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Group 2 innate lymphoid cells (ILC2s) are recognized as key controllers and effectors of type 2 inflammation. Mesenchymal stem cells (MSCs) have been shown to alleviate type 2 inflammation by modulating T lymphocyte subsets and decreasing TH2 cytokine levels. However, the effects of MSCs on ILC2s have not been investigated. In this study, we investigated the potential immunomodulatory effects of MSCs on ILC2s in peripheral blood mononuclear cells (PBMCs) from allergic rhinitis patients and healthy subjects. We further investigated the mechanisms involved in the MSC modulation using isolated lineage negative (Lin−) cells. PBMCs and Lin− cells were cocultured with induced pluripotent stem cell‐derived MSCs (iPSC‐MSCs) under the stimulation of epithelial cytokines IL‐25 and IL‐33. And the ILC2 levels and functions were examined and the possible mechanisms were investigated based on regulatory T (Treg) cells and ICOS‐ICOSL pathway. iPSC‐MSCs successfully decreased the high levels of IL‐13, IL‐9, and IL‐5 in PBMCs in response to IL‐25, IL‐33, and the high percentages of IL‐13+ILC2s and IL‐9+ILC2s in response to epithelial cytokines were significantly reversed after the treatment of iPSC‐MSCs. However, iPSC‐MSCs were found directly to enhance ILC2 levels and functions via ICOS‐ICOSL interaction in Lin− cells and pure ILC2s. iPSC‐MSCs exerted their inhibitory effects on ILC2s via activating Treg cells through ICOS‐ICOSL interaction. The MSC‐induced Treg cells then suppressed ILC2s by secreting IL‐10 in the coculture system. This study revealed that human MSCs suppressed ILC2s via Treg cells through ICOS‐ICOSL interaction, which provides further insight to regulate ILC2s in inflammatory disorders., Human mesenchymal stem cells (MSCs) either directly promote group 2 innate lymphoid cell (ILC2) activation through upregulated ICOS‐ICOSL interaction. MSCs exert inhibitory effects on ILC2s via activating regulatory T (Treg) cells through ICOS‐ICOSL interaction. Treg cells prevent the promotion of MSCs on ILC2s via decreased ICOS‐ICOSL interaction between MSCs and ILC2s. IL‐10 is required for the MSC‐induced Treg cells in suppressing ILC2s alongside ICOS‐ICOSL interaction.

Immunofibroblasts have been described within tertiary lymphoid structures (TLS) that regulate lymphocyte aggregation at sites of chronic inflammation. Here we report, for the first time, an immunoregulatory property of this population, dependent on inducible T-cell co-stimulator ligand and its ligand (ICOS/ICOS-L). During inflammation, immunofibroblasts, alongside other antigen presenting cells, like dendritic cells (DCs), upregulate ICOSL, binding incoming ICOS + T cells and inducing LTα3 production that, in turn, drives the chemokine production required for TLS assembly via TNFRI/II engagement. Pharmacological or genetic blocking of ICOS/ICOS-L interaction results in defective LTα expression, abrogating both lymphoid chemokine production and TLS formation. These data provide evidence of a previously unknown function for ICOSL-ICOS interaction, unveil a novel immunomodulatory function for immunofibroblasts, and reveal a key regulatory function of LTα3, both as biomarker of TLS establishment and as first driver of TLS formation and maintenance in mice and humans.

Inducible T cell co-stimulator (ICOS), an immune checkpoint protein expressed on activated T cells and its unique ligand, ICOSL, which is expressed on antigen-presenting cells and non-hematopoietic cells, have been extensively investigated in the immune response. Recent findings showed that a soluble recombinant form of ICOS (ICOS-Fc) can act as an innovative immunomodulatory drug as both antagonist of ICOS and agonist of ICOSL, modulating cytokine release and cell migration to inflamed tissues. Although the ICOS-ICOSL pathway has been poorly investigated in the septic context, a few studies have reported that septic patients have reduced ICOS expression in whole blood and increased serum levels of osteopontin (OPN), that is another ligand of ICOSL. Thus, we investigated the pathological role of the ICOS-ICOSL axis in the context of sepsis and the potential protective effects of its immunomodulation by administering ICOS-Fc in a murine model of sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in five-month-old male wild-type (WT) C57BL/6, ICOS-/-, ICOSL-/- and OPN-/- mice. One hour after the surgical procedure, either CLP or Sham (control) mice were randomly assigned to receive once ICOS-Fc, F119SICOS-Fc, a mutated form uncapable to bind ICOSL, or vehicle intravenously. Organs and plasma were collected 24 h after surgery for analyses. When compared to Sham mice, WT mice that underwent CLP developed within 24 h a higher clinical severity score, a reduced body temperature, an increase in plasma cytokines (TNF-α, IL-1β, IL-6, IFN-γ and IL-10), liver injury (AST and ALT) and kidney (creatinine and urea) dysfunction. Administration of ICOS-Fc to WT CLP mice reduced all of these abnormalities caused by sepsis. Similar beneficial effects were not seen in CLP-mice treated with F119SICOS-Fc. Treatment of CLP-mice with ICOS-Fc also attenuated the sepsis-induced local activation of FAK, P38 MAPK and NLRP3 inflammasome. ICOS-Fc seemed to act at both sides of the ICOS-ICOSL interaction, as the protective effect was lost in septic knockout mice for the ICOS or ICOSL genes, whereas it was maintained in OPN knockout mice. Collectively, our data show the beneficial effects of pharmacological modulation of the ICOS-ICOSL pathway in counteracting the sepsis-induced inflammation and organ dysfunction.

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.

The liver capacity to recover from acute liver injury is a critical factor in the development of acute liver failure (ALF) caused by viral infections, ischemia/reperfusion or drug toxicity. Liver healing requires the switching of pro-inflammatory monocyte-derived macrophages(MoMFs) to a reparative phenotype. However, the mechanisms involved are still incompletely characterized. In this study we investigated the contribution of T-lymphocyte/macrophage interaction through the co-stimulatory molecule Inducible T-cell co-stimulator (ICOS; CD278) and its ligand (ICOSL; CD275) in modulating liver repair. The role of ICOS/ICOSL dyad was investigated during the recovery from acute liver damage induced by a single dose of carbon tetrachloride (CCl4). Flow cytometry of non-parenchymal liver cells obtained from CCl4-treated wild-type mice revealed that the recovery from acute liver injury associated with a specific up-regulation of ICOS in CD8+ T-lymphocytes and with an increase in ICOSL expression involving CD11bhigh/F4-80+ hepatic MoMFs. Although ICOS deficiency did not influence the severity of liver damage and the evolution of inflammation, CCl4-treated ICOS knockout (ICOS-/-) mice showed delayed clearance of liver necrosis and increased mortality. These animals were also characterized by a significant reduction of hepatic reparative MoMFs due to an increased rate of cell apoptosis. An impaired liver healing and loss of reparative MoMFs was similarly evident in ICOSL-deficient mice or following CD8+ T-cells ablation in wild-type mice. The loss of reparative MoMFs was prevented by supplementing CCl4-treated ICOS-/- mice with recombinant ICOS (ICOS-Fc) which also stimulated full recovery from liver injury. These data demonstrated that CD8+ T-lymphocytes play a key role in supporting the survival of reparative MoMFs during liver healing trough ICOS/ICOSL-mediated signaling. These observations open the possibility of targeting ICOS/ICOSL dyad as a novel tool for promoting efficient healing following acute liver injury.

With the rapid increase in the incidence of allergic diseases, the mechanisms underlying the development of these diseases have received a great deal of attention, and this is particularly true in regard to the role of ICOS in allergic diseases. Current studies have revealed that ICOS affects the functional activity of multiple immune cells that modulate the adaptive immune system. Additionally, ICOS also plays a crucial role in mediating cellular immunity and coordinating the response of the entire immune system, and thus, it plays a role in allergic reactions. However, the ICOS/ICOS-ligand (ICOS-L) axis functions in a dual role during the development of multiple allergic diseases. In this review, we explore the role of ICOS/ICOSL in the context of different immune cells that function in allergic diseases, and we summarize recent advances in their contribution to these diseases.

The role of ICOS and its ligand (ICOSL) have both been shown to be essential for proper humoral responses as well as autoimmune Ab development in mouse models of lupus. In this paper, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using B6mir146a−/− mice and a model of lymphoproliferative disease using the well-characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared with control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA Ab production. In line with this, we found a significant reduction in follicular helper T cells and germinal center B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only show a role of B cell ADAM10 in control autoimmunity but also increase our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity.

Genome-wide association studies have identified ICOSLG, which encodes the inducible costimulator ligand (ICOSLG or ICOSL) as a susceptibility locus for inflammatory bowel disease. ICOSL has been implicated in the enhancement of pattern recognition receptor signaling in dendritic cells, induction of IL-10 production by CD4 T cells, and the generation of high-affinity antibodies to specific antigens—all of which can potentially explain its involvement in gastrointestinal inflammation. Here, we show that murine ICOSL deficiency results in significant enrichment of IL-10–producing CD4 T cells particularly in the proximal large intestine. Transient depletion of IL-10–producing cells from adult ICOSL-deficient mice induced severe colonic inflammation that was prevented when mice were first treated with metronidazole. ICOSL-deficient mice displayed reduced IgA and IgG antibodies in the colon mucus and impaired serum antibody recognition of microbial antigens, including flagellins derived from mucus-associated bacteria of the Lachnospiraceae family. Confirming the synergy between ICOSL and IL-10, ICOSL deficiency coupled with CD4-specific deletion of the Il10 gene resulted in juvenile onset colitis that was impeded when pups were fostered by ICOSL-sufficient dams. In this setting, we found that both maternally acquired and host-derived antibodies contribute to the life anti-commensal antibody repertoire that mediates this protection in early life. Collectively, our findings reveal a partnership between ICOSL-dependent anti-commensal antibodies and IL-10 in adaptive immune regulation of the microbiota in the large intestine. Furthermore, we identify ICOSL deficiency as an effective platform for exploring the functions of anti-commensal antibodies in host–microbiota mutualism.

18 p.-8 fig.-1 tab., The interaction between the T‐lymphocyte costimulatory molecule ICOS and its ligand (ICOS‐L) is needed for efficient immune responses, but expression levels are tightly controlled, as altered expression of ICOS or ICOS‐L may lead to immunodeficiency, or favor autoimmune diseases and tumor growth. Using cells of mouse B cell lymphoma (M12.C3) and melanoma (B16), or hamster CHO cells transfected with various forms of mouse ICOS‐L, and ICOS+ T cell lines, we show that, within minutes, ICOS induces significant downmodulation of surface ICOS‐L that is largely mediated by endocytosis and trans‐endocytosis. So, after interaction with ICOS+ cells, ICOS‐L was found inside permeabilized cells, or in cell lysates, with significant transfer of ICOS from ICOS+ T cells to ICOS‐L‐expressing cells, and simultaneous loss of surface ICOS by the T cells. Data from cells expressing ICOS‐L mutants show that conserved, functionally important residues in the cytoplasmic domain of mouse ICOS‐L (Arg300, Ser307 and Tyr308), or removal of ICOS‐L cytoplasmic tail have minor effect on its internalization. Internalization was dependent on temperature, and was partially dependent on actin polymerization, the GTPase dynamin, protein kinase C, or the integrity of lipid rafts. In fact, a fraction of ICOS‐L was detected in lipid rafts. On the other hand, proteinase inhibitors had negligible effects on early modulation of ICOS‐L from the cell surface. Our data add a new mechanism of control of ICOS‐L expression to the regulation of ICOS‐dependent responses., Supported by Grants PI13/01809 (to J.M.R.), PI13/02153 and PI16CIII/00012 (to P.P.) from “Acción Estratégica en Salud, Plan Estatal I+D+i”, Ministerio de Economía, Industria y Competitividad(MINECO, Spain) and by the Associazione Italiana Ricerca sul Cancro(Grant IG20714, AIRC, Milan), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017-0535) (to U.D.).

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses., eLife, 10, ISSN:2050-084X

Elevated gene expression of the costimulatory receptor Icos is a hallmark of CD8(+) tissue-resident memory (Trm) T cells. Here we examined the contribution of ICOS in Trm cell differentiation. Upon transfer into WT mice, Icos(−/−) CD8(+) T cells exhibited defective Trm generation but produced recirculating memory populations normally. ICOS-deficiency or ICOS-L blockade compromised establishment of CD8(+) Trm cells but not their maintenance. ICOS ligation during CD8(+) T cell priming did not determine Trm induction; rather effector CD8(+) T cells showed reduced Trm differentiation after seeding into Icosl(−/−) mice. Icos(YF/YF) CD8(+) T cells were compromised in Trm generation, indicating a critical role for PI3K signaling. Modest transcriptional changes in the few Icos(−/−) Trm cells suggest that ICOS-PI3K signaling primarily enhances the efficiency of CD8(+) T cell tissue residency. Thus, local ICOS signaling promotes production of Trm cells, providing insight into the contribution of costimulatory signals in the generation of tissue-resident populations.

The inducible co-stimulator (ICOS) is a member of the CD28/B7 superfamily, and delivers a positive co-stimulatory signal to activated T cells upon binding to its ligand (ICOS-L). Dysregulation of this pathway has been implicated in autoimmune diseases and cancer, and is currently under clinical investigation as an immune checkpoint blockade. Here, we describe the molecular interactions of the ICOS/ICOS-L immune complex at 3.3 Å resolution. A central FDPPPF motif and residues within the CC’ loop of ICOS are responsible for the specificity of the interaction with ICOS-L, with a distinct receptor binding orientation in comparison to other family members. Furthermore, our structure and binding data reveal that the ICOS N110 N-linked glycan participates in ICOS-L binding. In addition, we report crystal structures of ICOS and ICOS-L in complex with monoclonal antibodies under clinical evaluation in immunotherapy. Strikingly, antibody paratopes closely mimic receptor-ligand binding core interactions, in addition to contacting peripheral residues to confer high binding affinities. Our results uncover key molecular interactions of an immune complex central to human adaptive immunity and have direct implications for the ongoing development of therapeutic interventions targeting immune checkpoint receptors., The inducible co-stimulator (ICOS) is a member of the CD28/B7 superfamily, binding its ligand (ICOS-L) on activated T cells. The structure of the ICOS/ICOS-L complex reveals a distinct receptor binding orientation. The structures of ICOS and ICOS-L in complex with potentially therapeutic antibodies suggest the structural basis of such antibodies’ efficacies and high binding affinities.

CTLA4-Ig/abatacept dampens activation of naive T cells by blocking costimulation via CD28. It is an approved drug for rheumatoid arthritis but failed to deliver efficacy in a number of other autoimmune diseases. One explanation is that activated T cells rely less on CD28 signaling and use alternate coreceptors for effector function. ICOS is critical for activation of T-dependent humoral immune responses, which drives pathophysiology of IgG-mediated autoimmune diseases. In this study, we asked whether CD28 and ICOS play nonredundant roles for maintenance of T-dependent responses in mouse models. Using a hapten–protein immunization model, we show that during an ongoing germinal center response, combination treatment with CTLA4-Ig and ICOS ligand (ICOSL) blocking Ab completely dissolves ongoing germinal center responses, whereas single agents show only partial activity. Next, we took two approaches to engineer a therapeutic molecule that blocks both pathways. First, we engineered CTLA4-Ig to enhance binding to ICOSL while retaining affinity to CD80/CD86. Using a library approach, binding affinity of CTLA4-Ig to human ICOSL was increased significantly from undetectable to 15–42 nM; however, the affinity was still insufficient to completely block binding of ICOSL to ICOS. Second, we designed a bispecific costimulation inhibitor with high-affinity CTLA4 extracellular domains fused to anti-ICOSL Ab termed bifunctional costimulation inhibitor. With this bispecific approach, we achieved complete inhibition of CD80 and CD86 binding to CD28 as well as ICOS binding to ICOSL. Such bispecific molecules may provide greater therapeutic benefit in IgG-mediated inflammatory diseases compared with CTLA4-Ig alone.

13 p.-7 fig.-1 tab., Inducible T-cell costimulator (ICOS) upon binding to its ligand (ICOSL) mediates adaptive immunity and antitumor response. Thus, antitumor therapies targeting the ICOS/ICOSL pathway hold great promise for cancer treatment. In this regard, ICOSL triggering by a soluble recombinant form of ICOS (ICOS-Fc) hampered adhesiveness and migration of dendritic, endothelial, and tumor cells in vitro. Furthermore, in vivo treatment with ICOS-Fc previously showed the capability to inhibit lung metastatization of ICOSL+ B16-F10 melanoma cells when injected intravenously in mice, but it failed to block the growth of established subcutaneous B16-F10 murine tumors. Thus, we asked whether passive targeting of solid tumors with ICOS-Fc-loaded biocompatible and biodegradable nanoparticles (NPs) could instead prove effectiveness in reducing tumor growth. Here, ICOS-Fc was loaded in two types of polymer nanoparticles, i.e. cross-linked β-cyclodextrin nanosponges (CDNS) and poly(lactic-co-glycolic acid) (PLGA) NPs and in vitro characterized. In vivo experiments showed that treatment of C57BL6/J mice with ICOS-Fc loaded into the two nanoformulations inhibits the growth of established subcutaneous B16-F10 tumors. This anticancer activity appears to involve both anti-angiogenic and immunoregulatory effects, as shown by decreased tumor vascularization and downmodulation of IL-10 and Foxp3, two markers of regulatory T cells (Tregs). Overall, the substantial in vivo anticancer activity of ICOS-Fc-loaded CDNS and PLGA NPs against different components of the tumor microenvironment makes these nanoformulations attractive candidates for future combination cancer therapy., This work was supported by the Associazione Italiana Ricerca sul Cancro (IG 20714, AIRC, Milano), Fondazione Amici di Jean (Torino), and Fondazione Cariplo (2017–0535).

Background Allergic asthma is characterized by chronic inflammation and remodelling of the airways, associated with dysregulated type 2 immune responses and allergen‐specific IgE. T follicular helper cells (TFH) are crucial in T‐dependent B‐cell responses and have been implicated in allergic airway disease (AAD). TFH, unlike other CD4+ T cells, are uniquely reliant on continuous ICOS signalling to maintain their phenotype after T‐cell priming; therefore, disrupting this signal can impair TFH responses. However, the contribution of TFH to disease during chronic aero‐allergen exposure and the therapeutic potential of targeting these cells have not been evaluated. Methods To establish AAD, female BALB/c mice were repeatedly exposed to house dust mite or Alternaria alternata three times a week for up to 5 weeks. To examine the impact of TFH on AAD, mice were allergen exposed for 5 weeks and co‐administered anti‐ICOS Ligand‐targeted antibodies, three times a week for the last 2 weeks. Results TFH were first observed in the lung‐draining lymph nodes and with further exposure were also found locally within the lungs. TFH accumulated with sustained allergen exposure, alongside germinal centre (GC) B cells. Blockade of ICOS signalling after AAD establishment successfully depleted TFH but did not affect the differentiation of other CD4+ T‐cell subsets. This reduced GC responses, allergen‐specific IgE, inflammation, pulmonary IL‐13 and airway hyper‐responsiveness. Conclusions TFH are crucial in the regulation of AAD and the ICOS/ICOS‐L pathway could represent a novel therapeutic target in allergic asthma.

B-cell interaction with follicular helper T cells and subsequent differentiation of B cells into high-affinity APCs normally takes place in secondary lymphoid organs. The costimulator ICOS plays a key role in this process and is therefore considered as an attractive target to modulate exaggerated B-cell responses in autoimmune or allergic diseases. Inflamed tissues were recently recognized as additional sites of active T-cell/B-cell interaction. To analyze whether ICOS costimulation is also important there, we employed a mouse airway inflammation model that allows direct comparison of immune reactions in the lung-draining lymph node and the lung tissue as well as assessment of the relative importance of dendritic cells versus B cells as APCs. In both organs, ICOS regulated the pool size of antigen-specific T and B cells and B-cell differentiation into germinal center(-like) cells but not into antibody-secreting cells. In the lymph node, lack of ICOS costimulation drastically reduced the frequency of T follicular helper cells but did not affect production of T-helper cell type 2 (Th2) cytokines. Vice versa in the lung tissue, ICOS did not change PD-1 expression on infiltrating T cells but regulated Th2 cytokine production, a process for which ICOS ligand expression on B cells was of particular importance. Taken together, the results of this study show that ICOS differentially regulates effector T cells in secondary lymphoid organs and inflamed tissues but that blockade of the ICOS pathway is suitable to target T cell-dependent B cell responses at both sites.

Emerging data suggest that locoregional cancer therapeutic approaches with oncolytic viruses can lead to systemic anti-tumour immunity, although the appropriate targets for intratumoral immunomodulation using this strategy are not known. Here we find that intratumoral therapy with Newcastle disease virus (NDV), in addition to the activation of innate immunity, upregulates the expression of T-cell co-stimulatory receptors, with the inducible co-stimulator (ICOS) being most notable. To explore ICOS as a direct target in the tumour, we engineered a recombinant NDV-expressing ICOS ligand (NDV-ICOSL). In the bilateral flank tumour models, intratumoral administration of NDV-ICOSL results in enhanced infiltration with activated T cells in both virus-injected and distant tumours, and leads to effective rejection of both tumours when used in combination with systemic CTLA-4 blockade. These findings highlight that intratumoral immunomodulation with an oncolytic virus expressing a rationally selected ligand can be an effective strategy to drive systemic efficacy of immune checkpoint blockade., Oncolytic viruses induce a variety of immune targets in the infected tumours. Here, the authors show that Newcastle Disease Virus (NDV) upregulates the inducible co-stimulator (ICOS) on T cells and that intratumoral targeting of ICOS with engineered NDV in combination with CTLA-4 blockade induces systemic anti-tumour immunity in mice.

This study aimed to investigate the possible functions and mechanisms of positive and negative costimulatory molecules in the pathological process of myasthenia gravis (MG). The expression levels of membrane-bound inducible costimulator (ICOS) and programmed cell death 1 (PD-1) in peripheral blood T cells, their corresponding ligands ICOSL and PDL-1 on B cells, and their soluble forms (sICOS, sPD-1, sICOSL, and sPDL-1) in plasma were detected in patients with untreated-stage MG (USMG) and remission-stage MG (RSMG). The results showed that the expression levels of membrane-bound ICOS and PD-1 in the peripheral blood T cells of the USMG group and their corresponding ligands ICOSL and PD-L1 on B cells were significantly increased compared to those in the RSMG group and healthy controls (HCs). The levels of sICOSL and sPD-1 were significantly upregulated in USMG patients compared to those in the RSMG and HC groups, while the levels of sICOS and sPD-L1 were not different. The expression of PD-L1 on CD19+ B cells was positively correlated with the concentrations of AchR Ab in the USMG group. The expression of ICOS and PD-1 in CD4+ T cells and the expression of ICOSL and PD-L1 on CD19+ B cells were positively correlated with the quantitative myasthenia gravis (QMG) scores in the USMG group. Also, in the USMG group, the plasma levels of sICOSL and sPD-1 were positively correlated with the QMG scores. In addition, the percentage of peripheral blood follicular helper T (Tfh) cells in the USMG group was positively correlated with ICOS and PD-1 expression on CD4+ T cells and ICOSL and PD-L1 expression on CD19+ B cells. There were positive correlations between sICOSL and sPD-1 levels and the percentage of peripheral blood Tfh cells and plasma interleukin-21 (IL-21) levels in the USMG group. The results suggest that the positive ICOS/ICOSL and negative PD-1/PD-L1 costimulatory molecule pairs participate in the pathological process of MG. Abnormal sICOSL and sPD-1 expression might interfere with the normal signal transduction of ICOS and PD-1 on Tfh cells, causing excessive activation of Tfh cells and promotion of disease progression. sICOSL and sPD-1 have potential value in monitoring MG disease states.

26 p.-8 fig., Signaling through the inducible costimulator ICOS is required for the homeostasis and function of various immune cell populations, with an outstanding role in the generation and maintenance of germinal centers. Very recently, it has been suggested that the clinical phenotype of ICOS-deficient patients is much broader than initially anticipated and the innate immune response might be also affected. However, the role of the ICOS/ICOS-Ligand axis in the homeostasis and development of innate NK cells is not known, and reports on its participation in NK cell activation are scarce. NK cells may express low levels of ICOS that are markedly enhanced upon activation. We show here that ICOS-deficient (ICOS-KO) mice present low NK cell numbers and defects in the homeostasis of these cells, with delayed maturation and altered expression of the developmental NK cell markers CD122, NK1.1, CD11b or CD27. Our experiments in mixed bone marrow chimera mice indicate that, both, cell-intrinsic defects of ICOS-KO NK and deficiencies in the milieu of these mice contribute to the altered phenotype. ICOS-deficient NK cells show impaired production of IFN-γ and cytotoxicity, and a final outcome of defects in NK cell-mediated effector function during the response to poly(I:C) or vaccinia virus infection in vivo. Interestingly, we show that murine innate cells like IL-2-cultured NK and bone marrow-derived dendritic cells can simultaneously express ICOS and ICOS-Ligand; both molecules are functional in NK intracellular signaling, enhancing early phosphorylation of Akt and Erk, or IFN-γ secretion in IL-2-activated NK cells. Our study shows the functional importance of the ICOS/ICOS-L pair in NK cell homeostasis, differentiation and activity and suggests novel therapeutic targets for NK manipulation., This work was supported by grants from the Acción Estratégica en Salud (Instituto de Salud Carlos III, ISCIII, MINECO, Spain) (PI13/02153 and PI16CIII/00012 to P.P.; PI13/01809 to J.M.R.; and PI14/00049 to BdA); grants from Ministerio de Economía y Competitividad MINECO/FEDER, Spain (SAF2015-70880-R to M.L.G. and SAF2014-58052 to D.L.); and grants from Associazione Italiana per la Ricerca sul Cancro, AIRC, Milan (IG20714) and the Fondazione Amici di Jean, Torino, Italy (to U.D.).

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.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.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.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.

T cells undergo homeostatic expansion and acquire an activated phenotype in lymphopenic microenvironments. Restoration of normal lymphocyte numbers typically re-establishes normal homeostasis, and proinflammatory cytokine production returns to baseline. Mice deficient in guanine nucleotide exchange factor RasGRP1 exhibit dysregulated homeostatic expansion, which manifests as lymphoproliferative disease with autoantibody production. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development, as well as during germinal center responses, suggesting that T cell–independent and T cell–dependent mechanisms are responsible. Examination of whether a particular T cell subset is involved in the breach of B cell tolerance revealed increased Th17 cells in Rasgrp1-deficient mice relative to control mice. Rasgrp1-deficient mice lacking IL-17R had fewer germinal centers, and germinal centers that formed contained fewer autoreactive B cells, suggesting that IL-17 signaling is required for a break in B cell tolerance in germinal centers. Interestingly, a fraction of Th17 cells from Rasgrp1-deficient mice were CXCR5+ and upregulated levels of CD278 coordinate with their appearance in germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278–CD275 interactions were required for the development of Tfh17 cells and for autoantibody, Rasgrp1-deficient mice were crossed with CD275-deficient mice. Surprisingly, mice deficient in RasGRP1 and CD275 formed Tfh17 cells and germinal centers and produced similar titers of autoantibodies as mice deficient in only RasGRP1. Therefore, these studies suggest that requirements for Tfh cell development change in lymphopenia-associated autoimmune settings.

Graft-versus-host disease is a complication in patients undergoing hematopoietic stem cell transplantation. Graft-versus-host disease includes acute graft-versus-host disease and chronic graft-versus-host disease. Host APCs (e.g., dendritic cells and macrophages), effector T cells (e.g., Th1, Th17, and abnormal Th17:regulatory T cell ratio), B cells, and NK cells are implicated in graft-versus-host disease physiopathology. Proinflammation cytokines (e.g., IL-17, IL-1β, and TNF-α) are increased in graft-versus-host disease. Costimulatory molecules play an important role in inducing graft-versus-host disease. Pattern-recognition receptors, such as TLRs and nucleotide-binding oligomerization domain-like receptors, are critically involved in the pathogenesis of graft-versus-host disease. Complement system C3 mediates Th1/Th17 polarization in human T cell activation and skin graft-versus-host disease. Accumulation of CD26 T cells in graft-versus-host disease target organs was found. As a therapeutic target, soluble CD83 molecules or antibodies have been demonstrated to have therapeutic effects against graft-versus-host disease, and signaling molecules promote the inflammatory and immune process of graft-versus-host disease. These immune cells and molecules could be the predictors of graft-versus-host disease development and the drug targets of the treatments for graft-versus-host disease. This article focuses on major advances on cellular and molecular mechanisms in graft-versus-host disease.

Lymphocyte costimulation plays a central role in immunology, inflammation, and immunotherapy. The inducible T cell costimulator (ICOS) is expressed on T cells following peptide: MHC engagement with CD28 costimulation. The interaction of ICOS with its sole ligand, the inducible T cell costimulatory ligand (ICOSL; also known as B7-related protein-1), triggers a number of key activities of T cells including differentiation and cytokine production. Suppression of T cell activation can be achieved by blocking this interaction and has been shown to be an effective means of ameliorating disease in models of autoimmunity. In this study, we isolated specific anti-ICOSL new antigen receptor domains from a synthetic phage display library and demonstrated their ability to block the ICOS/ICOSL interaction and inhibit T cell proliferation. Anti-mouse ICOSL domains, considered here as surrogates for the use of anti-human ICOSL domains in patient therapy, were tested for efficacy in a collagen-induced mouse model of rheumatoid arthritis where they significantly decreased the inflammation of joints and delayed and reduced overall disease progression and severity.

Polymorphisms within a gene’s 3′-UTR may modulate posttranscriptional regulation of gene expression, and may explain individual sensitivity of chemotherapy. To investigate the correlation between single nucleotide polymorphisms (SNPs) in 3′-UTRs of B7/CD28 family genes and the response of capecitabine-based chemotherapy in colon cancer, 16 SNPs were identified in 274 advanced colon cancer patients. Statistical analysis indicated that ICOS rs1559931, rs4404254, and rs4675379 were in complete linkage disequilibrium and significantly associated with chemotherapy response. Heterozygous patients with rs1559931 G/A (31.34% vs 48.29%; P = 0.016), rs4404254 T/C (30.43% vs 48.77%; P = 0.011), or rs4675379 G/C (28.13% vs 49.04%; P = 0.004) genotypes showed poorer response to chemotherapy compared to wildtype patients. Moreover, three SNPs, including ICOSL rs15927, ICOSL rs3804033 and CD28 rs3181113, were significantly associated with the occurrence of side effects of chemotherapy. In addition, patients with ICOSL rs15927 G/G (78.26%), ICOSL rs3804033 G/G (76.00%), or CD28 rs3181113 T/T (82.05%) were more prone to enduring adverse events compared to patients bearing other polymorphisms. Taken together, our findings demonstrated that polymorphisms in the 3′-UTRs of genes in the ICOS/CD28-ICOSL pathway may influence the efficacy and occurrence of adverse events of capecitabine-based chemotherapy in advanced colon cancer patients.

Activation of the inducible costimulator (ICOS) signaling pathway in T cells is difficult to assess with bioassays, because most T cell lines do not constitutively express ICOS. Additionally, engagement of ICOS by its natural ligand B7 related protein 1 (B7RP1) is insufficient to elicit ICOS signaling, but requires simultaneous costimulation of the T cell receptor (TCR) to be effective. Here we describe a genetically engineered human T cell line that expresses a chimeric receptor (ICOS-CD3) consisting of full-length human ICOS fused at its C-terminal end to the cytoplasmic domain of human CD3 zeta. When engaged by B7RP1, ICOS-CD3 initiated signaling independently of TCR costimulation and induced substantially more IL-2 secretion in Jurkat T cells compared to wildtype ICOS. We demonstrate that this signaling-enhanced chimeric receptor can be used in simple and sensitive bioassays to detect bioactive B7RP1, anti-B7RP1 drugs, and the presence of corresponding neutralizing anti-drug antibodies.

The inducible T cell costimulator (ICOS) is a potent promoter of organ inflammation in murine lupus. ICOS stimulates T follicular helper cell differentiation in lymphoid tissue, suggesting that it might drive autoimmunity by enhancing autoantibody production. Yet the pathogenic relevance of this mechanism remains unclear. It is also unknown whether other ICOS-induced processes might contribute to lupus pathology. Here we show that selective ablation of ICOS ligand (ICOSL) in CD11c(+) cells, but not in B cells, dramatically ameliorates kidney and lung inflammation in lupus-prone MRL.Fas(lpr) mice. Autoantibody formation was largely unaffected by ICOSL deficiency in CD11c(+) cells. However, ICOSL display by CD11c(+) cells in inflamed organs had a nonredundant role in protecting invading T cells from apoptosis by elevating activity of the PI3K-Akt signaling pathway, thereby facilitating T cell accrual. These findings reveal a mechanism that locally sustains organ inflammation in lupus.

SummaryAllergic asthma is caused by Th2-cell-type cytokines in response to allergen exposure. Type 2 innate lymphoid cells (ILC2s) are a newly identified subset of immune cells that, along with Th2 cells, contribute to the pathogenesis of asthma by producing copious amounts of IL-5 and IL-13, which cause eosinophilia and airway hyperreactivity (AHR), a cardinal feature of asthma. ILC2s express ICOS, a T cell costimulatory molecule with a currently unknown function. Here we showed that a lack of ICOS on murine ILC2s and blocking the ICOS:ICOS-ligand interaction in human ILC2s reduced AHR and lung inflammation. ILC2s expressed both ICOS and ICOS-ligand, and the ICOS:ICOS-ligand interaction promoted cytokine production and survival in ILC2s through STAT5 signaling. Thus, ICOS:ICOS-ligand signaling pathway is critically involved in ILC2 function and homeostasis.

ICOS signaling is required for inhibition of the transcription factor Klf2, which controls expression of genes expressed by follicular T helper (Tfh) cells. When ICOS signaling is blocked, Tfh cells lose expression of characteristic Tfh genes and revert to an effector phenotype, resulting in disruption of the germinal center response., The co-stimulators ICOS (inducible T cell co-stimulator) and CD28 are both important for T follicular helper (TFH) cells, yet their individual contributions are unclear. Here, we show that each molecule plays an exclusive role at different stages of TFH cell development. While CD28 regulated early expression of the master transcription factor Bcl-6, ICOS co-stimulation was essential to maintain the phenotype by regulating the novel TFH transcription factor Klf2 via Foxo1. Klf2 directly binds to Cxcr5, Ccr7, Psgl-1, and S1pr1, and low levels of Klf2 were essential to maintain this typical TFH homing receptor pattern. Blocking ICOS resulted in relocation of fully developed TFH cells back to the T cell zone and reversion of their phenotype to non-TFH effector cells, which ultimately resulted in breakdown of the germinal center response. Our study describes for the first time the exclusive role of ICOS and its downstream signaling in the maintenance of TFH cells by controlling their anatomical localization in the B cell follicle.

Systemic sclerosis (SSc), an autoimmune disorder, is characterized by vasculopathy, inflammation, progressive perivascular and interstitial fibrosis. Its pathogenesis is largely unknown, however strong evidences suggest that genetic predisposition may contribute to SSc development. Several gene polymorphisms involved in regulatory T cell function have been identified in many autoimmune diseases, including SSc. Moreover, dysregulation of co-stimulatory and/or co-inhibitory signals, including ICOS signalling, can lead to autoimmunity. The aim of the present study was to investigate the association of the FOXP3 rs2294020, ICOS rs6726035 and ICOSL rs378299 SNPs with both the susceptibility and the progression to SSc in an Italian case-series of patients. SNP genotyping results were successfully obtained from a total of 350 subjects including 166 individuals with SSc and 184 healthy controls. Although analysis tests did not show any significant associations between the SNPs under study and susceptibility to SSc, the occurrence of FOXP3 rs2294020 in female patients was associated with decreased time to progression from early to definite SSc (allelic model: HR = 1.43; CI = 1.03-1.99; p = 0.03; dominant model: HR = 1.54; CI = 1.04-2.28; p = 0.03). The inclusion of presence of ACA autoantibodies in the model did not significantly change the estimates. No conclusions can be drawn for the susceptibility to the disease or the time to progression in men due to the low statistical power. This study provides evidence of the association of rs2294020 with SSc evolution in female patients, modulating the time of progression from the diagnosis of early SSc to the diagnosis of definite SSc, while no effect on SSc susceptibility per se was found. rs2294020 may be considered a disease-modifying gene-variant rather than a disease-susceptibility SNP in SSc.

Background MicroRNAs (miRs) are involved in tumor progression by regulating tumor cells and tumor microenvironment. MiR21 is overexpressed in diffuse large B-cell lymphoma (DLBCL) and its biological impact on tumor microenvironment remains unclear. Methods MiR21 was assessed by quantitative RT-PCR in patients with newly diagnosed DLBCL. The mechanism of action of miR21 on lymphoma progression and tumor angiogenesis was examined in vitro in B-lymphoma cell lines and in vivo in a murine xenograft model. Results Serum miR21 was significantly elevated in patients and associated with advanced disease stage, International Prognostic Index indicating intermediate-high and high-risk, and increased tumor angiogenesis. When co-cultured with immune cells and endothelial cells, miR21-overexpressing B-lymphoma cells were resistant to chemotherapeutic agents, but sensitive to Bcl-2 inhibitor ABT-199, irrespective of Bcl-2 expression on lymphoma cells. In both co-culture systems of Bcl-2positive and Bcl-2negative B-lymphoma cells, miR21 induced inducible co-stimulator (ICOS) expression on regulatory T (Treg) cells. Through crosstalking with Treg cells by ICOS ligand (ICOSL), endothelial cells were activated, resulting in stimulation of Bcl-2 expression and vessel formation. ABT-199 directly targeted Bcl-2 on endothelial cells, induced endothelial cell apoptosis and inhibited tumor angiogenesis. In a murine xenograft model established with subcutaneous injection of B-lymphoma cells, ABT-199 particularly retarded the growth of miR21-overexpressing tumors, consistent with the induction of endothelial cell apoptosis and inhibition of tumor angiogenesis. Conclusions As a serum oncogenic biomarker of B-cell lymphoma, miR21 indicated B-lymphoma cell sensitivity to ABT-199 via ICOS/ICOSL-mediated interaction of Treg cells with endothelial cells. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0551-z) contains supplementary material, which is available to authorized users.

Mesenchymal stem cells (MSCs) can modulate lymphocyte proliferation and function. One of the immunomodulatory functions of MSCs involves CD4+CD25+FoxP3+ regulatory T cells (Tregs), which negatively regulate inflammatory responses. MSC-mediated Treg induction is supposed to be regulated by mechanisms requiring both soluble and cell contact-dependent factors. Although the involvement of soluble factors has been revealed, the contact-dependent mechanisms in MSC-mediated Treg induction remain unclear. We attempted to identify molecule(s) other than secreted factors that are responsible for MSC-mediated Treg induction and to uncover the underlying mechanisms. Under in vitro Treg-inducing conditions, ICOSL expression in MSCs coincided with Treg induction in co-cultures of MSCs with CD4+ T cells. When cultured in a transwell plate, MSCs failed to induce Tregs. Neutralization or knockdown of ICOSL significantly reduced Tregs and their IL-10 release. ICOSL overexpression in MSCs promoted induction of functional Tregs. ICOSL-ICOS signaling promoted Treg differentiation from CD4+ T cells through activation of the phosphoinositide 3-kinase-Akt pathway. MSCs primed with Interleukin-1β significantly induced Tregs through ICOSL upregulation. We demonstrated that the Treg-inducing activity of MSCs is proportionate to their basal ICOSL expression. This study provides evidence that ICOSL expression in human MSCs plays an important role in contact-dependent regulation of MSC-mediated Treg induction.

Group 2 innate lymphoid cells (ILC2s) are a recently identified group of cells with the potent capability to produce Th2-type cytokines such as interleukin (IL)-5 and IL-13. Several studies suggest that ILC2s play an important role in the development of allergic diseases and asthma. Activation of pulmonary ILC2s in murine models lacking T and B cells induces eosinophilia and airway hyper-reactivity (AHR), which are cardinal features of asthma. More importantly, numerous recent studies have highlighted the role of ILC2s in asthma persistence and exacerbation among human subjects, and thus, regulation of pulmonary ILC2s is a major area of investigation aimed at curbing allergic lung inflammation and exacerbation. Emerging evidence reveals that a group of regulatory T cells, induced Tregs (iTregs), effectively suppress the production of ILC2-driven, pro-inflammatory cytokines IL-5 and IL-13. The inhibitory effects of iTregs are blocked by preventing direct cellular contact or by inhibiting the ICOS-ICOS-ligand (ICOSL) pathway, suggesting that both direct contact and ICOS-ICOSL interaction are important in the regulation of ILC2 function. Also, cytokines such as IL-10 and TGF-β1 significantly reduce cytokine secretion by ILC2s. Altogether, these new findings uncover iTregs as potent regulators of ILC2 activation and implicate their utility as a therapeutic approach for the treatment of ILC2-mediated allergic asthma and respiratory disease.

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

SummaryInflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune homeostasis and cytokine secretion. Multiple loci are associated with IBD, but a functional explanation is missing for most. Here we found that pattern-recognition receptor (PRR)-induced cytokine secretion was diminished in human monocyte-derived dendritic cells (MDDC) from rs7282490 ICOSLG GG risk carriers. Homotypic interactions between the costimulatory molecule ICOS and the ICOS ligand on MDDCs amplified nucleotide-binding oligomerization domain 2 (NOD2)-initiated cytokine secretion. This amplification required arginine residues in the ICOSL cytoplasmic tail that recruited the adaptor protein RACK1 and the kinases PKC and JNK leading to PKC, MAPK, and NF-κB activation. MDDC from rs7282490 GG risk-carriers had reduced ICOSL expression and PRR-initiated signaling and this loss-of-function ICOSLG risk allele associated with an ileal Crohn’s disease phenotype, similar to polymorphisms in NOD2. Taken together, ICOSL amplifies PRR-initiated outcomes, which might contribute to immune homeostasis.

Purpose The interaction between the inducible costimulatory molecule (ICOS) and ICOS ligand (ICOSL) has been implicated in the differentiation and functions of T cells. The purpose of the present study was to determine the role of ICOS-ICOSL in the immune privilege of corneal allografts. Methods Expression of ICOS and ICOSL mRNA from mouse eyes was assessed by RT-PCR. Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of ICOS-/- BALB/c recipients and BALB/c wild-type (WT) recipients treated with anti-ICOSL mAb, and graft survival was assessed. A separate set of WT and ICOS-/- BALB/c mice received an anterior chamber injection of C57BL/6 splenocytes, and induction of allospecific anterior chamber-associated immune deviation (ACAID) was assessed. In vitro, cornea was incubated with T cells from WT and ICOS-/- BALB/c mice, and destruction of corneal endothelial cells (CECs) and the population of Foxp3+ CD25+ CD4+ T cells was assessed. Results Inducible costimulatory molecule ligand mRNA was constitutively expressed in the cornea, iris-ciliary body, and retina. Allograft survival in ICOS-/- recipients and WT recipients treated with anti-ICOSL mAb was significantly shorter than in control recipients. Anterior chamber-associated immune deviation was induced less efficiently in ICOS-/- mice. Destruction of CECs by alloreactive ICOS-/- T cells was enhanced compared with WT T cells. After coincubation with allogeneic corneal tissue, the proportion of regulatory T cells was significantly greater among WT T cells than in ICOS-/- T cells. Conclusions The expression of ICOSL in the cornea and the ICOS-mediated induction of Foxp3+ CD4+ regulatory T cells may contribute to successful corneal allograft survival.

SummaryT follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

Osteoblasts, osteocytes, and osteoclasts (OCs) are involved in the bone production and resorption, which are crucial in bone homeostasis. OC hyperactivation plays a role in the exaggerated bone resorption of diseases such as osteoporosis, rheumatoid arthritis, and osteolytic tumor metastases. This work stems from the finding that OCs can express B7h (ICOS-Ligand), which is the ligand of the ICOS T cell costimulatory molecule. Because recent reports have shown that, in endothelial, dendritic, and tumor cells, B7h triggering modulates several activities of these cells, we analyzed the effect of B7h triggering by recombinant ICOS-Fc on OC differentiation and function. The results showed that ICOS-Fc inhibits RANKL-mediated differentiation of human monocyte-derived OC-like cells (MDOCs) by inhibiting the acquirement of the OC morphology, the CD14− cathepsin K+ phenotype, and the expression of tartrate-resistant acid phosphatase, OSCAR, NFATc1, and DC-STAMP. Moreover, ICOS-Fc induces a reversible decrease in the sizes of cells and nuclei and cathepsin K expression in mature MDOCs. Finally, ICOS-Fc inhibits the osteolytic activities of MDOCs in vitro and the development of bone loss in ovariectomized or soluble RANKL-treated mice. These findings open a novel field in the pharmacological use of agonists and antagonists of the ICOS–B7h system.

Objective. Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptorligand pair. Here we investigated the expression of ICOS and ICOSL in patients with SSc. Methods. ICOS expression on peripheral blood T cells, and ICOSL expression on B cells and macrophages was determined by flow cytometry. Expression of ICOS and ICOSL was assessed by immunohistological staining and real-time PCR of lesional skin. Results. ICOS expression levels were specifically increased on both peripheral blood memory T cells and Tregs from early dcSSc patients compared with those from healthy controls. Mean ICOSL expression on B cells or macrophages was comparable between SSc patients and healthy controls. ICOS-expressing T cells, ICOSL-expressing macrophages and mRNA levels of ICOS and ICOSL were increased in the lesional skin of patients with early dcSSc. In vitro ICOS costimulation enhanced production of IFN-g, IL-4 and IL-17A from T cells in SSc patients vs normal controls. Soluble ICOS levels were significantly increased in SSc patients and were negatively associated with the presence of ACAs and positively associated with CRP values. Serum levels of soluble ICOS were more closely associated with clinical features compared with levels of soluble IL-2 receptor. Conclusion. Augmented ICOS signalling may contribute to the pathogenesis of SSc during early progressive disease. Soluble ICOS levels may be used as a serum marker for the activity and severity of SSc.

Late activator antigen-presenting cells promote Tfh differentiation of antigen-primed CD4+ T cells and antibody responses in influenza A virus infection., The humoral immune response to most respiratory virus infections plays a prominent role in virus clearance and is essential for resistance to reinfection. T follicular helper (Tfh) cells are believed to support the development both of a potent primary antibody response and of the germinal center response critical for memory B cell development. Using a model of primary murine influenza A virus (IAV) infection, we demonstrate that a novel late activator antigen-presenting cell (LAPC) promotes the Tfh response in the draining lymph nodes (dLNs) of the IAV-infected lungs. LAPCs migrate from the infected lungs to the dLN “late,” i.e., 6 d after infection, which is concomitant with Tfh differentiation. LAPC migration is CXCR3-dependent, and LAPC triggering of Tfh cell development requires ICOS–ICOSL–dependent signaling. LAPCs appear to play a pivotal role in driving Tfh differentiation of Ag-primed CD4+ T cells and antiviral antibody responses.

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell–mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.

Objective Inducible costimulator (ICOS)/B7h costimulation plays a crucial role in acute and chronic allograft rejection. To test the role of the ICOS signal in T-cell activation and expansion, we used ICOS-Fc–targeted B cells as donor antigen presenting cells to challenge the allogeneic response in vitro. Methods In vitro, the binding of ICOS-Fc with B7h on splenic B cells was confirmed by flow cytometry analysis. To evaluate the capacity of ICOS-Fc–targeted B cells to elicit an allogeneic response in vitro, we performed mixed lymphocyte reactions. Results The binding of B7h on splenic B cells by ICOS-Fc was confirmed at a saturating concentration of 100 μg/mL. Blockade of ICOS/B7h in direct allorecognition depressed proliferation of alloreactive T cells in vitro. Conclusions ICOS/B7h signal plays an important role in direct allorecognition, eliciting allogeneic responses in vitro.

Skin wound healing is mediated by inflammatory cell infiltration of the wound site. Inducible costimulator (ICOS), expressed on activated T cells, and its ligand, ICOS ligand (ICOSL), expressed on antigen-presenting cells, have been considered a single receptor–ligand pair. Although the ICOS-ICOSL pathway participates in adaptive immunity, its roles in skin wound healing, which is mediated by innate immune responses, remain unknown. To clarify these roles, repair of excisional wounds was examined in ICOS −/− mice, ICOSL −/− mice, and ICOS −/− ICOSL −/− mice. Each mutant strain showed similar, dramatic delays in wound healing, especially at early times. Knockout mice showed suppressed keratinocyte migration, angiogenesis, and granulation tissue formation, and diminished T-cell, macrophage, and neutrophil infiltration. The loss of ICOS and/or ICOSL resulted in marked suppression of cytokine expression in wounds, especially the Th2 cytokines interleukin (IL)-4, IL-6, and IL-10. T-cell transfer experiments and T-cell depletion therapy further clarified the important roles of ICOS expressed on T cells and its interaction with ICOSL. Application of IL-6, but not IL-4, to the wounds significantly increased the onset of early wound healing in mutant mice. Thus, our results indicate that ICOS-ICOSL costimulatory signaling has critical roles during wound healing, most likely by inducing IL-6 production.

OBJECTIVE: To evaluate the immunomodulation of inducible co-stimulator (ICOS) in cytokine-induced killer (CIK) cells against cholangiocarcinoma. METHODS: CIK cells were generated from normal peripheral blood mononuclear cells. Methyl thiazolyl tetrazolium assay was performed to assess proliferation of CIK-ICOS and controlled CIK cells; ELISA was used to analyze the expression of cytokines. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of ICOS ligand (ICOSL) in CIK cells and human cholangiocarcinoma cell line QBC939 cells. The cytotoxicity of CIK cells was determined either by lactate dehydrogenase-releasing assay in vivo or alteration of tumor size prior to and after the treatment of CIK cells in vivo. RESULTS: CIK-ICOS cells proliferated more and expressed higher secretion a level of interferon-γ than the controlled CIK. These cells exhibited higher cytotoxicity against cholangiocarcinoma cell lines at all efficacy: toxicity (E:T) ratios tested than the controlled CIK cells. More importantly, the anti-ICOSL antibody was able to attenuate the elevated cytotoxicity mediated by ICOS overexpression. When injected into cholangiocarcinoma xenografts in severe combined immunodeficiency mice, CIK-ICOS cells survived better than the controlled CIK cells around xenografts and significantly reduced the growth rate of cholangiocarcinoma, with least volume increase and more severe necrosis of the xenografts than controlled mice treated with saline, CIK or CIK-enhanced green fluorescent protein. CONCLUSION: ICOS can enhance the cytotoxic effect of CIK cells against cholangiocarcinoma both in vitro and in vivo. This effect is mediated by ICOS-augmented cytokine secretion and cell proliferation, and in part through ICOS-ICOSL interaction.

Summary CD28 and CTLA-4 are cell surface cosignaling molecules essential for the control of T cell activation upon the engagement of their ligands B7-1 and B7-2 from antigen-presenting cells. By employing a receptor array assay, we have demonstrated that B7-H2, best known as the ligand of inducible costimulator, was a ligand for CD28 and CTLA-4 in human, whereas these interactions were not conserved in mouse. B7-H2 and B7-1 or B7-2 interacted with CD28 through distinctive domains. B7-H2-CD28 interaction was essential for the costimulation of human T cells' primary responses to allogeneic antigens and memory recall responses. Similar to B7-1 and B7-2, B7-H2 costimulation via CD28 induced survival factor Bcl-xL, downregulated cell cycle inhibitor p27 kip1 , and triggered signaling cascade of ERK and AKT kinase-dependent pathways. Our findings warrant re-evaluation of CD28 and CTLA-4's functions previously attributed exclusively to B7-1 and B7-2 and have important implications in therapeutic interventions against human diseases.

NOD mice spontaneously develop insulin-dependent diabetes around 10-40 wk of age. Numerous immune gene variants contribute to the autoimmune process. However, genes that direct the autoimmune response toward β cells remain ill defined. In this study, we provide evidence that the Icos and Icosl genes contribute to the diabetes process. Protection from diabetes in ICOS(-/-) and ICOSL(-/-) NOD mice was unexpectedly associated with the development of an autoimmune disorder of the neuro-muscular system, characterized by myositis, sensory ganglionitis and, to a reduced extent, inflammatory infiltrates in the CNS. This syndrome was reproduced upon adoptive transfer of CD4(+) and CD8(+) T cells from diseased donors to naïve NOD.scid recipients. Our data further show that protection from diabetes results from defective activation of autoimmune diabetogenic effector T cells in ICOS(-/-) NOD mice, whereas acceleration of diabetes in BDC2.5 ICOS(-/-) NOD mice is induced by a dominant defect in Treg. Taken together, our findings indicate that costimulation signals play a key role in regulating immune tolerance in peripheral tissues and that the ICOS/ICOSL costimulatory pathway influences the balance between Treg and diabetogenic effector T cells.

Natural killer T (NKT) cells modulate immune responses against pathogens and tumours, as well as immunological tolerance. We show here that CYLD, a tumour suppressor with deubiquitinase function, has a pivotal and cell-intrinsic function in NKT cell development. Unlike other known NKT regulators, CYLD is dispensable for intrathymic NKT cell maturation but is obligatory for the survival of immature NKT cells. Interestingly, CYLD deficiency impairs the expression of ICOS, a costimulatory molecule required for the survival and homeostasis of NKT cells, and this molecular defect is associated with attenuated response to an NKT-survival cytokine, IL-7, due to reduced expression of IL-7 receptor. We show, for the first time, that IL-7 induces the expression of ICOS in NKT cells, which is largely dependent on CYLD. Interestingly, loss of CYLD causes constitutive NF-kappaB activation in developing NKT cells, which contributes to their defective IL-7 response and attenuated ICOS expression. These findings establish CYLD as a critical regulator of NKT cell development and provide molecular insights into this novel function of CYLD.

Inducible co-stimulator (ICOS) interaction with its ligand (ICOSL) is involved in several T cell effector functions. While blockade of ICOS:ICOSL interaction in chronic graft versus host disease (GVHD) seems beneficial, results for acute GVHD remain controversial. To further elucidate its role in acute GVHD, C57BL/6 mice were reconstituted with allogeneic spleen cells in the absence or presence of ICOSL-blocking mAb. Mice reconstituted with allogeneic spleen cells experienced severe GVHD and died untreated within 6-9 days after transplantation. Mice treated with an anti-ICOSL mAb starting from day 3 after transplantation gained weight again and survived for at least additional 12 days, although the treatment was already stopped at day 11 after transplantation. In contrast, the anti-ICOSL treatment starting from day 0 did not prevent GVHD. The difference between therapeutic (day 3) and prophylactic (day 0) anti-ICOSL treatment was independent of CD25+CD4+ regulatory T cells since their depletion did not abrogate the therapeutic effect of ICOSL blockade. Microarray analysis revealed IFN-gamma and chemokine up-regulation in spleen cells of prophylactically treated mice, emphasizing kinetic dependence of acute GVHD modulation via blockade of ICOS:ICOSL interaction.

The CD28/B7 and ICOS/B7-H2 pathways are both critical for costimulating T cell immune responses. Deficiency in either pathway results in defective T cell activation, cytokine production and germinal center formation. However, the relative importance and contribution of each pathway towards T cell-mediated immunity is still not clear. To address this issue, we compared T cell responses of WT, CD28 knockout (KO), B7-H2 KO, and CD28-B7-H2 double KO (DKO) mice in a model of delayed-type hypersensitivity (DTH). While CD28 KO mice have partially compromised DTH response, DKO mice exhibited greatly diminished response, suggesting that the ICOS/B7-H2 pathway could partially compensate for the costimulation of DTH. Surprisingly, B7-H2 KO mice had comparable DTH response as WT mice, indicating that the ICOS/B7-H2 pathway is secondary to the CD28/B7 pathway in costimulating DTH. Interestingly, prolonging the period of sensitization could overcome the compromised DTH response in CD28 KO but not DKO mice, revealing a novel form of functional redundancy of ICOS/B7-H2 costimulation that is dependent on time to take effect. Taken together, our data reveal a functional hierarchy of the CD28/B7 and ICOS/B7-H2 pathways and delineated their relative contributions to the elicitation of a DTH response.

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.