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Sjögren's disease (SjD) is a chronic autoimmune disease characterized by focal lymphocytic inflammation in lacrimal and salivary glands. We recently identified IL-27 as a requisite signal for the spontaneous SjD-like manifestations in nonobese diabetic (NOD) mice. Here, we define T cell-intrinsic effects of IL-27 in lacrimal gland disease in NOD mice. IL-27 receptor was required by both CD4 T effector (Te) cells and CD8 T cells to mediate focal inflammation. Intrinsic IL-27 signaling was associated with PD-1 and ICOS expressing T follicular helper (Tfh)-like CD4 Te cells within lacrimal glands, including subsets defined by CD73 or CD39 expression. CD8 T cells capable of IL-27 signaling also expressed PD-1 with subsets expressing ICOS and CD73 demonstrating a T follicular cytotoxic (Tfc)-like cell phenotype and others expressing a CD39 hi exhausted-like phenotype. These findings suggest IL-27 is a key early signal driving a follicular-type response in lacrimal gland inflammation in NOD mice., Competing Interests: Declaration of competing interest All authors declare that no conflicts of interest exist., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The first-in-human phase I/II ICONIC trial evaluated an investigational inducible costimulator (ICOS) agonist, vopratelimab, alone and in combination with nivolumab in patients with advanced solid tumors., Patients and Methods: In phase I, patients were treated with escalating doses of intravenous vopratelimab alone or with nivolumab. Primary objectives were safety, tolerability, MTD, and recommended phase II dose (RP2D). Phase II enriched for ICOS-positive (ICOS+) tumors; patients were treated with vopratelimab at the monotherapy RP2D alone or with nivolumab. Pharmacokinetics, pharmacodynamics, and predictive biomarkers of response to vopratelimab were assessed., Results: ICONIC enrolled 201 patients. Vopratelimab alone and with nivolumab was well tolerated; phase I established 0.3 mg/kg every 3 weeks as the vopratelimab RP2D. Vopratelimab resulted in modest objective response rates of 1.4% and with nivolumab of 2.3%. The prospective selection for ICOS+ tumors did not enrich for responses. A vopratelimab-specific peripheral blood pharmacodynamic biomarker, ICOS-high (ICOS-hi) CD4 T cells, was identified in a subset of patients who demonstrated greater clinical benefit versus those with no emergence of these cells [overall survival (OS), P = 0.0025]. A potential genomic predictive biomarker of ICOS-hi CD4 T-cell emergence was identified that demonstrated improvement in clinical outcomes, including OS (P = 0.0062)., Conclusions: Vopratelimab demonstrated a favorable safety profile alone and in combination with nivolumab. Efficacy was observed only in a subset of patients with a vopratelimab-specific pharmacodynamic biomarker. A potential predictive biomarker of response was identified, which is being prospectively evaluated in a randomized phase II non-small cell lung cancer trial. See related commentary by Lee and Fong, p. 3633., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: ICOS and its ligand ICOSL are immune receptors whose interaction triggers bidirectional signals that modulate the immune response and tissue repair., Aim: The aim of this study was to assess the in vivo effects of ICOSL triggering by ICOS-Fc, a recombinant soluble form of ICOS, on skin wound healing., Methods: The effect of human ICOS-Fc on wound healing was assessed, in vitro, and, in vivo, by skin wound healing assay using ICOS -/- and ICOSL -/- knockout (KO) mice and NOD-SCID-IL2R null (NSG) mice., Results: We show that, in wild type mice, treatment with ICOS-Fc improves wound healing, promotes angiogenesis, preceded by upregulation of IL-6 and VEGF expression; increases the number of fibroblasts and T cells, whereas it reduces that of neutrophils; and increases the number of M2 vs. M1 macrophages. Fittingly, ICOS-Fc enhanced M2 macrophage migration, while it hampered that of M1 macrophages. ICOS -/- and ICOSL -/- KO, and NSG mice showed delayed wound healing, and treatment with ICOS-Fc improved wound closure in ICOS -/- and NSG mice., Conclusion: These data show that the ICOS/ICOSL network cooperates in tissue repair, and that triggering of ICOSL by ICOS-Fc improves cutaneous wound healing by increasing angiogenesis and recruitment of reparative macrophages.

Effectiveness of seasonal influenza vaccination varies between individuals and might be affected by vaccination history among other factors. Here we show, by monitoring frequencies of CD4 T cells specific to the conserved hemagglutinin epitope HA 118-132 and titres of IgG against the corresponding recombinant hemagglutinin protein, that antigen-specific CD4 T cell and antibody responses are closely linked to pre-existing immunity and vaccine history. Upon immunization, a strong early reaction is observed in all vaccine naïve participants and also in vaccine experienced individuals who have not received the respective seasonal vaccine in the previous year. This response is characterized by HA 118-132 specific CD4 T cells with a follicular helper T cell phenotype and by ascending titers of hemagglutinin-specific antibodies from baseline to day 28 following vaccination. This trend was observed in only a proportion of those participants who received the seasonal vaccine the year preceding the study. Regardless of history, levels of pre-existing antibodies and CD127 expression on CD4 T cells at baseline were the strongest predictors of robust early response. Thus, both pre-existing immunity and vaccine history contribute to the response to seasonal influenza vaccines., (© 2021. The Author(s).)

Objective: Accumulating studies have identified self-DNA as driving IgG anti-double-stranded DNA (anti-dsDNA) in lupus, though the underpinning mechanisms of this process remain largely undefined. Here, we explored the activity of transcription factor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) in the differentiation and function of self-DNA-specific follicular helper T (Tfh) cells in lupus., Methods: B6, TCRα -/- , CD4 -/- , RORγt fl/fl CD4Cre, RORγt +/+ CD4Cre, Bcl-6 fl/fl CD4Cre, Bcl-6 +/+ CD4Cre, IL-17 -/- , and ICOS -/- mice were immunized with normal self-DNA, immunogenic self-DNA, and pathogen DNA to induce the production of Tfh cells and IgG anti-dsDNA. Tfh cells with or without interleukin-17 (IL-17) were evaluated for their role in supporting the generation of IgG. NSG mice were reconstituted with immune cells and circulating DNA from human subjects for translational studies. IL-17-positive Tfh cells were analyzed for their correlation with IgG anti-dsDNA levels as well as their response to circulating self-DNA in lupus patients., Results: Unlike normal self-DNA, immunogenic self-DNA and pathogen DNA efficiently induced IgG responses. Immunogenic self-DNA induced IgG in a CD4+ T cell-dependent manner, which was abrogated by RORγt deficiency. In contrast, RORγt was not required for the generation of pathogen DNA-induced IgG. Further analyses identified RORγt as essential for the differentiation and function of Tfh cells in response to immunogenic self-DNA, assigning IL-17 as a feature cytokine. These IL-17-positive Tfh cells functioned independent of inducible costimulator (ICOS), critically supporting IgG generation. Targeting immunogenic self-DNA-specific Tfh cells by RORγ knockdown and IL-17 blockade ameliorated IgG response and lupus nephritis in a humanized mouse model. The presence of IL-17-positive Tfh cells was associated with IgG anti-dsDNA levels and were expanded by circulating immunogenic self-DNA in lupus patients., Conclusion: Immunogenic self-DNA instructs ICOS-dispensable IL-17-positive Tfh cells via RORγt to produce an IgG anti-dsDNA response. As such, IL-17-positive Tfh cells are a promising therapeutic target for lupus patients., (© 2021, American College of Rheumatology.)

A unique population of Foxp3+ regulatory T cells (TRs) resides in visceral adipose tissue (VAT) that regulates adipose inflammation and helps preserve insulin sensitivity. Inducible T cell co-stimulator (ICOS) is highly expressed on effector (e)TRs that migrate to nonlymphoid tissues, and contributes to their maintenance and function in models of autoimmunity. In this study, we report an unexpected cell-intrinsic role for ICOS expression and downstream phosphoinositide 3-kinase (PI3K) signaling in limiting the abundance, VAT-associated phenotype, and function of TRs specifically in VAT. Icos-/- mice and mice expressing a knock-in form of ICOS that cannot activate PI3K had increased VAT-TR abundance and elevated expression of canonical VAT-TR markers. Loss of ICOS signaling facilitated enhanced accumulation of TRs to VAT associated with elevated CCR3 expression, and resulted in reduced adipose inflammation and heightened insulin sensitivity in the context of a high-fat diet. Thus, we have uncovered a new and surprising molecular pathway that regulates VAT-TR accumulation and function., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Mittelsteadt et al.)

Humoral immune responses are dysregulated with aging, but the cellular and molecular pathways involved remain incompletely understood. In particular, little is known about the effects of aging on T follicular helper (Tfh) CD4 cells, the key cells that provide help to B cells for effective humoral immunity. We performed transcriptional profiling and cellular analysis on circulating Tfh before and after influenza vaccination in young and elderly adults. First, whole-blood transcriptional profiling shows that ICOS + CD38 + cTfh following vaccination preferentially enriches in gene sets associated with youth versus aging compared to other circulating T cell types. Second, vaccine-induced ICOS + CD38 + cTfh from the elderly had increased the expression of genes associated with inflammation, including tumor necrosis factor-nuclear factor κB (TNF-NF-κB) pathway activation. Finally, vaccine-induced ICOS + CD38 + cTfh display strong enrichment for signatures of underlying age-associated biological changes. These data highlight the ability to use vaccine-induced cTfh as cellular "biosensors" of underlying inflammatory and/or overall immune health., Competing Interests: The authors declare no competing interests. E.J.W. consults or is an advisor for Merck, Elstar, Janssen, Related Sciences, Synthekine, and Surface Oncology, unrelated to the present study. E.J.W. is a founder of Surface Oncology and Arsenal Biosciences, unrelated to the present study. E.J.W. is an inventor on a patent (US patent no. 10,370,446) submitted by Emory University that covers the use of PD-1 blockade to treat infections and cancer., (© 2021 The Authors.)

Background: Chronic graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality in transplant patients. We have previously shown that 3 doses of an anti-inducible costimulator (ICOS) mAb transiently ameliorated symptoms and extended survival of dogs affected by chronic GVHD over that of control dogs. The purpose of this study was to specifically correlate changes in T-cell populations in the peripheral blood with anti-ICOS treatment and chronic GVHD progression and regression to reach a better understanding of the mechanism of the disease and prioritize future studies., Methods: Peripheral blood cells from canines transplanted with DLA-mismatched bone marrow and peripheral blood mononuclear cells to generate chronic GVHD were analyzed by flow cytometry using a panel of antibodies specific to helper and cytolytic T cells., Results: Chronic GVHD was specifically associated with an increase in CD4+ICOS+ cells, ICOS+ cells expressing IL-17A, and CD8+ cells generating granzyme B. Treatment with anti-ICOS mAb at onset of chronic GVHD symptoms specifically targeted IL-17A+-expressing cells, transiently relieved symptoms, and lengthened survival but was unable to reduce the percentage of CD8+ T-cells expressing granzyme B., Conclusions: These studies suggested a role for both CD4+ and CD8+ T cells in pathogenesis of chronic GVHD in the canine model. We propose that future studies should focus on further extending survival by developing a treatment that would control both CD4+ and CD8+ T cells., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor β chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1β induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1β to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1βR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.

Purpose: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Noninvasive molecular imaging of CAR T cells by PET is a promising approach with the ability to provide spatial, temporal, and functional information. Reported strategies rely on the incorporation of reporter transgenes or ex vivo biolabeling, significantly limiting the application of CAR T-cell molecular imaging. In this study, we assessed the ability of antibody-based PET (immunoPET) to noninvasively visualize CAR T cells., Experimental Design: After analyzing human CAR T cells in vitro and ex vivo from patient samples to identify candidate targets for immunoPET, we employed a syngeneic, orthotopic murine tumor model of lymphoma to assess the feasibility of in vivo tracking of CAR T cells by immunoPET using the 89 Zr-DFO-anti-ICOS tracer, which we have previously reported., Results: Analysis of human CD19-CAR T cells during activation identified the Inducible T-cell COStimulator (ICOS) as a potential target for immunoPET. In a preclinical tumor model, 89 Zr-DFO-ICOS mAb PET-CT imaging detected significantly higher signal in specific bone marrow-containing skeletal sites of CAR T-cell-treated mice compared with controls. Importantly, administration of ICOS-targeting antibodies at tracer doses did not interfere with CAR T-cell persistence and function., Conclusions: This study highlights the potential of ICOS-immunoPET imaging for monitoring of CAR T-cell therapy, a strategy readily applicable to both commercially available and investigational CAR T cells. See related commentary by Volpe et al., p. 911 ., (©2020 American Association for Cancer Research.)

The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells (Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOS high T cells but was also associated with increased secretion of proinflammatory cytokines from ICOS low effector T cells (T eff ). In syngeneic mouse tumor models, KY1044 depleted ICOS high Treg and increased the intratumoral T Eff :Treg ratio, resulting in increased secretion of IFNγ and TNFα by T Eff cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response., (©2020 American Association for Cancer Research.)

Objectives: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients., Methods: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production., Results: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4 + CXC-motif chemokine receptor 5 (CXCR5) + programmed cell death protein 1 (PD1) + ICOS + Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4 + CD45RO + ICOS + PD1 + cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5 + CD4 + PD1 + ICOS + Foxp3 - Tfh-cells and a uniquely expanded population of CXCR5 - CD4 + PD1 hi ICOS + Foxp3 - Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α)., Conclusions: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS., Competing Interests: Competing interests: BF: Consultancy for Novartis, Roche, and BMS. SJB: Consultancy for Astrazeneca, Biogen, BMS, Celgene, Medimmune, MTPharma, Novartis, Ono, UCB, xtlbio. MB: consultancy and/or unrestricted grant support from Medimmune, GSK, Janssen, UCB. GC and JC are AstraZeneca employees and own company stocks., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Objective To detect the expression of inducible co-stimulator and inducible co-stimulator ligand (ICOS/ICOSL) on peripheral lymphocytes in patients with Graves' disease (GD) and its clinical significance. Methods A total of 105 untreated GD patients and 67 healthy controls were enrolled in our study. 19 GD patients treated with anti-thyroid drugs were followed up in the outpatient department until TRAb were negative. The peripheral blood mononuclear cells (PBMCs) were separated and analyzed with PCR. ICOS expression on CD4 + /CD8 + T cells and ICOSL expression on CD19 + B cells were detected by flow cytometry. The correlations between the expression of ICOS/ICOSL with thyroid parameters and TRAb were analyzed. Results The expression of both ICOS and ICOSL mRNA in PBMCs of the untreated GD patients were significantly higher than those in the healthy controls. Compared with healthy controls, ICOS expression on CD4 + T cells and ICOSL expression on CD19 + B cells were up-regulated significantly in the GD patients. Correlation analysis showed that ICOS expression on CD4 + T cells was positively correlated with the level of free triiodothyronine (FT3) in the GD patients. In the group of GD patients with high TRAb titers in serum, ICOSL expression on CD19 + B cells notably increased as compared with that in the patients with low TRAb. ICOS expression on CD4 + T cells and ICOSL expression on CD19 + B cells were recovered in the GD patients with negative TRAb. Conclusion The expression of ICOS on the surface of CD4 + T cells and ICOSL on CD19 + B cells in peripheral blood of patients with Graves's disease are significantly increased.With the decrease of TRAb level, the expression levels of ICOS on the surface of CD4 + T cells and ICOSL on CD19 + B cells are restored.

Background: Pemphigus vulgaris (PV) is an autoimmune bullous disease mediated by autoantibodies against desmoglein 3 (DSG3). Inducible costimulator (ICOS) is a costimulatory receptor expressed on T cells and influences the activity of T follicular helper (T FH ) cells in various autoimmune diseases, but the roles of ICOS and T FH cells in PV remain unclear., Objective: We examined the immunological characteristics, antigen specificity, and pathogenicity of CD4 + T-cell subpopulations, as well as the therapeutic effect of anti-ICOS blocking antibodies in PV., Methods: A mouse model of PV was established by adoptive transfer of immune cells from the skin-draining lymph nodes or spleens of DSG3-expressing skin-grafted Dsg3 -/- mice into Rag1 -/- mice. The T FH cells and CD4 + T cells in PBMCs from PV patients were examined by flow cytometry., Results: Among CD4 + T cells from the mouse model, ICOS-positive T FH cells were associated with B-cell differentiation and were required for disease induction. Using an MHC class II tetramer, DSG3-specific ICOS + T FH cells were found to be associated with anti-DSG3 antibody production and expanded in the absence of B cells. In human PV, the frequency of ICOS + CXCR5 + PD-1 + memory CD4 + T cells correlated with the autoantibody level. Treatment with anti-ICOS blocking antibodies targeting ICOS + T FH cells decreased the anti-DSG3 antibody level and delayed disease progression in vivo., Conclusions: Mouse Dsg3-specific ICOS + T FH cells and human ICOS + CXCR5 + PD-1 + T H cells are associated with the anti-DSG3 antibody response in PV. ICOS expressed on CXCR5 + PD-1 + T H cells may be a therapeutic target for PV., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.

Inducible co-stimulator-positive (ICOS) and programmed cell death 1-positive (PD-1) are important markers for follicular helper T cells (Tfh); however, their roles and clinical values in ulcerative colitis (UC) remain unknown. In this study, we recruited 68 UC patients and 34 healthy controls. Circulating ICOS + , PD-1 + and ICOS + PD-1 + Tfh subsets were analyzed by flow cytometry. Twelve active UC patients achieving remission after treatment with 5-aminosalicylic acid were followed-up and Tfh subset changes were analyzed. Serum immunoglobulin (Ig)G, C-reactive protein (CRP), interleukin (IL)-4 and IL-21 levels and B cell subsets were analyzed and Mayo scores were calculated. Correlation analyses were performed between Tfh subsets and the clinical indicators. Receiver operating characteristic (ROC) curves were generated to evaluate the efficiency of Tfh subsets for disease monitoring. We found that levels of ICOS + , PD-1 + and ICOS + PD-1 + Tfh cells were significantly increased in active UC and significantly decreased when achieving clinical remission. Activated ICOS + PD-1 + Tfh cells were positively correlated with serum CRP and Mayo scores. Furthermore, ICOS + PD-1 + Tfh cells were significantly correlated with circulating new memory B cells and plasmablasts, as well as serum IgG, IL-4 and IL-21. ROC analyses showed that when ICOS + PD-1 + Tfh cells were used in combination with PD-1 + Tfh cells, the diagnostic efficacy in distinguishing active UC from stable remission patients was higher than that of any one used alone, with area under curve (AUC) value 0·931. Our findings suggest that increased ICOS + PD-1 + Tfh cells are associated with the activation of B cells in the pathogenesis of UC, and may be a potential biomarker for UC disease monitoring., (© 2020 British Society for Immunology.)

Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors., Competing Interests: All authors were employees of Jounce Therapeutics during the time of study conduct. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Recent studies have reported the pathological effect of ICOS + T cells, but ICOS signals also widely participate in anti-inflammatory responses, particularly ICOS + regulatory T (Treg) cells. The ICOS signaling pathway endows Tregs with increased generation, proliferation, and survival abilities. Furthermore, there is enough evidence to suggest a superior capacity of ICOS + Tregs, which is partly attributable to IL-10 induced by ICOS, yet the associated mechanism needs further investigation. In this review, we discuss the complicated role of ICOS + Tregs in several classical autoimmune diseases, allergic diseases, and cancers and investigate the related therapeutic applications in these diseases. Moreover, we identify ICOS as a potential biomarker for disease treatment and prognostic prediction. In addition, we believe that anti-ICOS/ICOSL monoclonal antibodies exhibit excellent clinical application potential. A thorough understanding of the effect of ICOS + Tregs and the holistic role of ICOS toward the immune system will help to improve the therapeutic schedule of diseases., (Copyright © 2020 Li and Xiong.)

Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated through complex immunologic pathways. Among RA patients receiving low-dose methotrexate (MTX) monotherapy, approximately one-half exhibit a meaningful clinical response within the first 6 months of starting treatment. Whether baseline immune phenotypes differ between subsequent MTX responders and nonresponders is unknown. This study utilized comprehensive T cell immunophenotyping to identify specific immunologic pathways associated with MTX-nonresponsive joint inflammation in patients with RA., Methods: In total, 32 patients with recent-onset RA were treated with MTX therapy. After 6 months, 15 patients were categorized as responders and 17 as nonresponders. Comprehensive blood T cell immunophenotyping, using multiparameter immunofluorescence flow cytometry analyses, was performed at baseline and following 6 months of treatment., Results: Baseline measures of disease activity (Disease Activity Score in 28 joints [DAS28], C-reactive protein level, and erythrocyte sedimentation rate) did not differ between MTX responders and nonresponders following MTX treatment. Frequencies of CD4+ and CD8+ T cells were skewed to favor higher CD4:CD8 T cell ratios in MTX responders compared to nonresponders (P < 0.05). The proportion of inducible costimulator-expressing Treg cells was significantly greater among MTX nonresponders. Interleukin-13 (IL-13)-producing, but not interferon-γ- or IL-17-producing, CD4+ effector memory T (Tem) cells were significantly more frequent in MTX nonresponders (P < 0.05). The ratio of IL-13+:IL-17+ Tem cells among CD4+ Tem cells was 1.9-fold higher in MTX nonresponders compared to responders (P < 0.05). Both the CD4:CD8 T cell ratio and the frequency of IL-13+CD4+ Tem cells correlated with changes in the DAS28 score following MTX treatment, whereas T cell expression of immune checkpoint inhibitor markers (CTLA-4, programmed death 1, and T cell immunoglobulin and mucin domain-containing protein 3) did not differ between MTX responders and nonresponders., Conclusion: We observed a bias toward type 2-polarized T cell inflammatory responses in the peripheral blood of MTX-nonresponsive RA patients. Targeting the IL-13+CD4+ T cell pathway could be a new therapeutic strategy in RA patients whose disease remains resistant to MTX., (© 2020, American College of Rheumatology.)

Background: The clinical benefit of immunotherapy has been limited to a small subset of patients with cancer. Several clinical trials with immune checkpoint inhibitors in multiple cancers have shown some improvement in obese patients. However, how obesity regulates the immune microenvironment remains unclear., Methods: Bioinformatic analysis was used to discover immune microenvironmental-related genes associated with body mass index (BMI). The expression of ICOS in tumor tissues was detected using western blot, immunohistochemistry, quantitative real-time polymerase chain reaction (RT-qPCR) and flow cytometry. RT-qPCR was used to measure the expression of miR-27a-3p. The interaction between miR-27a-3p and ICOS was confirmed by dual-luciferase reporter assay. Functional testing of T cells based on proliferation and interferon (IFN)-gamma secretion was performed using ELISA and flow cytometry., Results: ICOS, an immune microenvironment-related gene, was significantly upregulated in obese patients with lung adenocarcinoma (LUAD). MiR-27a-3p showed a negative correlation with ICOS and suppressed the expression of ICOS. We determined that dipocyte-derived exo-miR-27a-3p could alter the tumor microenvironment by inhibiting ICOS + T cell proliferation and IFN-gamma secretion in vitro., Conclusions: Adipocyte-derived exo-miR-27a-3p can inhibit ICOS + T cell proliferation and IFN-gamma secretion. The upregulation of ICOS + T cell functions caused by the downregulation of miR-27a-3p in adipose tissue derived exosomes is one of the potential mechanisms for the improved efficacy of immunotherapy in obese LUAD patients., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Chimeric antigen receptor-T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response.

Follicular helper T cells (Tfhs) are essential for germinal center (GC) B cell maturation and antibody development. However, the intrinsic mechanisms that regulate Tfh differentiation are largely unknown. Here, we demonstrate that the frequencies of Tfhs and GC B cells, as well as interleukin-21 (IL-21) and anti-ovalbumin (OVA) antibodies, are markedly decreased in forkhead box O3 (Foxo3) knockout mice immunized with OVA. Using mixed bone marrow chimeras and lymphocyte-repopulated Rag1 -/- mice proves that wild-type (WT), but not Foxo3-deficient T cells provoke GC B cell maturation and antibody production. Deficiency of Foxo3 inhibits inducible T cell co-stimulator (ICOS)-induced Tfh differentiation. Chromatin immunoprecipitation assay results suggest that Foxo3 is able to bind to the IL-21 promoter and regulate IL-21 secretion. In conclusion, our study unveils a critical role of Foxo3 in the regulation of Tfh differentiation and IL-21 production. Modulating Foxo3 activity may be beneficial for enhancing or preventing antibody-mediated immune responses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The co-stimulatory molecule ICOS is associated with the induction and regulation of T helper cell responses, including the differentiation of follicular helper T (Tfh) cells and the formation and maintenance of memory T cells. However, the role of ICOS signaling in secondary immune responses is largely unexplored. Here we show that memory T cell formation and maintenance are influenced by persistent infection with P. chabaudi chabaudi AS infection, as memory T cell numbers decline in wild-type and Icos-/- mice after drug-clearance. Following drug-clearance Icos-/- mice display a relapsing parasitemia that occurs more frequently and with higher peaks compared to wild-type mice after re-challenge. The secondary immune response in Icos-/- mice is characterized by significant impairment in the expansion of effector cells with a Tfh-like phenotype, which is associated with a diminished and delayed parasite-specific Ab response and the absence of germinal centers. Similarly, the administration of an anti-ICOSL antagonizing antibody to wild-type mice before and after reinfection with P. c. chabaudi AS leads to an early defect in Tfh cell expansion and parasite-specific antibody production, confirming a need for ICOS-ICOSL interactions to promote memory B cell responses. Furthermore, adoptive transfer of central memory T (TCM) cells from wild-type and Icos-/- mice into tcrb-/- mice to directly evaluate the ability of TCM cells to give rise to Tfh cells revealed that TCM cells from wild-type mice acquire a mixed Th1- and Tfh-like phenotype after P. c. chabaudi AS infection. While TCM cells from Icos-/- mice expand and display markers of activation to a similar degree as their WT counterparts, they displayed a reduced capacity to upregulate markers indicative of a Tfh cell phenotype, resulting in a diminished humoral response. Together these findings verify that ICOS signaling in memory T cells plays an integral role in promoting T cell effector responses during secondary infection with P. c. chabaudi AS., Competing Interests: The authors have declared that no competing interests exist.

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2 INFLAM ) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2 INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers., Competing Interests: Declaration of Interests Although not related to this study, in the last 12 months D.A. has contributed to scientific advisory boards at Genentech, Pfizer, Takeda, FARE, and the KRF. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Humanized NOD/SCID/IL-2 receptor γ-chain null (huNSG) mice recapitulate some features of human T-cell populations that can be exploited in basic and pre-clinical research. CXCR5 + T CD8 + T-cells play an important role in the control of viral infections and tumors. Indeed, they have been associated with low-level HIV replication, making them a possible novel correlate of protection, and potentially useful in the eradication of HIV reservoirs. Here, by flow cytometry, we evaluated the reconstitution of CXCR5 + CD8 + T-cells in huNSG mice engrafted with CD34 + hematopoietic stem cells. This population was readily generated in huNSG mice, and where particularly confined to spleen and lymph nodes. These cells exhibited a follicular-like phenotype, with expression of Programmed Death (PD)-1, Inducible T-cell costimulatory (ICOS), and absence of CCR7. Moreover, CXCR5 + CD8 + T-cells had a higher expression of interleukin (IL)-21 and a higher cytotoxic potential compared with CXCR5 - cells. HIV infection did not affect the frequencies of CXCR5 + CD8 + T-cells in secondary lymphoid organs. Finally, taking advantage of the high proportion of naïve T-cells in huNSG mice, we evaluated the in vitro response of splenic T-cells to the follicular profile-polarizing cytokines Transforming Growth Factor (TGF)-β1 and IL-23. After in vitro treatment, there was an increase in CXCR5 + CD8 + T-cells, which exhibited high levels of PD-1, CD40 L and low expression of CCR7. Thus, there is a reconstitution of CXCR5 + CD8 + T-cells in huNSG mice, supporting the use of this model for exploring the biology and role of this cell population in healthy and diseased conditions., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Introduction : The promotion of antitumor response by targeting co-stimulatory B7 superfamily members has become evident to create a new wave of cancer immunotherapy. Inducible Co-Stimulator (ICOS), which is expressed on activated T cells, gained interest in the translational medicine community. Areas covered : We performed an extensive literature review using the keywords 'ICOS' and 'cancer', and the Clinicaltrials.gov database for early phase clinical trials targeting ICOS. In this review, we highlight the dual role of ICOS in oncogenesis in different malignancies. We summarize the current state of knowledge about ICOS/ICOSL pathway targeting by immunotherapies. Expert opinion : Due to its multifaceted link with anti-tumor immunity, both antagonist and agonist antibodies might be of interest to target the ICOS/ICOSL pathway for tumor treatment. Indeed, ICOS activation might potentiate the effect of an inhibitory checkpoint blockade, while its neutralization could decrease the function of immunosuppressive Tregs and inhibit lymphoid tumor cells expressing Tfh markers.

Liver kinase B1 (Lkb1) in dendritic cells (DCs) plays a key role in maintaining immunity homeostasis and adaptive immunity by controlling the CD4 + Foxp3 + T regulatory cell (CD4 + Tregs) pool and T cells activation. However, the function of Lkb1 in DCs for the regulation of CD8 + Foxp3 + T regulatory cells (CD8 + Tregs) has not been addressed. Herein, we found that Lkb1-deficient DCs could lead to excessive CD8 + Tregs expansion in multiple organs. We found that OX40 expression was significantly higher in Lkb1-deficient DCs compared with that in wild-type (WT) mice, suggesting a potential pathway of CD8 + Treg expansion. Moreover, we found that CD8 + Tregs from mice with conditional deletion Lkb1 in DCs (KO) displayed an activated phenotype and expressed higher levels of specific markers, including ICOS and CD103. Interestingly, compared with the WT mice without lipopolysaccharide(LPS) treatment, we found that CD8 + Tregs population increased in the WT mice with LPS treatment which can selectively delete Lkb1 protein in DCs. However, there was no significant difference in CD8 + Tregs population in the KO mice between LPS treatment group and non-LPS treatment. Collectively, our findings identified Lkb1 in DCs as a crucial regulator of CD8 + Treg expansion., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Immunotherapy, especially immune checkpoint inhibition, has provided powerful tools against cancer. We aimed to detect the expression of common immune checkpoints and evaluate their prognostic values in nasopharyngeal carcinoma (NPC)., Methods: The expression of 9 immune checkpoints consistent with 13 features was detected in the training cohort (n = 208) by immunohistochemistry and quantified by computational pathology. Then, the LASSO cox regression model was used to construct an immune checkpoint-based signature (ICS), which was validated in a validation cohort containing 125 patients., Results: High positive expression of PD-L1 and B7-H4 was observed in tumour cells (TCs), whereas PD-L1, B7-H3, B7-H4, IDO-1, VISTA, ICOS and OX40 were highly expressed in tumour-associated immune cells (TAICs). Eight of the 13 immune features were associated with patient overall survival, and an ICS classifier consisting of 5 features (B7-H3TAIC, IDO-1TAIC, VISTATAIC, ICOSTAIC, and LAG3TAIC) was established. Patients with high-risk scores in the training cohort had shorter overall (P < 0.001), disease-free (P = 0.002), and distant metastasis-free survival (P = 0.004), which were confirmed in the validation cohort. Multivariate analysis revealed that the ICS classifier was an independent prognostic factor. A combination of the ICS classifier and TNM stage had better prognostic value than the TNM stage alone. In addition, the ICS classifier was significantly associated with survivals in patients with high EBV-DNA load., Conclusions: We determined the expression status of nine immune checkpoints consistent with 13 features in NPC and further constructed an ICS prognostic model, which might add prognostic value to the TNM staging system.

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.

Clinically, most patients with primary nephrotic syndrome (PNS) have low serum IgG levels, which is an important factor in infection and in PNS relapse.To some extent, the mechanisms involved remain largely unknown. Here, we aimed to investigate the pathogenesis of the decreased IgG levels in PNS. Peripheral blood was collected from patients with PNS and closely age- and sex-matched healthy individuals. The frequency, phenotype and molecular function of different circulating B cell and T follicular helper cell (TFH) subsets were examined by flow cytometry. The function of the CD40/CD40 L interaction in immunoglobulin class-switch recombination (CSR) was evaluated by assessing the induction of activation-induced deaminase (AID) expression with CD40 L stimulation. We revealed an increase in the levels of circulating total plasmablasts, plasma cells and mature-naive B cells and a decrease in the levels of germinal centre-like B cells and CD19 + IgG + B cells in PNS. In addition, although the expression of CD86 on the surface of B cells and the expression of the inducible costimulator (ICOS) on the surface of TFH cells both were increased, the expression of CD40 L on the surface of TFH cells was decreased. Moreover, upon stimulation with CD40 L in vitro, the mRNA expression of AID in peripheral blood mononuclear cells (PBMCs) was decreased in patients with PNS compared with that in healthy controls. Our results indicate that the immunoglobulin CSR of B cells was partly dysfunctional and provide insights into the potential involvement of impaired TFH cell-dependent B cell responses in the pathogenesis of low IgG levels through downregulating CD40 L expression on TFH cells in PNS., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cancer-immunotherapy targeting programmed cell death 1 (PD-1) activates tumor-specific T cells and provides clinical benefits in various cancers. However, the molecular basis of PD-1 function is still enigmatic. Especially, it is unclear which signaling pathway PD-1 primarily targets. Besides, the capacity of PD-1 to inhibit the T cell receptor (TCR)-dependent activation of T cells in the presence of co-stimulation is also controversial. Here we used co-culture systems of T cells and antigen-presenting cells with targeted deletion and overexpression of co-receptors and ligands and examined the inhibitory potency of PD-1 against T cell activation upon TCR stimulation with CD28 and ICOS co-stimulation. As an unambiguous criterion of T cell activation, we used the acquisition of cytokine production capacity, which represents one of the most important functions of T cells. PD-1 inhibited functional T cell activation upon TCR stimulation in the absence as well as in the presence of CD28 co-stimulation, indicating that PD-1 can directly inhibit TCR signal. Notably, CD28 co-stimulation rather attenuated the efficiency of PD-1 in inhibiting TCR-dependent functional T cell activation. In addition, PD-1 inhibited TCR-dependent functional T cell activation with ICOS co-stimulation as efficiently as that with CD28 co-stimulation. Furthermore, we found that the maintenance of antigen-induced follicular helper T (T FH ) cells that required ICOS co-stimulation was persistently restrained by PD-1 in vivo . These findings indicate that PD-1 primarily targets TCR signal in the inhibition of functional T cell activation. Thus, PD-1 functions as the rheostat of T cell activation rather than an inhibitor of a specific stimulatory co-receptor.

Control of chronic CNS infection with the parasite Toxoplasma gondii requires ongoing T cell responses in the brain. Immunosuppressive cytokines are also important for preventing lethal immunopathology during chronic infection. To explore the loss of suppressive cytokines exclusively during the chronic phase of infection, we blocked IL-10R in chronically infected mice. Consistent with previous reports, IL-10R blockade led to severe, fatal tissue destruction associated with widespread changes in the inflammatory response, including increased APC activation, expansion of CD4 + T cells, and neutrophil recruitment to the brain. We then sought to identify regulatory mechanisms contributing to IL-10 production, focusing on ICOS, a molecule implicated in IL-10 production. Unexpectedly, ICOS ligand (ICOSL) blockade led to a local expansion of effector T cells in the brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL blockade led to changes in T cells associated with their proliferation and survival. We observed increased expression of IL-2-associated signaling molecules CD25, STAT5 phosphorylation, Ki67, and Bcl-2 in T cells in the brain, along with decreased apoptosis. Interestingly, increases in CD25 and Bcl-2 were not observed following IL-10R blockade. Also, unlike IL-10R blockade, ICOSL blockade led to an expansion of both CD8 + and CD4 + T cells in the brain, with no expansion of peripheral T cells or neutrophil recruitment to the brain and no severe tissue destruction. Overall, these results suggest that IL-10 and ICOS differentially regulate T cell responses in the brain during chronic T. gondii infection., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Preeclampsia (PE) is characterized by hypertension and proteinuria. It occurs in an around 3% to 5% of all pregnancies worldwide. The fetus is kind of semiallograft to the maternal host; immune system components encounter fetal antigens and develop adverse immune responses. Recently, it has been observed that the immune system plays an important role in PE. In the current study, we have tried to investigate the role of follicular helper T (Tfh) cells in the pathogenesis of PE. Blood samples of 49 PE women and 50 healthy controls were collected. Peripheral blood mononuclear cells were isolated, cells were cultured, and then RNA was extracted. Autoantibody and secretory cytokine levels were analyzed by ELISA. Tfh frequency and transcription levels of the related molecules and cytokine were assessed by flow cytometry and real-time PCR, respectively. The frequency of circulating Tfh cell in PE women was significantly higher compared with the healthy pregnant woman (Tfh cells with CD4 + ICOS + , P = 0.0064 and Tfh cells with CD4 + CXCR5 + , P = 0.029). Moreover, mRNA expression levels of CXCR5, BCL6, IL-21, and IL-6 ( P = 0.0006, P = 0.008, P = 0.0063, and P = 0.027, respectively) were upregulated in PE patients. Furthermore, IL-6 ( P = 0.0014) and IL-21 ( P = 0.0059) levels in both group were assayed and the results showed increased in patient group. We also measured autoantibody levels including antiphospholipid antibodies ( P = 0.0001), anticardiolipin antibodies ( P = 0.0004), anti-TPO ( P = 0.0008), anti-TG ( P = 0.001) in circulation of PE group, which were higher than the control group. This study provided insights into the involvement of Tfh cells in etiology and pathogenesis of PE, probably by developing autoantibodies., (© 2018 Wiley Periodicals, Inc.)

The ICOS pathway has been implicated in the development and functions of regulatory T (Treg) cells, including those producing IL-10. Treg cell-derived IL-10 is indispensable for the establishment and maintenance of intestinal immune homeostasis. We examined the possible involvement of the ICOS pathway in the accumulation of murine colonic Foxp3- and/or IL-10-expressing cells. We show that ICOS deficiency does not impair induction of IL-10 by intestinal CD4 T cells but, instead, triggers substantial reductions in gut-resident and peripherally derived Foxp3 + Treg cells. ICOS deficiency is associated with reduced demethylation of Foxp3 CNS2 and enhanced loss of Foxp3. This instability significantly limits the ability of ICOS-deficient Treg cells to reverse ongoing inflammation. Collectively, our results identify a novel role for ICOS costimulation in imprinting the functional stability of Foxp3 that is required for the retention of full Treg cell function in the periphery., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Objective: ICOS/ICOSL plays a crucial part in various disease-mediated immune responses. However, the exact role of ICOS/ICOSL in type 2 diabetes mellitus (T2DM) development remains unexplored. This study aims to investigate the role of ICOS/ICOSL in the pathogenesis of T2DM., Materials and Methods: Human peripheral blood T-lymphocytes (CD3) and umbilical vein endothelial cells (HUVECs) were treated with high-glucose (HG) or advanced glycation end products (AGEs). A portion of CD3 cells was co-cultured with HUVECs and treated with different mediums or anti-ICOS mAbs. The ICOS/ICOSL and caspase-3 protein expression was measured by Western blotting. ELISA (enzyme-linked immunosorbent assay), MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and NOx production assays were respectively used to detect cytokines level, cell viability and the production of NOx., Results: HG and AGEs significantly upregulated ICOS/ICOSL expressions in T cells and HUVECs. T cell contact with HUVECs secreted more IFN-γ, IL-4, and IL-10 compared to non-contact cells, while cytokines from IL-6-, IL-1β-, and CM- (the conditioned medium) treated cells did not differ from the control. A significant increase of IL-8 and IL-6 was found in HUVECs under both contact and non-contact conditions vs. control cells. Similar results were also observed in the comparison between CM1- (T cell condition medium) or CM2- (co-culture condition medium) treated cells and control cells. However, CM1 and CM2 treatment significantly inhibited cell viability and increased caspase-3 and NOx production; blocking ICOS/ICOSL remarkably decreased cytokines secretion, enhanced cell viability and reduced caspase-3 and NOx production., Conclusions: HG and AGEs cause T cell inflammatory response and vascular endothelial dysfunction by upregulating ICOS/ICOSL, which may be one of the possible mechanisms of cardiovascular complications development in T2DM patients.

Despite genetic and epidemiological evidence strongly supporting an autoimmune basis for narcolepsy type 1, the mechanisms involved have remained largely unknown. Here, we aimed to investigate whether the frequency and function of circulating follicular helper and follicular regulatory T cells are altered in narcolepsy type 1. Peripheral blood mononuclear cells were collected from 32 patients with narcolepsy type 1, including 11 who developed disease after Pandemrix ® vaccination, and 32 age-, sex-, and HLA-DQB1*06:02-matched healthy individuals. The frequency and phenotype of the different circulating B cell and follicular T cell subsets were examined by flow cytometry. The function of follicular helper T cells was evaluated by assessing the differentiation of naïve and memory B cells in a co-culture assay. We revealed a notable increase in the frequency of circulating B cells and CD4 + CXCR5 + follicular T cells in narcolepsy patients compared to age-, sex- and HLA-matched healthy controls. However, the inducible T-cell costimulator molecule, ICOS, was selectively down-regulated on follicular T cells from patients. Reduced frequency of activated ICOS + and PD1 high blood follicular T cells was also observed in the narcolepsy group. Importantly, follicular T cells isolated from patients with narcolepsy type 1 had a reduced capacity to drive the proliferation/survival and differentiation of memory B cells. Our results provide novel insights into the potential involvement of T cell-dependent B cell responses in the pathogenesis of narcolepsy type 1 in which down-regulation of ICOS expression on follicular helper T cells correlates with their reduced function. We hypothesize that these changes contribute to regulation of the deleterious autoimmune process after disease onset., (Copyright © 2018. Published by Elsevier Ltd.)

Objective: Follicular T helper (Tfh) cells are involved in the pathogenesis of Hashimoto's thyroiditis (HT), while follicular T regulatory (Tfr) cells inhibit Tfh cells, which mediate B cell responses. However, the role of Tfr cells in HT remains unclear., Methods: Forty-six healthy controls (HCs) and 84 HT patients were enrolled in the study. The percentage of Treg cells, CXCR5 - Treg cells, and Tfr cells; the Tfr/Tfh ratio; and the percentage of ICOS, PD-1, CTLA-4, CXCR3 and CCR6 in Tfr cells were investigated; furthermore, the associations between the percentage of Tfr cells or the Tfr/Tfh ratio and the autoantibody indices were investigated., Results: Compared with that in the HCs, the percentage of Treg cells in the HT patients was not significantly changed, but the percentage of CXCR5 - Tfr cells was decreased. In contrast, both the percentage of Tfr cells and the Tfr/Tfh ratio were significantly increased in the HT patients. Among the Tfr cells, the percentage of Th2-like Tfr cells was increased in the HT patients, while the percentage of Th17-like Tfr cells was decreased. Moreover, the percentages of ICOS and PD-1 on Tfr cells were significantly increased in the HT patients, while the percentage of CTLA-4 on Tfr cells was significantly decreased. However, the percentage of ICOS, PD-1 and CTLA-4 on Treg or CXCR5 - Treg cells was not significantly changed. Last, no association was found between either the percentage of Tfr cells or the Tfr/Tfh ratio and the antithyroglobulin and antithyroid peroxidase antibody levels in the HT patients., Conclusions: In the HT patients, the circulating Tfr cell percentage and Tfr/Tfh ratio were significantly increased, but the humoral immune function of Tfr cells might be impaired.

As a critical linker between mTORC1 and mTORC2, Akt is important for the cell metabolism. The role of Akt in the function and development of B and T cells is well characterized, however, the role of Akt for development and function of iNKT cells is unknown. iNKT cells bridge the adaptive and innate immunity, and in this study, we found that the differentiation of NKT17 cells and IL17 production of NKT17 cells were disrupted in Akt2 KO mice. ICOS has been demonstrated to be critical for the differentiation of NKT17 cells and we found that ICOS mRNA and protein expression was reduced in Akt2 KO iNKT cells. As a consequence, phosphorylation of FoxO-1 was downregulated in Akt2 KO thymocytes but the sequestration of FoxO-1 in the nucleus of Akt2 KO iNKT cells was increased. The negative feedback loop between ICOS and FoxO-1 has been demonstrated in CD4 + T follicular helper cells. Therefore our study has revealed a new intracellular mechanism in which Akt2 regulates ICOS expression via FoxO-1 and this signaling axis regulates the differentiation and function of NKT17 cells. This study provides a new linker between cell metabolism and function of iNKT cells.

Background: While vaccination remains the most effective method to control hepatitis B virus (HBV) infection, 5-10% of recipients exhibit non-responsiveness to the HB vaccine. Immunological analysis of strong, weak or absent protective antibody responses to the HB vaccine should provide insights into the mechanisms that contribute to non-responsiveness., Results: We investigated the potential involvement of follicular helper T (Tfh) cells in the immune response to HB vaccine, and associations between the miR-17-92 cluster and Tfh cells. We recruited 12 adults who had completed the HB vaccination course during childhood. Following a booster dose of HB vaccine, hepatitis B surface antibody (HBsAb) titers, percentage of PD-1+ICOS+ circulating Tfh (cTfh) and plasma cells, and expression of miR-17-92 were assessed at baseline (before immunization) and after vaccination on days 7 and 14. Notably, the HBsAb level gradually increased after HB vaccination while the proportion of PD-1+ICOS+ cTfh cells was significantly increased on day 7 relative to baseline, so as plasma cells. Expression of miR-18a and miR-17 within the miR-17-92 cluster and HBsAb titers in CD4+ T cells were positively correlated with the PD-1+ICOS+ cTfh cells proportions after HB vaccination., Conclusions: The increase in HBsAb titers was positively associated with expression of all the components of the miR-17-92 cluster except miR-19a. Our findings indicate that the miR-17-92 cluster contributes to antibody production, and miR-18a and miR-17 are involved in Tfh cells differentiation after HB vaccination.

The mechanistic/mammalian target of rapamycin (mTOR) has emerged as a critical integrator of signals from the immune microenvironment capable of regulating T cell activation, differentiation, and function. The precise role of mTOR in the control of regulatory T cell (Treg) differentiation and function is complex. Pharmacologic inhibition and genetic deletion of mTOR promotes the generation of Tregs even under conditions that would normally promote generation of effector T cells. Alternatively, mTOR activity has been observed to be increased in Tregs, and the genetic deletion of the mTOR complex 1 (mTORC1)-scaffold protein Raptor inhibits Treg function. In this study, by employing both pharmacologic inhibitors and genetically altered T cells, we seek to clarify the role of mTOR in Tregs. Our studies demonstrate that inhibition of mTOR during T cell activation promotes the generation of long-lived central Tregs with a memory-like phenotype in mice. Metabolically, these central memory Tregs possess enhanced spare respiratory capacity, similar to CD8 + memory cells. Alternatively, the generation of effector Tregs (eTregs) requires mTOR function. Indeed, genetic deletion of Rptor leads to the decreased expression of ICOS and PD-1 on the eTregs. Overall, our studies define a subset of mTORC1 hi eTregs and mTORC1 lo central Tregs., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Objective: To explore the role of inducible costimulatory molecule (ICOS) signaling pathway-mediated Th17 cells polarization in renal damage in essential hypertension., Methods: Four-week-old spontaneously hypertensive rats (SHR) were randomly divided into control (SHR-C) group and intervention (SHR-I) group and subjected to intraperitoneal injections of PBS and ICOS monoclonal antibody for 2 weeks, respectively. Blood pressure of the rats was monitored using noninvasive tail artery blood pressure measuring instrument. The percentage of Th17 cells in the splenocytes was analyzed using flow cytometry, and the expression levels of IL-17A mRNA in the rat's kidneys were detected using RT-PCR. The levels of IL-17A and TGF-β1 in the plasma and kidneys were dynamically detected using ELISA and immunohistochemistry, respectively. Renal pathological changes in the rats were detected using Masson staining., Results: At the age of 10 and 30 weeks, the rats in SHR-C group had a significantly higher blood pressure than those in SHR-I group (P<0.05 or 0.01). In rats in SHR-C group, Th17 cells percentage in the splenocytes and IL-17A mRNA level in the kidney was significantly higher than those in SHR-I group from the age of 6 weeks (P<0.05). The expressions of IL-17A and TGF-β1 in the plasma and kidney were significantly higher in SHR-C group than that in SHR-I group at 6 weeks (P<0.05). Compared with those in SHR-C group, the rats in SHR-I group showed significant alleviation of renal fibrosis from the age of 30 weeks (P<0.05)., Conclusion: The ICOS signaling pathway-mediated Th17 cells polarization plays an important role in renal fibrosis in hypertensive rats.

ICOS is a key costimulatory receptor facilitating differentiation and function of follicular helper T cells and inflammatory T cells. Rheumatoid arthritis patients were shown to have elevated levels of ICOS + T cells in the synovial fluid, suggesting a potential role of ICOS-mediated T cell costimulation in autoimmune joint inflammation. In this study, using ICOS knockout and knockin mouse models, we found that ICOS signaling is required for the induction and maintenance of collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. For the initiation of CIA, the Tyr 181 -based SH2-binding motif of ICOS that is known to activate PI3K was critical for Ab production and expansion of inflammatory T cells. Furthermore, we found that Tyr 181 -dependent ICOS signaling is important for maintenance of CIA in an Ab-independent manner. Importantly, we found that a small molecule inhibitor of glycolysis, 3-bromopyruvate, ameliorates established CIA, suggesting an overlap between ICOS signaling, PI3K signaling, and glucose metabolism. Thus, we identified ICOS as a key costimulatory pathway that controls induction and maintenance of CIA and provide evidence that T cell glycolytic pathways can be potential therapeutic targets for rheumatoid arthritis., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Objective: To investigate whether the balance of blood follicular helper T (Tfh) cells and T follicular regulatory (Tfr) cells can provide information about ectopic lymphoid neogenesis and disease activity in primary Sjögren's syndrome (SS)., Methods: We prospectively recruited 56 patients clinically suspected of having SS. Sixteen of these patients subsequently fulfilled the American-European Consensus Group criteria for SS and were compared to 16 patients with non-SS sicca syndrome. Paired blood and minor salivary gland (MSG) biopsy samples were analyzed to study Tfr cells and subsets of Tfh cells in both compartments., Results: Patients with primary SS had normal Tfh cell counts in peripheral blood; however, activated programmed death 1-positive (PD-1+) inducible costimulator-positive (ICOS+) Tfh cells in peripheral blood were strongly associated with disease activity assessed by the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (r = 0.8547, P = 0.0008). Conversely, the blood Tfr cell:Tfh cell ratio indicated ectopic lymphoid structure formation in MSGs, being strongly associated with B cell, CD4+ T cell, and PD-1+ICOS+ T cell infiltration in MSGs, and was especially increased in patients with focal sialadenitis. Further analysis showed that the blood Tfr cell:Tfh cell ratio allowed discrimination between SS patients and healthy donors with excellent accuracy and was a strong predictor of SS diagnosis (odds ratio [OR] 12.96, P = 0.028) and the presence of focal sialadenitis (OR 10, P = 0.022) in patients investigated for sicca symptoms, thus highlighting the potential clinical value of this marker., Conclusion: The blood Tfr cell:Tfh cell ratio and PD-1+ICOS+ Tfh cells constitute potential novel biomarkers for different features of primary SS. While the blood Tfr cell:Tfh cell ratio is associated with ectopic lymphoid neogenesis, activated Tfh cells indicate disease activity., (© 2018, American College of Rheumatology.)

The aim of this study was to investigate the role of miR-192 in differentiation of T follicular helper cells in childhood asthma. Blood samples were taken from eighteen children with acute asthma attacks and fifteen healthy children (HC). Quantitative real-time PCR and Western blotting were used to detect the expression levels of miR-192, C-X-C chemokine receptor type 5 (CXCR5), B-cell lymphoma 6 (BCL-6) and inducible T-cell costimulator (ICOS). The flow cytometry was performed to detect the proportion of CD4 + CXCR5+ Tfh cells on CD4 + T lymphocytes. The enzyme-linked immunosorbent assay (ELISA) was carried out to determine the plasma concentrations of total IgE and IL-21. The effect of miR-192 on the T follicular helper cells differentiation by targeting CXCR5 was determined by dual-luciferase reporter assay. Children with asthma had lower levels of miR-192 than HC. The proportion of CD4 + CXCR + Tfh cells was significantly higher in the acute asthma group than HC. Similarly, the plasma concentration of total IgE and IL-21 in the acute group markedly increased compared with the HC, and IgE concentration was positively correlated with the proportion of CD4 + CXCR5 + Tfh cells. Furthermore, the expression levels of CXCR5, Bcl-6 and ICOS were significantly higher in the acute group than in the HC. While the proportion of CD4 + CXCR5 + Tfh cells, IL-21, CXCR5, Bcl-6 and ICOS were obviously lower in the CD4 + T cells transfected with miR-192 plasmid than that in miR-192 + CXCR5 group and control group. In conclusion, miR-192 blocks the activation pathway of Tfh cells by targeting CXCR5, which is a reasonable cellular target for therapeutic intervention.

Introduction: The recent success of checkpoint-inhibitors in cancer treatment paved the way for the development of new strategies of agonist and antagonist agents against B7 superfamily members. Inducible Co-Stimulator (ICOS), a co-stimulatory receptor for T-cell enhancement, arouses interest. Areas covered: We performed an extensive literature search with PUBMED using the keywords 'ICOS' and 'cancer' to discuss its involvement in oncogenesis, its expression in different malignancies, and its targeting in relevant preclinical studies. We also searched the Clinicaltrials.gov database for recent updates on early phase clinical trials. Expert opinion: ICOS/ICOSL axis has a dual effect and might participate in anti-tumour T cell response as well as a pro-tumour response due to its connection with regulatory T-cells (Tregs) suppressive activity. Therefore, both antagonist and agonist antibodies might be of interest in the targeting ICOS/ICOSL pathway for cancer treatment. In preclinical studies, ICOS agonist monoclonal antibodies (mAbs) have shown to potentiate the effect of inhibitory checkpoint blockade. In contrast, antagonistic anti-ICOS mAbs could not only inhibit lymphoid tumour cells expressing ICOS, but also dampen immunosuppressive Tregs. Two agonist and one antagonist mAbs are evaluated in phase I/II trials. Efficacy, safety, and combination strategies with anti-ICOS agonist or antagonist have yet to be specified.

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3'-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3'-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3'-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

In murine model systems inducible costimulator (ICOS) signaling has been implicated in the formation of chronic graft-versus-host disease (GVHD). Previously, we showed that chronic GVHD can be reproducibly produced in the dog hematopoietic cell transplantation (HCT) model and that ICOS expression is upregulated on T cells in dogs with chronic GVHD. The goal of the present study was to determine whether administration of a short course of anti-canine ICOS mAb could alter the rapid and progressive course of chronic GVHD. Five dogs underwent HCT from dog leukocyte antigen mismatched unrelated donors after total body irradiation. Postgrafting immunosuppression consisted of methotrexate (days 1, 3, 6, and 11) and cyclosporine (days -1 through 78). Anti-ICOS mAb (3 injections, 72 hours apart) was administered upon diagnosis of GVHD. One dog failed to respond to anti-ICOS mAb therapy and succumbed to chronic GVHD in a time course similar to control untreated dogs. Overall, anti-ICOS-treated dogs experienced a significant prolongation in survival from the time of diagnosis of chronic GVHD compared with control dogs. Within the limitations of the number of study dogs we suggest that a short course of anti-ICOS mAb may be useful in the treatment of chronic canine GVHD., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Hepatitis B surface antigen (HBsAg) seroconversion in HBeAg -ve chronic hepatitis B (CHB) infection is rare, possibly due to poor antigen processing and impaired humoral response. We investigated the role of dendritic cells (DCs), T follicular helper (TFH) cells and plasma B cells in seroconversion., Methods: HBeAg -ve (n=135) CHB patients with raised ALT at baseline were followed up. Patients undergoing HBsAg seroconversion (Gr. I, n=11) were compared with non-converters with low (Gr. II, n=17, HBV DNA<2000 IU/mL) or high HBV DNA (Gr. III, HBV DNA >2000 IU/mL, n=17). We measured cell phenotypes (TFH, B and DCs), HBV specific T-cell functionality [using pooled overlapping surface and core peptides], IL21 levels and gene expression analysis by qRT-PCR., Results: Patients in Gr. I compared to Gr. II and III, had higher IL-21 levels (865 vs 276 vs 111 pg/mL, P=<.0001), TFH (CD4 + CXCR5 + ) cells (12.3 vs 4.67 vs 2.77, P=<.001), inducible T-cell co-stimulator (ICOS) expression on TFH cells (20.0 vs 13.0 vs 13.68, P=.01), HBsAg specific IL-17 (9.40 vs 2.33 vs 2.61, P=<.001) and TNF-α secreting TFH17 cells (82 vs 1.43 vs 2.33, P=<.001), plasma B (CD19 + CD38 + ) cells (15.0 vs 5.08 vs 5.57, P=<.001), myeloid (17.80 vs 5.39 vs 2.70, P=<.001) and plasmocytoid DCs (2.6 vs 0.43 vs 0.21, P=<.001). Plasma B-cell frequency (R 2 =.64, P=.01) and IL-21 levels (R 2 =.52, P=.003) correlated with anti-HBs titres in patients with HBsAg seroconversion., Conclusions: Dendritic cell and TFH cell mediated responses regulate humoral responses against HBV and play a major role in HBsAg seroconversion in CHB patients., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)