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NKG2D chimeric antigen receptor (CAR)-modified T cells (NKG2D CAR-T cells) have been reported to be preclinically efficient in several tumors, but little is known whether NKG2D CAR-T cells co-expressing IL21 (IL21-NKG2D CAR-T cells) display greater antitumor activity in multiple myeloma (MM). In this study, the lentivirus has been produced for expression of the IL21 sequence linked to the extracellular NKG2D sequence with the signal peptide linked through the CD8α hinge-transmembrane domain to the 4-1BB molecule fused with the CD3-ζ chain signaling domain, and the engineered IL21-NKG2D CAR-T cells and NKG2D CAR-T cells were constructed. The CAR expression on CAR-T cells was assessed by flow cytometry, and the killing effects of CAR-T cells on MM were assessed by the cytotoxicity assay and ELISA assay. Moreover, xenograft models were also established to evaluate the ability of IL21-NKG2D-CAR-T cells to eliminate MM in vivo. Our results indicated that NKG2D CAR-T cells had dramatic cytotoxicity on MM cells in vitro, and co-expression of IL-21 significantly increased the cytotoxicity of NKG2D CAR-T cells on MM cells. Remarkably, we found that dexamethasone enhanced the cytotoxicity of IL21-NKG2D CAR-T cells on MM cells. Furthermore, IL21-NKG2D CAR-T cells also displayed significant anti-myeloma activity in vivo. In conclusion, IL21-NKG2D CAR-T cells had dramatic cytotoxicity on MM cells in vitro and in vivo, and a system to apply IL21-NKG2D CAR-T cells and low dosage of dexamethasone for the future study of the targeted therapy for MM has been established., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: To elucidate the association between the changes in intracellular metabolism in the early stage of B cell activation and systemic lupus erythematosus (SLE) pathogenesis., Methods: CD19 + or CD19 + CD27 - (naïve) cells from the peripheral blood of healthy controls and lupus patients were cultured under different stimuli. The changes in intracellular metabolism and signalling pathways in these cells were evaluated., Results: Stimulation with CpG (Toll-like receptor 9 (TLR9) ligand) in vitro induced enhanced interleukin 21 (IL-21) receptor expression in CD19 + CD27 - cells after 24 hours. The addition of IL-21 to the CpG stimulation enhanced the extracellular acidification rate, which indicates glycolysis, within 30 min. IL-21 receptor (IL-21R) expression induced by CpG stimulation was selectively inhibited by 2-deoxy-D-glucose (hexokinase 2 (HK2) inhibitor) and heptelidic acid (glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitor). RNA immunoprecipitation with anti-GAPDH antibody revealed that CpG stimulation dissociated the binding between IL-21R messenger RNA (mRNA) and GAPDH under no stimulation. HK2 and GAPDH expression were higher in CD19 + CD27 - cells of lupus patients than in those of healthy controls, and GAPDH expression was correlated with the plasmocyte count and disease activity score., Conclusion: IL-21R mRNA-GAPDH binding dissociation associated with rapid glycolytic enhancement by the TLR9 ligand in B cells may induce plasmocyte differentiation through IL-21 signals and be involved in exacerbating SLE., Competing Interests: Competing interests: MU has received speaking fee and honoraria from GlaxoSmithKline. SI has received consulting fees, speaking fees and honoraria from Ono, Taisho, Janssen, UCB, AstraZeneca, Pfizer, GlaxoSmithKline, Astellas, Asahi Kasei, Teijin, Eli Lilly, Nippon-Shinyaku, Daiichi-Sankyo, AbbVie, Eisai, Mitsubishi-Tanabe and Chugai. KS has received speaking fee from Chugai, Astellas, AbbVie, Taisho, Janssen, Eli Lilly Japan, Ayumi and Gilead Sciences and has received research funding from UCB Japan. SK has received consulting fees, speaking fees and honoraria from Eli Lilly, Bristol-Myers and GlaxoSmithKline and has received research grants from Daiichi-Sankyo, AbbVie, Behringer-Ingelheim and Astellas. YM has received consulting fees, speaking fees and honoraria from Eli Lilly and has received research grants from GlaxoSmithKline. SN has received consulting fees, speaking fees, lecture fees and/or honoraria from AstraZeneca, GlaxoSmithKline, Pfizer, Bristol-Myers, Astellas, Asahi Kasei, AbbVie, Chugai, Sanofi, Eisai, Gilead Sciences, Mitsubishi-Tanabe, Janssen, Eli Lilly and Ayumi. YT has received consulting fees, speaking fees and honoraria from Behringer-Ingelheim, Eli Lilly, AbbVie, Gilead, AstraZeneca, Bristol-Myers, Chugai, Daiichi-Sankyo, Eisai, Pfizer, Mitsubishi-Tanabe and GlaxoSmithKline and has received research grants from Asahi Kasei, AbbVie, Chugai, Eisai, Takeda, Daiichi-Sankyo and Behringer-Ingelheim., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Children with recurrent/metastatic neuroblastoma (NB) have a dismal survival (<25%). Novel therapies are desperately needed. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed on NB. C021 is a selective oncolytic herpes simplex virus modified to overexpress human interleukin-21 (hIL-21), a cytokine that enhances natural killer (NK) cell cytotoxicity. In the current study, we successfully engineered ex - vivo -expanded NK cells to express a chimeric antigen receptor (CAR) against ROR1 using mRNA electroporation and investigated the efficacy of anti-ROR1-CAR-NK cells combined with C021 in targeting ROR1 + NB. We found that C021-infected NB cells secreted hIL-21 in vitro and in vivo . Compared to the non-cytokine-secreting parental virus C134, C021 significantly enhanced the in vitro cytotoxicity ( p  < 0.05) of anti-ROR1-CAR-NK cells with increased interferon (IFN)-γ ( p  < 0.05), granzyme B ( p  < 0.05), and perforin ( p  < 0.05) secretion against NB cells. Furthermore, the combination of C021 and anti-ROR1-CAR-NK cells significantly extended the survival of human NB xenografted NSG mice compared to controls (mock NK, ROR1-CAR-NK, C134, C021, C134+ROR1-CAR-NK, and C021+mock NK). Our results suggest that cytokine-secreting oncolytic virus in combination with CAR-NK cells is a novel, effective immunotherapeutic approach for high-risk NB., Competing Interests: This work was presented in part at the Pediatric Transplantation & Cellular Therapy Consortium (2023), Fort Worth, TX; International Society for Cell & Gene Therapy (2022), San Francisco, CA; and Transplantation & Cellular Therapy Meetings of the American Society for Transplantation and Cellular Therapy (2022), Salt Lake City, Utah. M.S.C. has served as a consultant for Jazz Pharmaceuticals, Omeros Pharmaceuticals, Servier Pharmaceuticals, Abbvie, and Novartis Pharmaceuticals; on speakers bureau for Jazz Pharmaceuticals, Servier Pharmaceuticals, Amgen, Inc., Sanofi, and Sobi; and on the advisory board for Astra Zeneca and has received research funding from Celularity, Merck, Miltenyi Biotec, Servier, Omeros, Jazz, and Janssen. D.A.L. reports personal fees and other fees from Kiadis Pharma, CytoSen Therapeutics, Courier Therapeutics, and Caribou Biosciences outside the submitted work. In addition, D.A.L. has a patent broadly related to NK cell therapy of cancer with royalties paid to Kiadis Pharma. T.P.C. recently served as a one-time consultant to Blueprint, Incyte, and Oncopeptides and a DSMB chair for SpringWorks and is a cofounder of Vironexis Biotherapeutics, Inc., (© 2024 The Authors.)

Objective: To investigate serum TL1A levels and their correlation with Th17 cells, IL-17, and IL-21 in children with Graves' disease (GD)., Methods: Thirty-seven children (12 males and 25 females) aged 9-14 years with newly diagnosed and untreated GD were enrolled in this study. Serum TL1A, IL-17, and IL-21 levels were measured using enzyme-linked immunosorbent assay (ELISA). The percentage of Th17 cells in peripheral blood was determined by flow cytometry. The correlation between serum TL1A levels and Th17 cells, IL-17, and IL-21 was analyzed using Pearson's correlation coefficient., Results: Serum TL1A levels and the percentage of Th17 cells were significantly higher in children with GD compared to healthy controls (P<0.05). Serum IL-17 and IL-21 levels were also significantly elevated in GD patients (P<0.05). Serum TL1A levels positively correlated with the percentage of Th17 cells (r=0.625, P<0.05), IL-17 (r=0.573, P<0.05), and IL-21 (r=0.542, P<0.05) in children with GD., Conclusion: Serum TL1A levels are increased in children with GD and positively correlate with Th17 cells, IL-17, and IL-21, suggesting that TL1A may play a role in the pathogenesis of GD by regulating Th17 cell differentiation and the production of IL-17 and IL-21., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hao, Yang, Lian, Yin, Xiao and Liu.)

Local delivery of mRNA-based immunotherapy offers a promising avenue as it enables the production of specific immunomodulatory proteins that can stimulate the immune system to recognize and eliminate cancer cells while limiting systemic exposure and toxicities. Here, we develop and employ lipid-based nanoparticles (LNPs) to intratumorally deliver an mRNA mixture encoding the cytokines interleukin (IL)-21 and IL-7 and the immunostimulatory molecule 4-1BB ligand (Triplet LNP). IL-21 synergy with IL-7 and 4-1BBL leads to a profound increase in the frequency of tumor-infiltrating CD8 + T cells and their capacity to produce granzyme B and IFN-γ, leading to tumor eradication and the development of long-term immunological memory. Mechanistically, the efficacy of the Triplet LNP depends on tumor-draining lymph nodes to tumor CD8 + T-cell trafficking. Moreover, we highlight the therapeutic potential of the Triplet LNP in multiple tumor models in female mice and its superior therapeutic efficacy to immune checkpoint blockade. Ultimately, the expression of these immunomodulators is associated with better overall survival in patients with cancer., Competing Interests: Competing interests: J.F., E.B., S.D.K., and F.L. are employees of etherna. J.F., E.B., S.D.K., and F.L. have applied for a patent related to the study (Compositions and methods for delivery of agents to immune cells; WO2023118411A1). S.D.K. and B.G.D.G. have applied for a patent related to the ionizable lipids used in this work (Ionizable lipids; WO2022136641A1). All other authors declare no potential conflicts of interest., (© 2024. The Author(s).)

Background: Hypoxia-induced pulmonary hypertension (HPH) is a subgroup of type 3 pulmonary hypertension that may cause early right ventricular failure and eventual cardiac failure, which lacks potential therapeutic targets. Our previous research demonstrated that T follicular helper (T FH ) cells that produce IL-21 were involved in HPH. However, the molecular mechanisms of T FH /IL-21-mediated pathogenesis of HPH have been elusive. Here we investigate the role of T FH cells and IL-21 in HPH., Methods: Studies were performed in C57BL/6 mice or IL-21 knockout mice exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in mouse lung and pulmonary arterial smooth muscle cells (PASMCs). M2 signature gene (Fizz1), M1 signature genes (iNos, IL-12β and MMP9), GC B cell and its marker GL-7, caspase-1, M2 macrophages, T FH cells, Bcl-6 and IL-21 level were measured. Proliferation rate of PASMCs was measured by EdU. Pyroptosis was assessed using Hoechst 33,342/PI double fluorescent staining., Results: In response to chronic hypoxia exposure-induced pulmonary hypertension, IL-21 -/- mice or downregulation of T FH cells in WT mice developed blunted pulmonary hypertension, attenuated pulmonary vascular remodelling. Furthermore, chronic hypoxia exposure significantly increased the germinal center (GC) B cell responses, which were not present in IL-21 -/- mice or downregulation of T FH cells in WT mice. Importantly, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Consistently, significantly enhanced expression of M2 macrophage marker Fizz1 were detected in the bronchoalveolar lavage fluid of HPH mice. Moreover, alveolar macrophages that had been cultivated with IL-21 promoted PASMCs proliferation and pyroptosis in vitro, while a selective CX3CR1 antagonist, AZD8797 (AZD), significantly attenuated the proliferation and pyroptosis of the PASMCs. Finally, ECM1 knockdown promoted IL-2-STAT5 signaling and inhibited Bcl-6 signaling to inhibit T FH differentiation in HPH., Conclusions: T FH /IL-21 axis amplified pulmonary vascular remodelling in HPH. This involved M2 macrophage polarization, PASMCs proliferation and pyroptosis. These data suggested that T FH /IL-21 axis may be a novel therapeutic target for the treatment of HPH., Competing Interests: Declarations. Ethics approval: All animals procedures were approved by the Laboratory Animal Ethics Committee of Hunan Provincial People’s Hospital (XSY-2021-42). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive., Methods: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion., Results: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells., Conclusions: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Solid tumors significantly impact global health, necessitating enhanced prevention, early diagnosis, and treatment approaches. Tumor immunotherapy, notably through programmed cell death protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1), offers new hope to patients with advanced tumors, although many still do not benefit. Interleukin-21 (IL-21), a cytokine produced by certain immune cells, performs various biological functions by activating the JAK/STAT signaling pathway. Currently, recombinant IL-21 demonstrates promising antitumor activity and acceptable toxicity in several clinical trials. However, challenges such as side effects, off-target reactions, and a short half-life limit the effectiveness of cytokine-based immunotherapies. Therefore, researching enhanced IL-21 treatment strategies in solid tumors is crucial. Integrating IL-21 with various treatment modalities, including immune checkpoint inhibitors, additional cytokines, vaccines, or radiotherapy, is essential for improving response rates and prolonging patient survival. This review explores the specific mechanisms of IL-21 in prevalent high-incidence tumors, examines improved strategies for IL-21 in solid tumors, and aims to provide a theoretical basis for developing targeted treatment strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, abnormal inflammation, and fibrosis of the skin and internal organs, notably the skin and lungs, significantly impairing quality of life. There is currently no cure for SSc, and its etiology remains largely unknown, presenting a primary barrier to effective treatment. We investigated the role of interleukin-21 (IL-21) in the pathogenesis of SSc., Methods: We assessed the expression levels of fibrosis-related genes in human dermal fibroblasts exposed to IL-21 and TGF beta. We also induced SSc in wild-type C57BL/6 mice and IL-21 knockout (KO) mice with a C57BL/6 background using bleomycin (Bleomycin). Histological analyses were conducted on skin and lung tissues from these mice. The distribution and expression levels of fibrosis-related proteins in the tissues were examined via immunohistochemistry and quantitative real-time PCR. Furthermore, we measured the frequency of Th1, Th2, and Th17 cells among splenocytes through flow cytometry., Results: IL-21 activation led to STAT3 phosphorylation more than TGF beta in dermal fibroblasts. In IL-21 KO mice with BLM-induced SSc, skin thickness and lung fibrosis were reduced. The absence of IL-21 in these mice resulted in suppressed expression of fibrosis-related genes, including Col1a1, Col1a2, Col3a1, CTGF, α-SMA, STAT3, and TGFβ, in the skin and lungs. It also led to a decreased frequency of Th1, Th2, and Th17 cells, as well as a lower Th17/Treg ratio among splenocytes, factors known to contribute to the development of SSc., Conclusions: IL-21 contributes to the development of SSc by promoting the expression of fibrosis-related genes and modulating the levels of CD4+ T cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Follicular helper T (Tfh) cells have been implicated in the pathophysiology of numerous diseases. This study investigated the hypothetical function of peripheral blood IL-21+ Tfh cells in the etiology of focal segmental glomerulosclerosis (FSGS). Tfh cell subsets were identified via flow cytometry in PBMCs from 15 patients with FSGS and 9 healthy controls (HCs). Moreover, a cytometric bead array (CBA) was used to determine the level of IL-21 in the serum. The proportions of IL-21+ cTfh cells, IL-21+ PD-1+ cTfh cells and serum IL-21 were lower in FSGS patients than in HCs. In FSGS patients, the serum IL-21 concentration was positively correlated with the frequency of IL-21+ cTfh cells and IL-21+ PD-1+ cTfh cells. The frequencies of IL-21+ cTfh cells and IL-21+ PD-1+ cTfh cells were negatively associated with 24-h urine protein but positively correlated with eGFR, serum albumin and serum IgG. CONCLUSIONS: An aberrant frequency of IL-21+ Tfh cells was detected in FSGS patients, which may provide a better understanding of FSGS pathogenesis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Interleukin-21 (IL-21) is a cytokine produced by various cell types, including T cells, natural killer cells, myeloid cells, and B cells, and has a broad range of potential applications in cancer therapy. To improve the therapeutic index, we explored the use of fusion technologies that involved linking other anticancer peptides to the IL-21 gene using specific linkers., Objectives: This study aimed to compare the anticancer potential of IL-21 and IL-21 fusion proteins., Methods: Antimicrobial peptides possessing anticancer properties were fused with IL-21 gene using a flexible linker (-GGGGS-), and the resulting construct was inserted into the pSecTag2a mammalian expression vector. The cassette was transfected into several cancer cell lines including H1 HeLa, HepG2, MCF-7, MDA-MB-231, HCT-116, HCC-1954, HEK-293, and SF-767. The cytotoxic effects of IL-21 and fusion proteins were evaluated using MTT, Caspase-3, LDH, and scratch assays., Results: The IL-21-Tachyplesin I fusion protein had the strongest antiproliferative activity against all tested cancer cells, followed by IL21-LPSBD2 and IL-21. In contrast, IL21-Cop A3, IL21-CSP I-Plus, and IL21-RGD Temporin-Las did not inhibit the viability of cancer cells., Conclusion: Fusion technology is a promising therapeutic technique that can be used to enhance the cytotoxicity and antiproliferative activity of anticancer proteins such as IL-21., Competing Interests: Declarations. Conflict of interest: The authors (s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Ethical approval: This article does not contain any studies involving human participants or animals performed by any of the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4 + T cell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling, resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML, serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR T cell treatment on LSCs in vitro. Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore, promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Current therapies for systemic lupus erythematosus that target a particular factor or cell type exhibit limited effectiveness. To address this limitation, our focus was on CD132, a subunit common to six inflammatory factor receptors implicated in SLE. Our study revealed heightened CD132 expression in SLE patients' lymphocytes, contributing to the production of pro-inflammatory cytokines and immunoglobulins. We developed a novel humanized anti-CD132 monoclonal antibody, named as 2D4. 2D4 efficiently blocked IL-21 and IL-15, with limited effectiveness against IL-2, thereby suppressing T and B cells without disrupting immune tolerance. In the mouse immunization model, 2D4 virtually inhibited T cell-dependent, antigen-specific B-cell response. In lupus murine models, 2D4 mitigated inflammation by suppressing multiple pro-inflammatory cytokines and anti-dsDNA antibody titers, also diminishing proteinuria and glomerulonephritis. Compared to Belimumab, 2D4 exhibited superior efficacy in ameliorating the inflammatory state and preserving renal function. Moreover, 2D4 exhibited the ability to inhibit the production of pro-inflammatory factors and autoantibodies in PBMCs from individuals with SLE, highlighting its therapeutic potential for SLE individuals. Potent, 2D4 has the potential to significantly improve clinical outcomes in SLE and other complex autoimmune disorders., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: Peripheral helper T (Tph) cells have an important role in the induction of humoral immune responses and autoantibody production. Accordingly, it is feasible that this lymphocyte subset has a relevant role in the pathogenesis of autoimmune thyroid diseases (AITD). In this study we aim to analyze the levels and function of Tph cells in blood samples from patients with AITD., Methods: We performed an observational study with cases and controls. Blood samples were obtained from nineteen patients with Hashimoto's thyroiditis (HT), twenty-four with Graves' disease (GD), and fifteen healthy controls. In addition, the levels of follicular T helper (Tfh) cells and Tph cells, the release of interleukin-21 (IL-21) by these lymphocytes and the number of plasmablasts were analyzed by multi-parametric flow cytometry analyses., Results: Increased percentages of Tfh and Tph lymphocytes were detected in patients with HT and GD. Furthermore, an enhanced synthesis of the cytokine IL-21 by these cells was observed. Accordingly, we detected significant higher percentages of plasmablasts in patients with GD, and these values tended to be also higher in HT patients. Moreover, significant positive associations were observed between the levels of Tfh or Tph and the number of plasmablast or anti-TSHR Ab titers in patients with AITD., Conclusion: Our data suggest that Tph lymphocytes may have a relevant role in the pathogenesis of AITD., (© 2024. The Author(s).)

Background: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5, and IL-13 cytokines produced by T H 2 lymphocytes or type 2 innate lymphoid cells (ILC2s). IL-2 family cytokines play a key role in the differentiation, homeostasis, and effector function of innate and adaptive lymphocytes., Objective: IL-9 and IL-21 boost activation and proliferation of T H 2 and ILC2s, but the relative importance and potential synergism between these γ common chain cytokines are currently unknown., Methods: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human subjects with allergic asthma compared with nonasthmatic controls. Here, we also measured IL-21 in both groups., Results: IL-9 played a central role in controlling innate IL-33-induced lung inflammation by promoting proliferation and activation of ILC2s in an IL-21-independent manner. Conversely, chronic house dust mite-induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, which controlled T H 2 activation, eosinophilia, total serum IgE, and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, as combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage samples we measured elevated IL-21 protein within the allergic asthmatic group compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets., Conclusions: IL-9 and IL-21 play important and nonredundant roles in allergic asthma by boosting ILC2s and T H 2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach., Competing Interests: Disclosure statement This work was funded by a Baekeland Mandate of Flanders Innovation & Entrepreneurship (VLAIO) (HBC.2019.2598) to F.B. B.N.L. acknowledges support from a European Research Council (ERC) advanced grant (789384 ASTHMA CRYSTAL CLEAR), a concerted research initiative grant from Ghent University (GOA, 01G010C9), a Fonds Wetenschappelijk Onderzoek (FWO) Methusalem grant (01M01521) and an FWO Excellence of Science (EOS) Consortium research grant (3G0H1222), and the Flanders Institute of Biotechnology (VIB). H.H. is supported by a concerted research initiative grant from Ghent University (GOA, 01G010C9) and FWO EOS Consortium research grant (3G0H1222). M.J.S. acknowledges support from FWO Vlaanderen (12Y5322N), Fund Suzanne Duchesne (managed by the King Baudouin Foundation), and Fondation ACTERIA. Disclosure of potential conflict of interest: N. P. Smith is a consultant for Hera Biotech. A.-C. Villani has financial interest in 10X Genomics. B. D. Medoff serves on advisory boards for Sanofi, Verona Pharma, and Apogee Therapeutics and has sponsored research agreements from Sanofi and Regeneron. R. Bigirimana is an employee of argenx. C. Blanchetot is a consultant and shareholder of argenx. B. N. Lambrecht receives consultancy fees from GSK Biologics, Novartis, Sanofi, AstraZeneca, ALK, OncoArendi, and argenx and has research grants from ALK, argenx, AstraZeneca, GSK, GSK Biologics, and Johnson & Johnson. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Systemic sclerosis (SSc), a rare and lethal autoimmune disorder where patients presents diverse clinical features, therefore unravelling a potential biomarker within a specific cohort is crucial for improving patient care, especially for rare diseases. This study sought to identify potential biomarkers in Tunisian SSc patients. Gene expression analysis of interleukins (IL)-21 and IL-22 in peripheral blood mononuclear cells, using quantitative real-time polymerase chain reaction (qrt-pcr), revealed upregulated IL-21 and downregulated IL-22 in SSc patients compared to healthy controls. Notably, IL-21 overexpression in patients correlated with pulmonary complications, a severe SSc manifestation. Interestingly, flow cytometry analysis displayed no difference in Th17 cells between groups, suggesting that Th17 might not be the primary drivers of cytokine dysregulation. The hypothesis was supported by qRT-PCR, which analysed two key genes: IL-17A and RORγt. Finally, we examined RNA sequencing data to further validate our hypothesis. Collectively, our study provides novel insights into the cytokine landscape of SSc in Tunisian patients, highlighting a dysregulation in IL-21 and IL-22 expression, and suggesting that IL-21 could be a potential biomarker of severity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

This study aimed to explore the molecular mechanism of neuronal cell adhesion molecule (NrCAM) by regulating Th17 cell differentiation in the pathogenesis of Graves' disease (GD). Naïve CD4 + T cells were isolated from peripheral blood mononuclear cells of GD patients and healthy control (HC) subjects. During the differentiation of CD4 + T cells into Th17 cells, NrCAM level in GD group was improved. Interference with NrCAM in CD4 + T cells of GD patients decreased the percentage of Th17 cells. NrCAM overexpression in CD4 + T cells of HC subjects increased the percentage of Th17 cells and upregulated p-IκBα, p50, p65, c-Rel protein expressions, and NF-κB inhibitor BAY11-7082 partially reversed NrCAM effect. NrCAM overexpression promoted the degradation of IκBα, and overexpression of small ubiquitin-related modifier 1 (SUMO-1) inhibited IκBα degradation. NrCAM overexpression reduced IκBα binding to SUMO-1. During Th17 cell differentiation in HC group, NrCAM overexpression increased IL-21 levels and secretion, and IL-21 neutralizing antibody reversed this effect. IL-21 level was decreased after p65 interference in CD4 + T cells of HC subjects. p65 interacts with IL-21 promoter region. In conclusion, NrCAM binds to SUMO-1 and increases phosphorylation of IκBα, leading to activation of NF-κB pathway, which promotes Th17 cell differentiation., (© 2024 The Scandinavian Foundation for Immunology.)

Background: De novo donor-specific antibodies (dnDSAs) significantly affect the long-term outcomes of liver transplantation (LT), highlighting the importance of risk prediction. Follicular helper T (Tfh) cells have been implicated in dnDSA formation after transplantation. Considering the influence of immune response gene polymorphisms on transplantation outcomes, we investigated the association between polymorphisms in Tfh cell-related genes and dnDSA formation after LT., Methods: Fifty-three living-donor LT patients were included in this study. Single nucleotide polymorphisms (SNPs) were identified in six Tfh cell-related genes crucial for differentiation and maturation (BCL6, CXCR5, CXCL13, ICOS, CD40L, and IL-21); their association with the development of dnDSA after LT was evaluated., Results: Among the 53 recipients, 9 developed dnDSAs. BCL6 and IL-21 SNPs showed potential associations with dnDSA formation, enabling risk stratification., Conclusions: Variations in Tfh cell-related genes may predispose individuals to dnDSA formation after LT, emphasizing the importance of genetic factors for predicting post-transplant complications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Introduction and Objective: Peripheral and follicular helper T lymphocytes (Tph and Tfh, respectively) have an important role in B cell immune responses and the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Although several studies on the number of Tph and Tfh cells in these conditions have been published, different phenotypes have been employed for their analysis. In this study, we assessed the levels and function of Tph and Tfh cells in blood samples from patients with RA and SLE by using an extended immunophenotype., Materials and Methods: In a cross-sectional pilot study, blood samples from twenty-seven patients with RA and fifteen with SLE, and twenty-six healthy controls were studied. The levels of Tph (CD4 + PD-1 + CXCR5 - CD38 + CD69 + ICOS + ) and Tfh (CD4 + PD-1 + CXCR5 + CD38 + CD69 + ICOS + ) cells were analyzed by flow cytometry. In addition, the function of Tph/Tfh cells was estimated by measuring the synthesis of IL-21 by these lymphocytes as well as the number of circulating plasmablasts (CD19 + CD27 + CD20 - CD38 hi )., Results: Increased percentages of Tph and Tfh lymphocytes were detected in patients with RA and SLE. Furthermore, the synthesis of IL-21 tended to be higher in both conditions, and higher levels of plasmablasts were detected in these patients, compared to controls. In patients with SLE, the number of Tph cells was associated with disease activity and with the levels of circulating plasmablasts, whereas in patients with RA a significant correlation between Tph cells and evolution time was observed., Discussion and Conclusions: Our data of Tph and Tfh lymphocytes, based in the analysis of an extended phenotype of these cells, provides further evidence on their involvement in the pathogenesis of RA and SLE., (Copyright © 2024 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.)

Antibody responses induced by current vaccines for influenza and SARS-CoV-2 often lack robust cross-reactivity. As hubs where diverse immune cells converge and interact, the alterations in the immune microenvironment within lymph nodes (LNs) are intricately linked to immune responses. Herein, we designed a lipid nanoparticle (LNP) loaded with circular RNA (circRNA) and targeted to LNs, in which CXCL13 was directly integrated into antigen-encoding circRNA strands. We demonstrated that CXCL13 alters the transcriptomic profiles of LNs, especially the upregulation of IL-21 and IL-4. Meanwhile, CXCL13 promotes the formation of germinal center and elicits robust antigen-specific T cell responses. With the codelivery of CXCL13 and the antigen, CXCL13 enhances cross-reactive antibodies against influenza virus and SARS-CoV-2, achieving protection against both homologous and heterologous influenza virus challenges in a mouse model. Notably, the targeted modification of LNP surfaces with antibodies helps address some of the challenges associated with lyophilized LNP vaccines, which is crucial for the long-term storage of LNP-circRNA vaccines. Overall, the circRNA-based antigen-CXCL13 coexpression system developed herein provides a simple and robust platform that enhances the magnitude and breadth of antibody responses against multiple viral glycoproteins, highlighting the potential utility of CXCL13 in inducing broad immune responses., Competing Interests: Competing interests statement:The authors declare no competing interest.

Objective: To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer., Methods: The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2. The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments. Lactate dehydrogenase (LDH) release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells, and the secretion levels of IFN-γ, IL-21 and CCL19 were determined with enzyme-linked immunospot assay (ELISPOT) and ELISA. A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site, and the fluorescence signals were captured using a fluorescent microscopy., Results: The NKP30 ligand B7H6, which was almost undetectable in normal tissues and blood cells, was highly expressed (over 90%) in lung cancer cells. Compared with NKP30 CAR-T cells and conventional T cells, IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells. The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ, IL-21 and CCL19. In the zebrafish models, CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells, but these differences were not statistically significant between the two CAR-T cells., Conclusion: Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.

B cells play a key role in humoral immune responses by producing antibodies. Although there are numerous research on memory B cells definition markers and cytokines on B cell development, different studies have yielded contradictory conclusions due to species studied, the different cells and stimulating agents used. In the current study, we conducted a detailed characterization of B cells in human CBMCs, PBMCs and tonsil, including expression of Igs, activation and memory markers. Furthermore, we found that considerable amounts of IgA and IgG were expressed by CD27 - B cells. These "Atypical" memory B cells corresponded to approximately 50% of IgG + and IgA + B cells in blood, this proportion even reached 90% in tonsil. In addition, we investigated the effect of IL-21 and TGF-β1 on the membrane-bound form and secreted form of Igs using PBMCs and purified blood B cells. There were actual differences between the effect of cytokines on Igs secretion and surface expression. Our study will be helpful to advance the knowledge and understanding of humoral memory., (© 2024. The Author(s).)

Rheumatoid arthritis (RA) is frequent, an imbalance between helper cells (Th) and regulatory T cells (Treg) is the fundamental immunological cause of RA. This study investigates how recombinant human programmed cell death 1 (PD-L1) protein affects circulating T follicular helper (cTfh), circulating T follicular regulatory (cTfr), and their equilibrium. Magnetic bead sorting was used to select CD4+CXCR5+T cells from RA patients' and healthy individuals' peripheral blood mononuclear cells for in vitro growth. Recombinant human PD-L1 protein stimulated CD4+CXCR5+T cells. Cell counting kit 8 (CCK-8), flow cytometry surface labeling, ELISA, and RT-PCR were used to measure CD4+CXCR5+T cell proliferation inhibition, cTfh and cTfr frequencies, IL-21 expression, and PI3K, AKT, Bcl-6, and Blimp-1 mRNA levels. The recombinant human PD-L1 protein dose-dependently inhibited the proliferation of CD4+CXCR5+T cells in active RA peripheral blood. However, it has a weaker inhibitory effect on healthy peripheral blood CD4+CXCR5+T cells. PD-L1 protein decreased cTfh in active RA peripheral blood CD4+CXCR5+T overall cultured cells but did not affect cTfr; The cTfr/cTfh ratio increased but did not affect the frequency of cTfh and cTfr in healthy persons' cultured CD4+CXCR5+T cells. PD-L1 protein reduced IL-21 in CD4+CXCR5+T cell culture supernatant from active RA peripheral blood. Recombinant human PD-L1 protein lowered PI3K, AKT, and Bcl-6 mRNA in active RA peripheral blood CD4+CXCR5+T cell culture, including significant differences. But Blinmp-1 mRNA variations were neither substantial nor statistically different. PD-1/PD-L1 limits cTfh proliferation, differentiation, and activation via the PI3K/AKT signaling pathway regulates its immunological balance with cTfr, and corrects the cTfr/cTfh imbalance by controlling their interaction.

Objective: To investigate the effect of feeder layer cells expressing interleukin (IL)-21 on the amplification of NK cells In Vitro ., Methods: The K562 cell line with IL-21 expression on its membrane was constructed by electroporation, and co-cultured with NK cells after inactivation. The proliferation of NK cells was observed. The killing function of the amplified NK cells In Vitro was evaluated by the lactate dehydrogenase (LDH) and interferon-γ (IFN-γ) release assay. A colorectal cancer xenograft model in NOD/SCID mice was established, and a blank control group, a NK cell group and an amplified NK cell group were set up to detect the tumor killing effect of amplified NK cells in vivo ., Results: K562 cells expressing IL-21 on the membrane were successfully constructed by electroporation. After co-culturing with K562 cells expressing IL-21 on the membrane for 17 days, the NK cells increased to 700 times, which showed an enhanced amplification ability compared with control group ( P < 0.001). In the tumor cell killing experiment In Vitro , there was no significant difference in the killing activity on tumor cells between NK cells and amplified NK cells, and there was also no significant difference in mice in vivo ., Conclusion: K562 cells expressing IL-21 on the membrane can significantly increase the amplification ability of NK cells In Vitro , but do not affect the killing function of NK cells In Vitro and in vivo . It can be used for the subsequent large-scale production of NK cells In Vitro .

Renal transplantation is the preferred treatment option for patients with end-stage renal disease (ESRD) in a clinical setting. Antibody mediated rejection (AMR) is one of the leading causes of graft dysfunction. To address the current shortcomings in the early diagnosis and treatment of AMR in clinical practice, this article analyzes the distribution of different circulating T follicular helper (cTfh) cell subtypes and B cell subpopulations in peripheral blood and detects the cytokine levels of chemokine ligand 13 (CXCL13), interleukin-21 (IL-21), and interleukin-4 (IL-4) related to cTfh cells in peripheral blood of kidney transplant recipients. Moreover, we also explore the correlation between cTfh cells, peripheral blood memory B cells, and AMR, their value as early predictive indicators of AMR, and explore potential therapeutic targets for AMR patients. Our results indicate that the proportion of cTfh cells increased at the onset of AMR, which plays an important role in antigen-specific B-cell immune regulation. Activation of cTfh cells in AMR patients correlates with phenotypes of memory B cells and plasma blasts. cTfh cells and memory B cells have promising diagnostic efficacies and predictive values for AMR. The proportion of cTfh cells to CD4 + T cells and the proportion of memory B cells to CD19 + B cells are correlated with serum creatinine levels, indicating that cTfh cells and memory B cells may be involved in the progression of AMR. In addition, the CXCL13, IL-21, and IL-4, which were associated with cTfh cells, may be involved in the onset of AMR., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Interleukin 27 (IL-27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL-27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL-27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL-21, anti-L. infantum antibodies and parasite loads. We performed functional assays followed by IL-27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD-1, CTLA-4, phospho-Stat-1/3, T-bet, GATA3 and nitric oxide production (NO). Both IL-27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL-21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti-L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL-27 and IL-21 participate of immune responses in CanL. IL-27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD-1, CTLA-4, T-bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL-27 are involved in CanL pathogenesis in the host, and may be targets for new therapies., (© 2024 John Wiley & Sons Ltd.)

IL-21/IL-21R signaling dysregulation is linked to multiple chronic intestinal inflammatory disorders in humans and animal models of human diseases. In addition to its critical requirement for the generation and development of germinal center B cells, IL-21/IL-21R signaling can also regulate the effector functions of a variety of T-cell subsets. The antibody-mediated abrogation of IL-21/IL-21R signaling led to the impaired expression of IFN-γ by mucosal CD4+ T cells from human subjects with colitis, suggesting an IL-21/IL-21R-triggered positive feedback loop of the TH1 immune response in the colon. Despite recent advances in our understanding of the mechanisms underpinning the regulation of proinflammatory immune responses by the IL-21/IL-21R signaling axis, it remains unclear how this pathway or its downstream molecules contribute to inflammation during bacterial-induced colitis. This study found that IL-21 enhances the surface expression of IL-12Rβ2, but not IL-12Rβ1, in CD4+ T cells, leading to TH1 differentiation and stability. Consistently, these findings also point to an indispensable role of the IL-12Rβ2 signaling axis in promoting proinflammatory immune responses during Citrobacter rodentium-induced colitis. Genetic deletion of the IL-12Rβ2 signaling pathway led to the attenuation of C. rodentium-induced colitis in vivo. The genetic deletion of the IL-12Rβ2 signaling pathway did not alter the host's ability to respond adequately to C. rodentium infection or the ability of Il12rb2-/- mice to express antigen-specific cytokines (IFN-γ, IL-17A). IL-21 is a pleiotropic cytokine exerting a wide range of immunomodulatory functions in multiple tissues, and its direct targeting may result in undesirable off-target consequences. These findings highlight the possibility for targeted manipulations of signaling cascades downstream of main regulators of proinflammatory responses to control invading pathogens while preserving the integrity of host immune responses. A better understanding of the novel mechanisms by which IL-21/IL-21R signaling regulates bacterial-induced colitis will provide insights into the development of new therapeutic and preventive strategies to harness IL-21/IL-21R signaling or its downstream molecules to treat infectious colitis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Liver failure (LF) is prevalent in China and is characterized by complex pathogenesis, challenging clinical management, poor prognosis, and rising incidence and mortality rates. The immune status is an important factor affecting LF prognosis. Interleukins (Ils) are a type of cytokine that act and interact with multiple cells, including immune cells. These signaling molecules play important roles in intercellular information transmission, including the regulation of immune cells; mediation of the activation, proliferation, and differentiation of T and B cells; and orchestration of the inflammatory response. To date, many studies have explored the correlation between IL expression and liver disease prognosis, but few studies have evaluated Ils as the prognostic biomarkers of LF. This article reviews the potential use of Ils as the prognostic biomarkers of LF. Particularly, it evaluates the predictive values of IL-21, IL-22, and IL-31, the three often overlooked yet promising prognostic biomarkers, in predicting susceptibility to LF. Harnessing biomarkers for early prognostic insights can facilitate tailored treatment strategies and enhance patient survival. Thus, this article focuses on the identification of IL-21, IL-22, and IL-33 as biomarkers in preclinical and clinical studies on LF and reviews their role as biomarkers in the pathogenesis and diagnosis of LF., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to declare., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Rheumatoid arthritis (RA) is a systemic autoimmune disease, mediated by a complex interaction between B cells and various subsets of T cells. Dysfunction of helper T (Th) and regulatory T (Treg) cells may contribute to the breakdown of self-tolerance and the progression of autoimmune disease. In this study, we investigated the activity of Th and Treg cells on the differentiation of autologous B cells in vitro using cell cultures from the peripheral blood of healthy controls (HCs) and RA patients. The expressions of programmed death 1 (PD-1) and IL-21 were monitored as activation markers for Th cells. Unstimulated Th cells from RA patients showed remarkably higher PD-1 expression than HC samples. Stimulation of Th cells from RA patients with Staphylococcus enterotoxin B (SEB) in the presence of B cells significantly induced their PD-1 and IL-21 expression at a considerably higher level in RA compared to HCs, and Treg cells did not affect IL-21 production. When monitoring B-cell differentiation, a significantly higher frequency of plasma cells was observed, even in unstimulated samples of RA patients compared to HCs. In the SEB-stimulated co-cultures of the RA samples, plasma cell frequency and IgG production were considerably higher than in HCs and were not significantly affected by Tregs. These findings demonstrate that Th cells are constitutively active in RA, and their hyperactivity upon interaction with diseased B cells may lead to uncontrolled antibody production.

Introduction: NK cells are dysfunctional in myasthenia gravis (MG), but the mechanism is unclear. This study aims to measure associations and underlying mechanisms between the NK cells and the development of MG., Methods: Twenty healthy controls (HCs) and 53 MG patients who did not receive glucocorticoids and immunosuppressants were collected. According to the Myasthenia Gravis Foundation of America (MGFA) classification, MG patients were categorized into MGFA I group (n = 18) and MGFA II-IV group (n = 35). Flow cytometry, cell sorting, ELISA, mRNA-sequencing, RT-qPCR, western blot, and cell culture experiments were performed to evaluate the regulatory mechanism of exhausted NK cells., Results: Peripheral NK cells in MGFA II-IV patients exhibit exhausted phenotypes than HCs, marked by the dramatic loss of total NK cells, CD56 dim CD16- NK cells, elevated PD1 expression, reduced NKG2D expression, impaired cytotoxic activity (perforin, granzyme B, CD107a) and cytokine secretion (IFN-γ). Plasma IL-6 and IL-21 are elevated in MG patients and mainly derived from the aberrant expansion of monocytes and Tfh cells, respectively. IL-6/IL-21 cooperatively induced NK-cell exhausted signature via upregulating SOCS2 and inhibiting the phosphorylation of STAT5. SOCS2 siRNA and IL-2 supplement attenuated the IL-6/IL-21-mediated alteration of NK-cell phenotypes and function., Discussion: Inhibition of IL-6/IL-21/SOCS2/STAT5 pathway and recovery of NK-cell ability to inhibit autoimmunity may be a new direction in the treatment of MG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Han, Bi, Yang, Lin, Li, Zhang and Bu.)

In a previous study, it has been shown that the population of Th17 lymphocytes was increased in patients with FMF. IL-21 and IL-23 play significant roles in the production and differentiation of Th17 cells. In this study, we aimed to evaluate serum levels of IL-21 and IL-23 in FMF patients both at diagnosis and after treatment, and to compare these levels with those of healthy controls. Twenty-seven newly diagnosed patients with FMF in attack-free periods and twenty-seven healthy volunteers enrolled in the study. The groups were comparable with respect to age and gender. IL-21 and IL-23 levels in serum samples from patients at the time of diagnosis, in remission after treatment, and from the control groups were analysed using the ELISA method. There was no significant difference between the cytokine levels of the patient group at the time of diagnosis and the cytokine levels of the control group (for IL-21, p: 0.28 and for IL-23, p: 0.56). Similarly, there was no significant difference between the patients' cytokine levels at the time of diagnosis and after treatment (for IL-21, p: 0.99 and for IL-23, p: 0.08). Interleukin levels at the time of diagnosis did not differ among patient groups based on the presence of clinical findings or the M694V genotype. Our results suggest that IL-21 and IL-23 do not play a role in the pathogenesis of the disease. However, while interpreting these findings, it should be considered that patients with active episodes were excluded and cytokine levels were not measured in tissue samples., (© 2024. The Author(s).)

To investigate the regulatory effects of T helper 9 (Th9) cytokines on the proliferation, apoptosis and immune escape of thyroid cancer cells. The survival rate of human thyroid cancer cell line TPC-1 after treatment with 0, 1, 2.5, 5, 10, 20 ng/ml IL-9 (or IL-21) was determined by CCK-8 method and suitable concentrations of IL-9 and IL-21 were screened out. The TPC-1 cells cultured in vitro were randomly grouped into control group, IL-9 group, IL-21 group and IL-9+IL-21 group. After treatment with IL-9 and IL-21 factors, the proliferation and apoptosis of TPC-1 cells in each group were detected by CCK-8 method and flow cytometry, respectively. The flow cytometry was applied to detect the proportion of Th9 and activated CD8+ T cells in human peripheral blood lymphocytes co-cultured with TPC-1 in each group. The expression of TPC-1 and IL-9R and IL-21R protein in each group and human peripheral blood lymphocytes. Compared with the control group, the cell viability PCNA and Bcl-2 protein expression in TPC-1 cells were lower in the IL-9 group, IL-21 group and IL-9+IL-21 group (P<0.05). The apoptosis rate, proportions of Th9 and activated CD8+ T cells, killing rate of human peripheral blood lymphocytes, the expression of Bax and caspase-3 proteins in TPC-1 cells, the expression of TPC-1 and human peripheral blood lymphocytes IL-9R and IL-21R proteins were all higher (P<0.05) in IL-9+IL-21 group compared with the IL-9 group and the IL-21 group. The cell viability, PCNA and Bcl-2 protein expression in TPC-1 cells in the IL-9+IL-21 group were all lower (P<0.05). Th9 cytokines can promote the differentiation of Th9 cells and CD8+ T cells, enhance their lethality, reduce the immune escape of thyroid cancer cells, and then inhibit their proliferation and promote their apoptosis., Competing Interests: Declarations. Ethical Approval: Not applicable. Consent to Participate: Informed consent was obtained from all individual participants included in the study. Consent for Publication: The participants have consented to the submission in this Journal. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Interleukin-21 (IL-21) is an important antitumor cytokine that contributes to the proliferation and differentiation of CD8 + T cells. It has been proven to enhance the response to immune checkpoint inhibitors (ICIs) in various solid tumors. However, its role in hepatocellular carcinoma (HCC) has not yet been clarified. In this research, we aimed to investigate the antitumor effect of IL-21 in HCC and its effect on ICI treatment. Through transcriptome sequencing analysis and immunohistochemistry validation, we found that patients with high IL-21 expression had a better prognosis. HCCs with high expression of IL-21 had higher infiltration of CD8 + T cells, increased expression of immune checkpoints, and an improved response to ICI treatment. In conclusion, IL-21 can enhance the efficacy of ICI treatment and improve the prognosis of patients by promoting the infiltration of CD8 + T cells and the expression of immune checkpoint-related genes., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

We explored the impact of immune dysregulation on pancreatic beta cell injury in HIV patients. Analyzing 105 participant samples, we observed lower IL-21 levels and elevated immune checkpoint levels (e.g. PD-1, CD27+, CD40+) in untreated HIV patients. Notably, soluble TIM-3 correlated positively with improved beta cell function and inversely with beta cell stress, suggesting its potential role in beta cell protection in untreated HIV., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by defective antibody production and impaired differentiation of B cells. B cell proliferation is an essential step for antibody synthesis. Depending on the nature of the stimulus, their response may be either T-cell-dependent or T-cell-independent., Methods: We studied 23 CVID patients and 14 healthy donors (HD). The patients were categorized based on their percentage of memory B cells. In addition to standard immunophenotyping of circulating human B and T cell subsets, an in vitro CFSE dilution assay was used to assess the proliferative capacity of B cells and to compare the activation of the T cell-dependent and T cell-independent response among the patients., Results: Patients with a reduction in memory B cells exhibited an increase in follicular T cells (Tfh) and showed low proliferation in response to PKW, CpG, and SAC stimuli (Condition II) ( p = 0.0073). In contrast, patients with a normal percentage of memory B cells showed a high expression of IL-21R and low proliferation in response to CPG (Condition III); IL-21, CD40L, and anti-IgM (Condition IV) stimuli ( p = 0.0163 and p = 0.0475, respectively)., Conclusion: Defective proliferation in patients depends on the type of stimulus used and the phenotypic characteristics of the patients. Further studies are necessary to understand the disease mechanisms, which may guide us toward identifying genetic defects associated with CVID.

The application of CAR-T cells in solid tumors poses several challenges, including poor T cell homing ability, limited infiltration of T cells and an immunosuppressive tumor environment. In this study, we developed a novel approach to address these obstacles by designing GPC3-specific CAR-T cell that co-express IL-21 and CXCL9 (21 × 9 GPC3 CAR-T cells) and blocking the PD-1 expression on it. The proliferation, cell phenotype, cytokine secretion and cell migration of indicated CAR-T cells were evaluated in vitro. The cytotoxic activities of genetically engineered CAR-T cells were accessed in vitro and in vivo. Compared to conventional GPC3 CAR-T cells, the 21 × 9 GPC3 CAR-T cells demonstrated superior proliferation, cytokine secretion and chemotaxis capabilities in vitro. Furthermore, when combined with PD-1 blockade, the 21 × 9 GPC3 CAR-T cells exhibited enhanced proliferation, cytokine secretion and enrichment of effector T cells such as CTL, NKT and TEM cells. In xenograft tumor models, the PD-1 blocked 21 × 9 GPC3 CAR-T cells effectively suppressed HCC xenograft growth and increased T cell infiltration. Overall, our study successfully generated GPC3 CAR-T cells expressing both IL-21 and CXCL9, demonstrated that combining PD-1 blockade can further enhance CAR-T cell function by promoting proliferation, cytokine secretion, chemotaxis and antitumor activity. These findings present a hopeful and potentially effective strategy for GPC3-positive HCC patients., (© 2024. The Author(s).)

Glioblastoma is the most common brain cancer, with a 5-year survival rate of less than 10%. This grim prognosis highlights the urgent need for novel therapeutic approaches. In this issue of Cancer Cell, Shanley et al. 1 report an innovative engineering strategy to supercharge NK cell immunity against glioblastoma., Competing Interests: Declaration of interests N.D.H. reports stock or other ownership in oNKo-Innate and serves on advisory boards for Bristol Myers Squibb and Syena. N.D.H. is an inventor on multiple patents related to using adoptive NK cell therapy for cancer and targeting IL-15R signaling for cancer therapy., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM., Competing Interests: Declaration of interests M.S., M. Daher, R.B., H.R., S.A., Y.L., N.U., E.L., E.J.S., K.R., and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Takeda Pharmaceutical. R.B., E.J.S., K.R., and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Affimed GmbH. K.R. participates on the Scientific Advisory Board for Avenge Bio, Virogin Biotech, Navan Technologies, Caribou Biosciences, Bit Bio Limited, Replay Holdings, oNKo Innate, and The Alliance for Cancer Gene Therapy ACGT. K.R. is the Scientific founder of Syena. E.J.S. has served on the Scientific Advisory Board for Adaptimmune, Axio, Celaid, FibroBiologics, Navan Technologies, New York Blood Center and Novartis. M Daher participates on the Scientific Advisory Board for Cellsbin. M.S., H.S., E.J.S., and K.R. have filed for a patent (20230074303); “Cell immunotherapy for the treatment of cancer.”, (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: T follicular helper (Tfh) cells drive humoral immunity by facilitating B cell responses, but the functional role of Tfh cells in the pathogenesis of idiopathic membranous nephropathy (IMN) remains unclear., Objectives: This study aimed to establish a rat experimental membranous nephropathy model, investigate the phenotypic characteristics of Tfh cells, and analyze a clinically significant correlation between Tfh cells., Material and Methods: Passive Heymann nephritis (PHN) rats were induced by immunizing Sprague Dawley rats with anti-Fx1A serum. The frequency of Tfh and B cell subsets was analyzed with flow cytometry (FC). The serum concentration of interleukin-21 (IL-21), the relative mRNA expression levels of IL-21 and B cell lymphoma 6 (Bcl-6) in spleen mononuclear cells (MNCs), and the kidney infiltration of CD4+ T cells and IL-21 were assessed. The potential correlations among these measures were analyzed., Results: In comparison with the control group, significantly increased percentages of Tfh cells, inducible T cell co-stimulator-positive (ICOS+) Tfh cells, and mRNA expression of Bcl-6 were detected in the spleen of PHN rats. Elevated IL-21 expression was detected in the serum and kidneys. Remarkably, the percentage of splenic ICOS+ Tfh cells was positively correlated with 24 h urine protein concentrations (r = 0.676, p = 0.011) in PHN rats., Conclusion: These data indicate that ICOS+ Tfh cells contribute to development of IMN, and they might be potential therapeutic targets for IMN.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy affecting the skin, entheses, and joints. Over the past decade, experimental evidence has revealed the activation of several immune cells and signaling cascades in modulating the pathophysiology of PsA. Recently, targeted therapies have been developed to combat the severity of disease. However, with diverse etiologies, flareups, and relapses, there has been an increased prevalence and mortality associated with PsA in recent years. Therefore, it is imperative to investigate new potential mediators and combination therapies to manage PsA pathogenesis. IL-21, an immunomodulatory cytokine, has pleiotropic effects on immune cells and the protein cascades involved in PsA pathogenesis. Recently, emerging evidence of increased IL-21 levels in patients with PsA has engendered much enthusiasm for its potential as a therapeutic target. Here, we unmasked IL-21 as a significant modulator of PsA pathogenesis and reviewed the comorbidities associated with the disease, further cataloging future therapeutic modalities to ameliorate PsA progression., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs., (© 2024. The Author(s).)

Non-small cell lung cancer (NSCLC) is a prevalent and aggressive global malignancy. Conventional surgical treatments, radiotherapy, chemotherapy, and targeted therapies often fall short in halting disease progression due to inherent limitations, resulting in suboptimal prognosis. Despite the advent of immunotherapy drugs offering new hope for NSCLC treatment, current efficacy remains insufficient to meet all patient needs. Therefore, actively exploring novel immunotherapeutic approaches to further reduce mortality rates in NSCLC patients has become a crucial focus of NSCLC research. This article aims to systematically review the anti-tumor effects of interleukin-21 and follicular helper T cells in NSCLC immunotherapy by summarizing and analyzing relevant literatures from both domestic and international sources, as well as exploring the potential for enhancing NSCLC treatment prospects through immune checkpoint regulation via immunotherapeutic means.
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Background: Follicular helper T cells (Tfh) are pivotal in B cell responses. Activation of the purinergic receptor P2X7 on Tfh cells regulates their activity. We investigated the ATP-P2X7R axis in circulating Tfh (cTfh) cells during Respiratory Syncytial Virus (RSV) infection., Methods: We analyzed two cohorts: children with RSV infection (moderate, n=30; severe, n=21) and healthy children (n=23). We utilized ELISA to quantify the levels of PreF RSV protein-specific IgG antibodies, IL-21 cytokine, and soluble P2X7R (sP2X7R) in both plasma and nasopharyngeal aspirates (NPA). Additionally, luminometry was employed to determine ATP levels in plasma, NPA and supernatant culture. The frequency of cTfh cells, P2X7R expression, and plasmablasts were assessed by flow cytometry. To evaluate apoptosis, proliferation, and IL-21 production by cTfh cells, we cultured PBMCs in the presence of Bz-ATP and/or P2X7R antagonist (KN-62) and a flow cytometry analysis was performed., Results: In children with severe RSV disease, we observed diminished titers of neutralizing anti-PreF IgG antibodies. Additionally, severe infections, compared to moderate cases, were associated with fewer cTfh cells and reduced plasma levels of IL-21. Our investigation revealed dysregulation in the ATP-P2X7R pathway during RSV infection. This was characterized by elevated ATP levels in both plasma and NPA samples, increased expression of P2X7R on cTfh cells, lower levels of sP2X7R, and heightened ATP release from PBMCs upon stimulation, particularly evident in severe cases. Importantly, ATP exposure decreased cTfh proliferative response and IL-21 production, while promoting their apoptosis. The P2X7R antagonist KN-62 mitigated these effects. Furthermore, disease severity positively correlated with ATP levels in plasma and NPA samples and inversely correlated with cTfh frequency., Conclusion: Our findings indicate that activation of the ATP-P2X7R pathway during RSV infection may contribute to limiting the cTfh cell compartment by promoting cell death and dysfunction, ultimately leading to increased disease severity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Russo, Raiden, Algieri, Bruera, De Carli, Sarli, Cairoli, De Lillo, Morales, Seery, Otero, Sananez, Simaz, Alfiero, Rubino, Moya, Aedo Portela, Herrero, Blanco, Salcedo Pereira, Ferrero, Geffner and Arruvito.)

T-cell-engaging bispecific antibody (T-BsAb, also known as BiTE) therapy has emerged as a powerful therapeutic modality against multiple myeloma. Given that T-BsAb therapy redirects endogenous T cells to eliminate tumor cells, reinvigorating dysfunctional T cells may be a potential approach to improve the efficacy of T-BsAb. While various immunostimulatory cytokines can potentiate effector T-cell functions, the optimal cytokine treatment for T-BsAb therapy is yet to be established, partly due to a concern of cytokine release syndrome driven by aberrant interferon (IFN)-γ production. Here, we functionally screen immunostimulatory cytokines to determine an ideal combination partner for T-BsAb therapy. This approach reveals interleukin (IL)-21 as a potential immunostimulatory cytokine with the ability to augment T-BsAb-mediated release of granzyme B and perforin, without increasing IFN-γ release. Transcriptome profiling and functional characterization strongly support that IL-21 selectively targets the cytotoxic granule exocytosis pathway, but not pro-inflammatory responses. Notably, IL-21 modulates multiple steps of cytotoxic effector functions including upregulation of co-activating CD226 receptor, increasing cytotoxic granules, and promoting cytotoxic granule delivery at the immunological synapse. Indeed, T-BsAb-mediated myeloma killing is cytotoxic granule-dependent, and IL-21 priming significantly augments cytotoxic activities. Furthermore, in vivo IL-21 treatment induces cytotoxic effector reprogramming in bone marrow T cells, showing synergistic anti-myeloma effects in combination with T-BsAb therapy. Together, harnessing the cytotoxic granule exocytosis pathway by IL-21 may be a potential approach to achieve better responses by T-BsAb therapy.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder and its characteristics include the immune system's invasion of the healthy lining of the joints and the articular structures degeneration. The IL-21 pro-inflammatory cytokine, and the reactive oxygen species (ROS) might have a role in the RA etiopathogenesis. The present study assessed the correlation of IL-21 with vitamin 25(OH)D and the ROS., Methods: The study included 120 RA patients and 60 healthy group. The RA patients were categorized based on rheumatoid factor (RF) seropositivity or seronegativity and the RA severity. Chemiluminescent immunoassay and 10% hematocrit were used to check levels of vitamin 25(OH)D and ROS, respectively. ELISA was used for the detection of IL-21 in the plasma., Results: The RA patients had a significantly reduced vitamin 25(OH)D level compared to the healthy controls. The IL-21 and ROS were however significantly increased in the RA patients compared to the controls. Further, the seropositive RF and the high RA severity patients had significant IL-21 and ROS increase in comparison with the seronegative RF and the low severity RA patients. Finally, IL-21 negatively correlated with vitamin 25(OH)D, but positively correlated with the ROS., Conclusion: This is the first investigation to confirm the relationship between IL-21 with vitamin 25(OH)D and the ROS among the RA patients. The findings indicate that vitamin 25(OH)D is reduced in the RA patients' serum. ROS and IL-21 are also associated with increased RA severity., (© 2024 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Objective: Chlamydia pneumoniae ( C. pneumoniae ) is a gram-negative intracellular bacterium that causes respiratory infections in humans. C. pneumoniae is responsible for cell activation and production of cytokines that may contribute to inflammatory responses in asthma. Cell-mediated immune responses are important for protective immunity; however, these responses may be impaired in asthma. In this study, we examined cytokine responses (IL-21, IL-12, IL-13) responsible for T helper (Th)1 versus Th2 responses in C. pneumoniae -stimulated PBMC from subjects with or without asthma. These cytokines could be potential biomarkers in the evaluation of past C. pneumoniae infection., Methods: Peripheral blood mononuclear cells (PBMC) (1×10 6 /mL) from stable adult asthmatic (N=6) and non-asthmatic subjects (N=6) were infected +/- C. pneumoniae TW-183 at a multiplicity of infection (MOI)=0.1, using dose responses (1:10, 1:100), and cultured 48 hrs. Cytokine responses (Interleukin (IL)-21, IL-12, IL-13) were measured in supernatants (ELISA)., Results: Cytokine responses (mean differences: unstimulated-stimulated cells) were significant for IL-12 (1:10, 1:100) ( P =0.0005, 0.0005) but not for IL-21 or IL-13 (Wilcoxon signed-rank test). Cytokine levels were higher in asthmatic subjects for IL-13 (mean differences: non-asthma-asthma) (unstimulated, 1:10, 1:100) (-210±167, -140±113, -89±59, respectively) ( P =0.05, 0.05, 0.05, respectively) compared with non-asthma. However, IL-21 and IL-12 responses were similar in both groups. When subjects were stratified according to C. pneumoniae IgG antibody status, no significant differences in cytokine responses were observed., Conclusion: Differential cytokine patterns in subjects with or without asthma may suggest a mechanism for the development of persistent infection with C. pneumoniae ., (© 2024 by the Association of Clinical Scientists, Inc.)

Objectives: To investigate the role of follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells in immune disorders of idiopathic membranous nephropathy (IMN)., Methods: Peripheral blood samples of 41 IMN patients and 30 healthy controls were collected. The percentages of B cells, Tfh cells and Tfr cells were determined by flow cytometry, and the concentrations of IL-6, IL-17A, and IL-21 were determined by enzyme linked immunosorbent assay (ELISA). Furthermore, sorted Tfh cells or Tfr cells were co-cultured with B cells in vitro to detect the cell function., Results: B cells, Tfh cells, Tfr cells, Tfr / Tfh ratio, IL-6, IL-17A, and IL-21 were significantly higher in IMN patients compared to controls. IMN patients had reduced percentages of CTLA-4+Tfr cells, increased percentages of PD-1+Tfr cells, and reduced CTLA-4+Tfr / PD-1+Tfr. In the co-culture system, IgG4, lactic acid, IL-6, IL-17A, and IL-21 were higher in Tfh cells derived from IMN patients, while IgG4, lactic acid, IL-6, IL-17A, and IL-21 were lower in Tfr cells derived from healthy patients., Conclusions: Tfh cells and Tfr cells are involved in immune disorders in IMN. This may be associated with abnormal expression of CTLA-4 and PD-1., (© 2023. The Author(s), under exclusive licence to Dr. K C Chaudhuri Foundation.)

Regular assessment of disease activity in relapsing-remitting multiple sclerosis (RRMS) is required to optimize clinical outcomes. Biomarkers can be a valuable tool for measuring disease activity in multiple sclerosis (MS) if they reflect the pathological processes underlying MS pathogenicity. In this pilot study, we combined multiple biomarkers previously analyzed in RRMS patients into an MS disease activity (MSDA) score to evaluate their ability to predict relapses and treatment response to glatiramer acetate (GA). Response Gene to Complement 32 (RGC-32), FasL, IL-21, SIRT1, phosphorylated SIRT1 (p-SIRT1), and JNK1 p54 levels were used to generate cut-off values for each biomarker. Any value below the cutoff for RGC-32, FasL SIRT1, or p-SIRT1 or above the cutoff for IL-21 or JNK1 p54 was given a +1 value, indicating relapse or lack of response to GA. Any value above the cutoff value for RGC-32, FasL, SIRT1, p-SIRT1 or below that for IL-21 or JNK1 p54 was given a -1 value, indicating clinical stability or response to GA. An MSDA score above +1 indicated a relapse or lack of response to treatment. An MSDA score below -1 indicated clinical stability or response to treatment. Our results showed that the MSDA scores generated using either four or six biomarkers had a higher sensitivity and specificity and significantly correlated with the expanded disability status scale. Although these results suggest that the MSDA test can be useful for monitoring therapeutic response to biologic agents and assessing clinically challenging situations, the present findings need to be confirmed in larger studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tatomir, Anselmo, Boodhoo, Chen, Mekala, Nguyen, Cuevas, Rus and Rus.)

Aberrant accumulation of circulating follicular helper T cells (cTfh) has been found in the peripheral blood mononuclear cells (PBMCs) of Graves' disease (GD) patients. However, the underlying mechanism that contributes to the imbalance of cTfh cells remains unknown. Previously, studies described a GD-related circular RNAs (circRNAs)-circZNF644 that might be associated with cTfh cells. This study aimed to investigate the role of circZNF644 on cTfh cells in GD patients. Here, we found that circZNF644 was highly stable expression in the PBMCs of GD patients, which was positively correlated with the serum levels of TSH receptor autoantibodies (TRAb). Knockdown of circZNF644 caused a reduction of the proportion of cTfh cells in vitro. Mechanistically, circZNF644 served as a ceRNA for miR-29a-3p to promote ICOS expression, resulting in increased cTfh cells. In the PBMCs of GD patients, circZNF644 expression was positively correlated with ICOS expression and the percentage of cTfh cells, but negatively related to miR-29a-3p expression. Additionally, a strong relationship between circZNF644 and IL-21 was revealed in GD patients, and silencing of circZNF644 inhibited IL-21 expression. Our study elucidated that elevated expression of circZNF644 is a key feature in the development of GD and may contribute to the pathogenic role of cTfh cells in GD., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2024 Yingzhao Liu et al.)