL-21-CXCL13-auto-antibody axis. Supporting this, a systemic increase in CXCL13 and Il21 expression in CD4 T cells correlated with irAE incidence in ICB-treated patients. These findings provide rationale for therapeutic usefulness of CXCL13 in irAE management.

Central Roles of OX40L-OX40 Interaction in the Induction and Progression of Human T Cell-Driven Acute Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is one of the major obstacles for the success of allogeneic hematopoietic stem cell transplantation. Here, we report that the interaction between OX40L and OX40 is of critical importance for both induction and progression of acute GVHD (aGVHD) driven by human T cells. Anti-human OX40L monoclonal antibody (hOX40L) treatment could thus effectively reduce the disease severity in a xenogeneic-aGVHD (x-aGVHD) model in both preventative and therapeutic modes. Mechanistically, blocking OX40L-OX40 interaction with an anti-hOX40L antibody reduces infiltration of human T cells in target organs, including liver, gut, lung, and skin. It also decreases IL-21- and TNF-producing T cell responses, while promoting regulatory T cell (Treg) responses without compromising the cytolytic activity of CD8+ T cells. Single blockade of hOX40L was thus more effective than dual blockade of IL-21 and TNF in reducing the severity of aGVHD as well as mortality. Data from this study indicate that OX40L-OX40 interactions play a central role in the pathogenesis of aGVHD induced by human T cells. Therapeutic strategies that can efficiently interrupt OX40L-OX40 interaction in patients might have potential to provide patients with an improved clinical benefit.

Act1 is a negative regulator in T and B cells via direct inhibition of STAT3

Although Act1 (adaptor for IL-17 receptors) is necessary for IL-17-mediated inflammatory responses, Act1- (but not Il17ra-, Il17rc-, or Il17rb-) deficient mice develop spontaneous SLE- and Sjögren’s-like diseases. Here, we show that Act1 functions as a negative regulator in T and B cells via direct inhibition of STAT3. Mass spectrometry analysis detected an Act1–STAT3 complex, deficiency of Act1 (but not Il17ra-, Il17rc-, or Il17rb) results in hyper IL-23- and IL-21-induced STAT3 activation in T and B cells, respectively. IL-23R deletion or blockade of IL-21 ameliorates SLE- and Sjögren’s-like diseases in Act1−/− mice. Act1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T–B cell interaction for B cell expansion and antibody production. Moreover, anti-IL-21 ameliorates the SLE- and Sjögren’s-like diseases in Act1-deficient mice. Thus, IL-21 blocking antibody might be an effective therapy for treating SLE- and Sjögren’s-like syndrome in patients containing Act1 mutation.

Allogeneic dendritic cells stimulated with antibodies against HLA class II polarize naive T cells in a follicular helper phenotype

Follicular helper T cells (Tfh) are crucial for the production of high-affinity antibodies, such as alloantibodies, by providing the signals for B-cell proliferation and differentiation. Here, we demonstrate that human allogeneic dendritic cells (DC) stimulated with antibodies against HLA class II antigens preferentially differentiate human naive CD4+ T cells into Tfh cells. Following coculture with DCs treated with these antibodies, CD4+ T cells expressed CXCR5, ICOS, IL-21, Bcl-6 and phosphorylated STAT3. Blockade of IL-21 abrogated Bcl-6, while addition of the IL-12p40 subunit to the coculture increased CXCR5, Bcl-6, phosphorylated STAT3 and ICOS, indicating that they were both involved in Tfh polarization. We further phenotyped the peripheral T cells in a cohort of 55 kidney transplant recipients. Patients with anti-HLA-II donor-specific antibodies (DSA) presented higher blood counts of circulating Tfh cells than those with anti-HLA-I DSAs. Moreover, there was a predominance of lymphoid aggregates containing Tfh cells in biopsies from patients with antibody-mediated rejection and anti-HLA-II DSAs. Collectively, these data suggest that alloantibodies against HLA class II specifically promote the differentiation of naive T cells to Tfh cells following contact with DCs, a process that might appear in situ in human allografts and constitutes a therapeutic target.

Disruption of Pathogenic Cellular Networks by IL-21 Blockade Leads to Disease Amelioration in Murine Lupus.

Systemic lupus erythematosus (lupus) is characterized by autoantibody-mediated organ injury. Follicular Th (Tfh) cells orchestrate physiological germinal center (GC) B cell responses, whereas in lupus they promote aberrant GC responses with autoreactive memory B cell development and plasma cell-derived autoantibody production. IL-21, a Tfh cell-derived cytokine, provides instructional cues for GC B cell maturation, with disruption of IL-21 signaling representing a potential therapeutic strategy for autoantibody-driven diseases such as systemic lupus erythematosus. We used blockade of IL-21 to dissect the mechanisms by which this cytokine promotes autoimmunity in murine lupus. Treatment of lupus-prone B6.Sle1.Yaa mice with an anti-IL-21 blocking Ab reduced titers of autoantibodies, delayed progression of glomerulonephritis and diminished renal-infiltrating Tfh and Th1 cells, and improved overall survival. Therapy inhibited excessive accumulation of Tfh cells coexpressing IL-21 and IFN-γ, and suppressed their production of the latter cytokine, albeit while not affecting their frequency. Anti-IL-21 treatment also led to a reduction in GC B cells, CD138hi plasmablasts, IFN-γ-dependent IgG2c production, and autoantibodies, indicating that Tfh cell-derived IL-21 is critical for pathological B cell cues in lupus. Normalization of GC responses was, in part, caused by uncoupling of Tfh-B cell interactions, as evidenced by reduced expression of CD40L on Tfh cells and reduced B cell proliferation in treated mice. Our work provides mechanistic insight into the contribution of IL-21 to the pathogenesis of murine lupus, while revealing the importance of T-B cellular cross-talk in mediating autoimmunity, demonstrating that its interruption impacts both cell types leading to disease amelioration.

Prophylactic and therapeutic neutralization of IL-21 bioactivity protects mice from lethality induced by human T cells in a xenogeneic mouse model of graft vs host disease

Abstract IL-21 is a pluripotent cytokine involved in both T and B cell activation/differentiation and plays a major role in Xenogeneic graft vs. host disease (XenoGVHD) where human lymphocytes are transferred into NOD/SCID/gc mice. In order to elucidate the underlying mechanisms of IL-21 action, a neutralizing anti-human (but not anti-mouse) IL-21 mAb, was administered prior to cell transfer and the resultant effects on disease progression determined. Strikingly, IL-21 neutralization halted T cell expansion, and decreased CD4/CD8 T cell ratio resulting in complete protection to wasting and lethality. Loss of IL-21R signaling significantly inhibited several cytokines including, GM-CSF, IL-10, TNF, IFNγ, IL-17A and IL-9, while surprisingly significantly increased IL-5. Both T cell expansion and serum cytokines dramatically increased after discontinuation of anti-IL-21 treatment, resulting in weight loss and death of the mice. Anti-IL-21 was protective when administered 1 or 2 weeks post-graft, where 100% and 40% survival was found, respectively. These data demonstrate that blockade of IL-21 did not simply result in loss of T cell survival, but rather inhibited homeostatic CD4 T cell proliferation and selectively impacted cytokine production switching the response from a Th1 pathogenic to a Th2 protective response. Thus, blockade of IL-21 was not globally eradicating T cell responses, but rather shaping the immune response resulting in protection from GVHD-induced lethality.

PU.1 Expression in T Follicular Helper Cells Limits CD40L-Dependent Germinal Center B Cell Development

PU.1 is an ETS family transcription factor that is important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development and in limiting Th2 cytokine production. Whether PU.1 has functions in other Th lineages is not clear. In this study, we examined the effects of ectopic expression of PU.1 in CD4(+) T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1(lck-/-)) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1(lck-/-) mice had increased numbers of Tfh cells, increased germinal center B cells (GCB cells), and increased Ab production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1(lck-/-) mice compared with control mice. Finally, although blockade of IL-21 did not affect GCB cells in Sfpi1(lck-/-) mice, anti-CD40L treatment of immunized Sfpi1(lck-/-) mice decreased GCB cell numbers and Ag-specific Ig concentrations. Together, these data indicate an inhibitory role for PU.1 in the function of Tfh cells, germinal centers, and Tfh-dependent humoral immunity.

Blockade of IL-21 promotes the induction of T regulatory cells in healthy controls and IBD patients (P5158)

Abstract T regulatory cells (Treg) play a critical role in maintaining immune homeostasis and tolerance to self-antigen. A subset of Treg induced by activation in the presence of TGF-β (induced or iTreg) has been shown to play a role in regulating immune responses and preventing autoimmune disease (AID). IL-21 is a pleiotropic cytokine involved in both innate and adaptive immune responses. Recent data in mouse models has shown that modulation of IL-21 function can influence Treg development and function, but the effect of IL-21 on human Treg is unknown. Therefore, we investigated the role of IL-21 in development of iTreg from T cells from healthy control (HC) and Crohn’s disease (CD) patients. Induced Treg were generated from CD4+CD25- T cells by activation in the presence of IL-2 and TGFβ. Exogenous IL-21 or blocking anti-IL-21 monoclonal antibody was also included on day 0 and cells were stained for intracellular IL-21 and FoxP3 expression on day 7. There was a significant reduction in the number of Foxp3+ T cells (p<0.03) in the presence of exogenous IL-21 and a significant increase when anti-IL-21 antibody was included (p<0.02). These effects on iTreg development were seen in both HC and CD patients. These data suggest targeting IL-21 with neutralizing antibodies can increase iTreg development and hence has promising potential as a novel therapeutic approach for the treatment of AID such as CD.

Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis with polyangiitis (GPA)

IntroductionThe present study aimed to explore a possible role for IL-21 producing Th-cells in the immunopathogenesis of granulomatosis with polyangiitis (GPA).MethodsPeripheral blood from 42 GPA patients in remission and 29 age-matched healthy controls (HCs) were stimulated in vitro, and the frequencies of IL-21 producing Th-cells were determined by flow cytometry. Since Th17-cells produce a low level of IL-21, IL-17 was also included in the analysis. Given that IL-21 is a hallmark cytokine for T follicular helper cells (TFH), we next evaluated the expression of their key transcription factor BCL-6 by RT-PCR and flow cytometry. To investigate the effect of IL-21 on autoantibody-production, PBMCs from GPA patients were stimulated in vitro with BAFF/IL-21 and total IgG and ANCA levels were measured in supernatants. In addition, the expression of IL-21-receptor on B-cells was analyzed.ResultsPercentages of IL-21 producing Th-cells were significantly elevated in GPA-patients compared to HCs, and were restricted to ANCA-positive patients. The expression of BCL-6 was significantly higher in ANCA-positive GPA-patients, as compared with ANCA-negative patients and HCs. IL-21 enhanced the production of IgG and ANCA in vitro in stimulated PBMCs from GPA patients. No difference was found in the expression of the IL-21-receptor on B-cells between ANCA-negative patients, ANCA-positive patients, and HCs.ConclusionThe increased frequency of circulating IL-21 producing Th-cells in ANCA-positive GPA patients and the stimulating capacity of IL-21 on ANCA-production suggest a role for these cells in the immunopathogenesis of GPA. Blockade of IL-21 could constitute a new therapeutic strategy for GPA.

Psoriasis, from Pathogenesis to Therapeutic Strategies: IL-21 as a Novel Potential Therapeutic Target

Psoriasis is a common (1-3% of the population worldwide), multifactorial, immune-mediated chronic skin disease. In psoriasis pathogenesis an over-reaction of local innate immune response initiates inflammation with subsequent involvement of adaptive immune response leading to the production of a panel of cytokines, chemokines and growth factors leading to epidermal hyperplasia. Recently, IL-21 has been involved in this process as this cytokine is overexpressed in psoriatic skin and can cause epidermal hyperplasia and inflammation when injected intradermally into mice. Moreover blockade of IL-21 with a human antibody against IL-21 reduces the epidermal thickness and the expression of Th1 and Th17 genes in the well-characterized model of human psoriasis-xenograft mouse. Therefore, the inhibition of this cytokine may be therapeutically effective in the treatment of psoriasis. Here we will review recent data on psoriasis pathogenesis focusing on the role of IL-21 as novel therapeutic target.

Interleukin‐21 modulates Th1 and Th17 responses in giant cell arteritis

Giant cell arteritis (GCA) is a large-vessel vasculitis of unknown origin. Recent findings indicate that at least 2 separate lineages of CD4+ T cells, Th1 and Th17 cells, participate in vascular inflammation. The pathways driving these T cell differentiations are incompletely understood, but may provide novel therapeutic targets. This study was undertaken to identify cytokines involved in the pathogenesis of GCA. Thirty GCA patients fulfilling the American College of Rheumatology criteria, with active disease or disease in remission, and 30 age-matched controls were included. Levels of 27 cytokines were determined in culture supernatants, and flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and immunohistochemical analysis of temporal artery samples were performed. Multiparametric analysis of cytokines produced by PBMCs associated with GCA disease activity identified a signature involving interleukin-2 receptor (IL-2R), IL-12, interferon-γ (IFNγ), IL-17A, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). An expansion of Th1 and Th17 cells and a decrease in Treg cells were observed in the peripheral blood of patients with active GCA. An expansion of IL-21-producing CD4+ T cells was also observed in patients with active GCA and correlated positively with Th17 and Th1 cell expansion. Immunohistochemical analysis revealed IFNγ, IL-17A, and IL-21 expression within inflammatory infiltrates. Stimulation of purified CD4+ T cells with IL-21 increased Th1 and Th17 cell frequencies and decreased FoxP3 expression. In contrast, blockade of IL-21 using IL-21R-Fc markedly decreased the production of IL-17A and IFNγ and increased FoxP3 expression. Our findings indicate that IL-21 plays a critical role in modulating Th1 and Th17 responses and Treg cells in GCA, and might represent a potential target for novel therapy.

IL-21R is required for the systemic accumulation of activated B and T lymphocytes in MRLlpr mice (117.9)

Abstract IL-21 is a pleiotropic cytokine that can influence the activation, differentiation and expansion of B and T cells. Lupus prone MRL/MpJ-Faslpr/lpr/J (MRLlpr) mice have been shown to develop renal pathology and autoantibodies in an IL-21 dependent manner. We generated MRLlpr mice deficient in IL-21 receptor (MRLlpr.IL-21R-/-) to assess the impact of loss of IL-21R signaling on the systemic B and T cell lymphoaccumulation and activation that occurs in MRLlpr mice. Consistent with therapeutic blockade of IL-21, MRLlpr.IL21R-/- mice developed reduced proteinuria, renal pathology and serum IgG autoantibody levels. Lymphoaccumulation was significantly reduced in MRLlpr.IL-21R-/- mice, as evidenced by decreased lymphadenopathy and splenomegaly. Importantly, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R deficient MRLlpr mice. In addition, activated CD4+ CD44+ CD62Llo T cells failed to accumulate. We also demonstrate that T extrafollicular helper cells, which are a subset of activated CD4+ T cells that are reported to be the primary inducers of antibody production in MRLlpr mice, require IL-21R for their generation. T helper cell differentiation was also found to be impacted by loss of IL-21R, as IFN-γ producing CD4+ T cells were significantly reduced in MRLlpr.IL21R-/- mice. Together, our data highlight that IL-21 promotes multiple pathogenic B and T cell effector responses in MRLlpr mice.

Targeting interleukin-21 in inflammatory diseases

Introduction: IL-21, a new member of the type 1 cytokine superfamily, is produced by various subsets of CD4+ T cells and binds to a composite receptor that consists of a specific receptor, termed IL-21 receptor and the common γ-chain subunit. Initially considered to be a critical regulator of T and B cell function, IL-21 is now known to regulate the activity of many other cell types, including both immune and non-immune cells.Areas covered: In this review, we discuss the biological features of IL-21 and summarize recent advances in the pathogenic role of IL-21 in chronic inflammatory diseases. Moreover, we discuss why IL-21 blockers can have a place in the therapeutic armamentarium for patients with immune-mediated diseases and the potential risks of such treatments.Expert opinion: Data emerging from studies in human and experimental models of autoimmunity suggest that IL-21 is critically involved in the initiation and/or progression of inflammatory reactions where self-reactive immune cells or antibodies cause damage in tissue. Thus, theoretically, targeting IL-21 could help attenuate the activation of inflammatory pathways and facilitate the resolution of tissue damaging immune responses. However, one should also take into consideration some potential risks that could derive from the blockade of IL-21.

Regulation of human Th9 differentiation by type I interferons and IL‐21

Interleukin (IL)-9-producing CD4(+) T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-beta and IL-4 directed human naive CD4(+) T cells to produce IL-9. Whereas IL-4 suppressed TGF-beta-induced Foxp3 expression, TGF-beta failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-3. Addition of IL-1 beta, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-gamma and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4(+) T cells.

Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21

Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

IL‐21 Modulates CD4+ CD25+ Regulatory T‐Cell Homeostasis in Experimental Autoimmune Encephalomyelitis

The homeostasis of CD4+ CD25+ regulatory T cells (Tregs) depends on the cytokine interleukin (IL)-2. As IL-21 shares sequence homology with IL-2 and the IL-21 receptors contain a gamma-chain common to IL-2, we hypothesized that IL-21 could also affect the homeostasis of Tregs. We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), an animal model of relapsing-remitting human multiple sclerosis. We show that blockade of IL-21 in SJL/J mice before and after the induction of EAE enhances the influx of inflammatory cells into the central nervous system (CNS). The blockade of IL-21 leads to proliferation of proteolipid peptide (PLP(139-151))-autoreactive CD4+ T cells, which are capable to cause severe EAE in adoptively transferred recipient mice. Conversely, Tregs from mice where IL-21 was blocked, lose their capacity to prevent EAE induced PLP(139-151)-reactive T cells. Notably, direct effects of IL-21 on Tregs are confirmed by studies of blockade of IL-21 in mice expressing a green fluorescent protein 'knocked' into a Foxp3 allele, in which a reduction of the number of Tregs and a downregulation of their frequency and expression of Foxp3 are observed. These data suggest a role of the IL-21/IL-21R axis in the homeostasis of Tregs in CNS autoimmunity.