MiR-155-targeted IcosL controls tumor rejection.

Elevated levels of miR-155 in solid and liquid malignancies correlate with aggressiveness of the disease. In this manuscript, we show that miR-155 targets transcripts encoding IcosL, the ligand for Inducible T-cell costimulator (Icos), thus impairing the ability of T cells to recognize and eliminate malignant cells. We specifically found that overexpression of miR-155 in B cells of Eµ-miR-155 mice causes loss of IcosL expression as they progress toward malignancy. Similarly, in mice where miR-155 expression is controlled by a Cre-Tet-OFF system, miR-155 induction led to malignant infiltrates lacking IcosL expression. Conversely, turning miR-155 OFF led to tumor regression and emergence of infiltrates composed of IcosL-positive B cells and Icos-positive T cells forming immunological synapses. Therefore, we next engineered malignant cells to express IcosL, in order to determine whether IcosL expression would increase tumor infiltration by cytotoxic T cells and reduce tumor progression. Indeed, overexpressing an IcosL-encoding cDNA in MC38 murine colon cancer cells before injection into syngeneic C57BL6 mice reduced tumor size and increased intratumor CD8+ T cell infiltration, that formed synapses with IcosL-expressing MC38 cells. Our results underscore the fact that by targeting IcosL transcripts, miR-155 impairs the infiltration of tumors by cytotoxic T cells, as well as the importance of IcosL on enhancing the immune response against malignant cells. These findings should lead to the development of more effective anticancer treatments based on maintaining, increasing, or restoring IcosL expression by malignant cells, along with impairing miR-155 activity.

The impact of ICOSL/ICOS pathway-regulated long non-coding RNAs on liver fibrosis in mice infected with Schistosoma japonicum.

BACKGROUND: The primary pathogenic mechanism of schistosomiasis-associated liver fibrosis involves the deposition of schistosome eggs, leading to the formation of liver egg granulomas and subsequent liver fibrosis. Hepatic stellate cells are abnormally activated, resulting in excessive collagen deposition and fibrosis development. While specific long non-coding RNAs (lncRNAs) have been associated with fibrotic processes, their roles in schistosomiasis-associated liver fibrosis remain unclear. METHODS: Our previous research indicated that downregulating the ICOSL/ICOS could partially alleviate liver fibrosis. In this study, we established a schistosomiasis infection model in C57BL/6 and ICOSL knockout (KO) mice, and the liver pathology changes were observed at various weeks postinfection (wpi) using hematoxylin and eosin and Masson's trichrome staining. Within the first 4 wpi, no significant liver abnormalities were observed. However, mice exhibited evident egg granulomas and fibrosis in their livers at 7 wpi. Notably, ICOSL-KO mice had significantly smaller pathological variations compared with simultaneously infected C57BL/6 mice. To investigate the impact of lncRNAs on schistosomiasis-associated liver fibrosis, quantitative real-time polymerase chain reaction (RT-qPCR) was used to monitor the dynamic changes of lncRNAs in hepatic stellate cells of infected mice. RESULTS: The results demonstrated that lncRNA-H19, -MALAT1, -PVT1, -P21 and -GAS5 all participated in liver fibrosis formation after schistosome infection. In addition, ICOSL-KO mice exhibited significantly inhibited expression of lncRNA-H19, -MALAT1 and -PVT1 after 7 wpi. In contrast, they showed enhanced expression of lncRNA-P21 and -GAS5 compared with C57BL/6 mice, influencing liver fibrosis development. Furthermore, small interfering RNA transfection (siRNA) in JS-1 cells in vitro confirmed that lncRNA-H19, -MALAT1, and -PVT1 promoted liver fibrosis, whereas lncRNA-P21 and -GAS5 had the opposite effect on key fibrotic molecules, including α- smooth muscle actin and collagen I expression. CONCLUSIONS: This study uncovers that ICOSL/ICOS may play a role in activating hepatic stellate cells and promoting liver fibrosis in mice infected with Schistosoma japonicum by dynamically regulating the expression of specific lncRNAs. These findings offer potential therapeutic targets for schistosomiasis-associated liver fibrosis.

ICOSLG acts as an oncogene to promote glycolysis, proliferation, migration, and invasion in gastric cancer cells.

Gastric cancer (GC) has emerged as one of the most common malignancies in gastrointestinal system. Inducible T-cell costimulator ligand (ICOSLG) was found to be highly expressed in various cancers, which contributes to disease progression. This study aims to investigate the role of ICOSLG and its potential mechanism of action in dictating the aggressiveness of GC cell. ICOSLG and miR-331-3p expression patterns in cancerous and para-cancerous tissues from GC patients were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The miRNAs targeting ICOSLG were predicted by "miRDB", "starBase," and "TargetScan" databases. The interplay of ICOSLG and miR-331-3p in dictating the aggressiveness and glycolysis of GC cells was investigated by CCK-8 proliferation assay and Transwell migration/invasion assays, as well as the detection of glucose uptake, lactate production and ATP levels. The tumorigenesis of GC cells after ICOSLG silencing was examined in the nude mice. ICOSLG was highly expressed in GC tissues, and GC patients with high ICOSLG expression showed a poorer prognosis than the low-expression group. Further, high ICOSLG level was correlated with more advanced TNM stages, more lymph-node metastases, and poorer tumor differentiation. ICOSLG knockdown inhibited the proliferation, migration, invasion and tumor formation of GC cells, which was concomitant with reduced glucose consumption, lactate production, and ATP levels. In contrast, ICOSLG overexpression enhanced the aggressiveness of GC cells, and this effect was abrogated after the treatment with glycolysis inhibitor. We further found that miR-331-3p was a negative regulator of ICOSLG4, and miR-331-3p overexpression reduced ICOSLG4 expression and suppressed the aggressive phenotype induced by ICOSLG4 in GC cells. Together, these findings indicate that ICOSLG4, as an oncogene, is upregulated to promote glycolysis and the malignant phenotype in GC cells. miR-331-3p, which is downregulated in GC tissues, functions as a negative regulator of ICOSLG4. Targeting miR-331-3p/ICOSLG4 axis could potentially suppress GC progression.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

Expression and prognosis of inducible T-cell co-stimulator and its ligand in Chinese stage I-III lung adenocarcinoma patients.

BACKGROUND: Immunotherapy has become the fastest-adopting treatment paradigm for lung cancer with improved survival. By binding with its ligand (inducible T-cell co-stimulator and its ligand [ICOSL]), an inducible T-cell co-stimulator (ICOS) could contribute to reversing immunosuppression and improving immune response and thus be a potential target for cancer immunotherapy. METHODS: We selected 54 formalin-fixed, paraffin-embedded tumor tissues from cases with stage I-III lung adenocarcinoma cancer. Immunohistochemical expression of ICOS and ICOSL was evaluated. The correlation with clinical parameters in Chinese patients was also compared with TCGA results. RESULTS: The positive rates of ICOS and ICOSL were 68% and 81.5%, respectively, in lung tumor tissues. Of these, 9 cases had a low expression of ICOS, and 22 cases had a high expression of ICOS; ICOSL expression was low in 20 cases and high in 24 cases. According to the International Association for the Study of Lung Cancer (8th edition), phase I lesions were detected in 21 cases, phase II lesions in 15 cases, and phase III lesions in 18 cases. The median survival time of all patients was 44.5 months, and the median disease-free survival was 32 months. Univariate analysis showed that the factors significantly associated with overall survival were tumor size, regional lymph node involvement, stage, and expression level of ICOS/ICOSL. Survival analysis using log-rank test indicated that the lower ICOS+ cell infiltration may predict poor prognosis, whereas lower ICOSL protein expression may be associated with better prognosis, but ICOSL data need further validation in larger samples due to inconsistency in TCGA mRNA prediction. CONCLUSION: ICOS/ICOSL might be associated with prognosis of lung cancer, and ICOS and its ligand may be potential therapeutic targets in non-small cell lung cancer.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Pharmacokinetics and Pharmacokinetic/Pharmacodynamic Properties of Rozibafusp Alfa, a Bispecific Inhibitor of BAFF and ICOSL: Analyses of Phase I Clinical Trials.

Rozibafusp alfa (AMG 570) is a first-in-class bispecific IgG2-peptide fusion designed to inhibit inducible T-cell costimulator ligand (ICOSL) and B-cell activating factor (BAFF). The pharmacokinetics (PK) and pharmacodynamics (PD) of rozibafusp alfa were investigated in two randomized, placebo-controlled clinical studies: a phase Ia single ascending-dose study (7-700 mg subcutaneously (s.c.)) in healthy subjects and a phase Ib multiple ascending-dose study (70-420 mg s.c. every 2 weeks (q2w)) in patients with rheumatoid arthritis. Rozibafusp alfa exhibited nonlinear PK and dose-related and reversible dual-target engagement. Maximal reduction of naïve B cells from baseline (> 40%), reflective of BAFF inhibition, was achieved with rozibafusp alfa exposure (area under the concentration-time curve from time 0 to time infinity (AUCinf ) and AUC within a dosing interval from day 0 to day 14 (AUCtau )) above 51 and 57 days•µg/mL for the single-dose (≥ 70 mg) and multiple-dose studies (≥ 70 mg q2w), respectively. ICOSL receptor occupancy on circulating B cells, a surrogate PD end point for ICOSL inhibition, was directly related to drug concentration. PK/PD analysis showed > 90% RO at rozibafusp alfa ≥ 22.2 µg/mL (≥ 420-mg single dose or ≥ 210 mg q2w multiple dose), with saturation occurring at higher drug concentrations. These results informed the design and dose selection of a phase IIb study assessing the safety and efficacy of rozibafusp alfa in patients with active systemic lupus erythematosus.

ICOS-Fc as innovative immunomodulatory approach to counteract inflammation and organ injury in sepsis.

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-1ß, 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.

Inducible T-cell co-stimulator (ICOS) and ICOS ligand are novel players in the multiple-myeloma microenvironment.

The inducible T-cell co-stimulator (ICOS) is a T-cell receptor that, once bound to ICOS ligand (ICOSL) expressed on several cell types including the B-cell lineage, plays a decisive role in adaptive immunity by regulating the interplay between B and T cells. In addition to its immunomodulatory functions, we have shown that ICOS/ICOSL signalling can inhibit the activity of osteoclasts, unveiling a novel mechanism of lymphocyte-bone cells interactions. ICOS and ICOSL can also be found as soluble forms, namely sICOS and sICOSL. Here we show that: (i) levels of sICOS and sICOSL are increased in multiple myeloma (MM) compared to monoclonal gammopathy of undetermined significance and smouldering MM; (ii) levels of sICOS and sICOSL variably correlate with several markers of tumour burden; and (iii) sICOS levels tend to be higher in Durie-Salmon stage II/III versus stage I MM and correlate with overall survival as an independent variable. Moreover, surface ICOS and ICOSL are expressed in both myeloma cells and normal plasma cells, where they probably regulate different functional stages. Finally, ICOSL triggering inhibits the migration of myeloma cell lines in vitro and the growth of ICOSL+ MOPC-21 myeloma cells in vivo. These results suggest that ICOS and ICOSL represent novel markers and therapeutic targets for MM.

Terapia com células CAR-T: reprogramação celular para o combate de neoplasias malignas / CAR-T cell therapy: cell reprogramming to combat malignant neoplasms

As células CAR-T são linfócitos geneticamente modificados para reconhecerem um espectro amplo de antígenos de superfície celulares. Além disso, atacam células tumorais malignas, que expressam esses antígenos, por meio da ativação da coestimulação citoplasmática, secreção de citocinas, citólise de células tumorais e proliferação de células T. O objetivo desse estudo é abordar a imunoterapia com células CAR-T, a fim de explicar seu conceito, processo de fabricação e papel no tratamento de neoplasias hematológicas e tumores sólidos. Foi realizada uma revisão através do portal PubMed, utilizando como descritores: "car-t cell therapy" e "neoplasms", determinados com base nos "Descritores em Ciências da Saúde". Foram obtidos, inicialmente, 10 artigos, os quais foram lidos integralmente para a confecção dessa revisão. Além disso, foram adicionados 3 ensaios clínicos atualizados sobre o tema. Na terapia com células CAR-T, as células T são coletadas do paciente, geneticamente modificadas para incluir receptores de antígeno específicos e, posteriormente, expandidas em laboratórios e transfundidas de volta para o paciente. Assim, esses receptores podem reconhecer células tumorais que expressam um antígeno associado a um tumor. A terapia com células CAR-T é mais conhecida por seu papel no tratamento de malignidades hematológicas de células B, sendo a proteína CD19 o alvo antigênico mais bem estudado até o momento. Entretanto, estudos estão sendo feitos para verificar a eficácia desse tratamento, também, em tumores sólidos. Portanto, apesar de inicialmente ser indicada apenas para um grupo seleto de pessoas, essa terapia tem demonstrado grande potencial para atuar em um espectro maior de pacientes.

The CAR-T cells are lymphocytes genetically modified to recognize a broader spectrum of cell surface antigens. In addition, they attack malignant tumor cells, which express these antigens, by activating cytoplasmic co-stimulation, cytokine secretion, tumor cell cytolysis and T cell proliferation. The aim of this study is to address immunotherapy with CAR-T cells, in order to explain its concept, manufacturing process and role in the treatment of hematological neoplasms and solid tumors. This is a literature review conducted through the PubMed portal, that uses the terms "car-t cell therapy" and "neoplasms" as descriptors, determined based on the DeCS (Descritores em Ciências da Saúde). To prepare this review, initially 10 articles were found and read in full. In addition, 3 updated clinical trials on the subject were added. For CAR-T cell therapy, T cells are collected from the patient, genetically modified to include specific antigen receptors, and later expanded in laboratories and transfused back to the patient. Thus, these receptors can recognize tumor cells that express a tumor-associated antigen. CAR-T cell therapy is best known for its role in the treatment of B cell hematological malignancies, with the CD19 protein being the most studied antigenic target to date. However, studies are being conducted to verify the effectiveness of this treatment, also, in solid tumors. Therefore, despite being formulated only for a selected group of patients, this therapy has great potential to act on a broader spectrum of patients.

Inducible T-Cell Costimulator Mediates Lymphocyte/Macrophage Interactions During Liver Repair.

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.

TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Mifepristone inhibited the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis.

BACKGROUND: The immune mechanism was shown to be involved in the development of adenomyosis. The aim of the current study was to evaluate the expression of the immune checkpoints B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. METHODS: The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis patient samples treated with or without mifepristone was determined by immunohistochemistry analysis. RESULTS: In adenomyosis patient samples, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phases. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phases. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria in both the proliferative and secretory phases. In the secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesions was significantly higher than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 was elevated in the proliferative phase compared with that in the secretory phase, while in the ectopic endometria, B7-H4 expression was decreased in the proliferative phase compared with the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis tissues after treatment with mifepristone. CONCLUSIONS: The expression of the immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is upregulated in adenomyosis tissues and is downregulated with mifepristone treatment. The data suggest that B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis.

AIRE-overexpressing BMDCs suppress TFH cells through ICOSL to prevent and attenuate autoimmune diabetes in NOD mice.

The strong genetic association between autoimmune regulator (AIRE) and autoimmune diseases indicates its critical role in immune tolerance. AIRE deficiency is thought to promote the development of follicular helper T (TFH) cells, which are considered to be essential in B cell proliferation. Excessive TFH cell generation is a key step towards the development of autoimmune diseases, including type 1 diabetes. However, the potential mechanism by which AIRE contributes to the generation and function of the TFH cell population has remained elusive. We show that AIRE reduced TFH cell generation by inhibiting the expression of inducible costimulatory ligand (ICOSL), interleukin (IL)-6 and IL-27 in dendritic cells (DCs). To understand the precise impact of AIRE-overexpressing bone marrow-derived DCs (AIRE-BMDCs) on type 1 diabetes progression and the associated molecular mechanisms, we transferred AIRE-BMDCs to recipient NOD mice and found that transplantation of AIRE-BMDCs can prevent or delay the onset of diabetes, attenuate diabetes after the establishment of overt hyperglycaemia, and lead to the inhibition of autoreactive pathological TFH cells and germinal centre (GC) B cells. To further determine the potential mechanism underlying this TFH cell depletion, BMDCs were cotransferred with recombinant mouse ICOSL (ICOSLG protein). We demonstrated that NOD mice were more susceptible to diabetes when they received AIRE-BMDCs and ICOSLG than when they received only mock-vehicle BMDCs (GFP-BMDCs). In addition, we did not observe the reversal of diabetes in any mice subjected to this cotransfer system. A single cycle of ICOSLG treatment temporarily promoted TFH cell proliferation and GC development. Our results reveal a mechanistic role of AIRE-BMDCs in the initiation of TFH cell differentiation, and the AIRE-mediated decrease in ICOSL expression in BMDCs plays a critical role. The effect of decreased ICOSL expression in type 1 diabetes will guide the design and evaluation of parallel studies in patients.

Activated neutrophils polarize protumorigenic interleukin-17A-producing T helper subsets through TNF-α-B7-H2-dependent pathway in human gastric cancer.

RATIONALE: Neutrophils constitute massive cellular constituents in inflammatory human gastric cancer (GC) tissues, but their roles in pathogenesis of inflammatory T helper (Th) subsets are still unknown. METHODS: Flow cytometry analysis and immunohistochemistry were used to analyze the responses and phenotypes of neutrophils in different samples from 51 patients with GC. Kaplan-Meier plots and Multivariate analysis for the survival of patients were used by log-rank tests and Cox proportional hazards models. Neutrophils and CD4+ T cells were purified and cultured for ex vivo, in vitro and in vivo regulation and function assays. RESULTS: GC patients exhibited increased tumoral neutrophil infiltration with GC progression and poor patient prognosis. Intratumoral neutrophils accumulated in GC tumors via CXCL6/CXCL8-CXCR1-mediated chemotaxis, and expressed activated molecule CD54 and co-signaling molecule B7-H2. Neutrophils induced by tumors strongly expressed CD54 and B7-H2 in both dose- and time-dependent manners, and a close correlation was obtained between the expressions of CD54 and B7-H2 on intratumoral neutrophils. Tumor-derived tumor necrosis factor-α (TNF-α) promoted neutrophil activation and neutrophil B7-H2 expression through ERK-NF-κB pathway, and a significant correlation was found between the levels of TNF-α and CD54+ or B7-H2+ neutrophils in tumor tissues. Tumor-infiltrating and tumor-conditioned neutrophils effectively induced IL-17A-producing Th subset polarization through a B7-H2-dependent manner ex vivo and these polarized IL-17A-producing Th cells exerted protumorigenic roles by promoting GC tumor cell proliferation via inflammatory molecule IL-17A in vitro, which promoted the progression of human GC in vivo; these effects could be reversed when IL-17A is blocked. Moreover, increased B7-H2+ neutrophils and IL-17A in tumors were closely related to advanced GC progression and predicted poor patient survival. CONCLUSION: We illuminate novel underlying mechanisms that TNF-α-activated neutrophils link B7-H2 to protumorigenic IL-17A-producing Th subset polarization in human GC. Blocking this pathological TNF-α-B7-H2-IL-17A pathway may be useful therapeutic strategies for treating GC.

ICOSL in host defense at epithelial barriers: lessons from ICOSLG deficiency.

Autosomal recessive mutations in Inducible T Cell Costimulator Ligand (ICOSLG) result in a combined immunodeficiency syndrome of humans, saliently marked by recurrent respiratory tract infections and significant disease with DNA-based viruses at epithelial barriers, including human papillomavirus (HPV). These features are also seen in persons with loss of function of the complementary gene, ICOS. The infection phenotypes associated with these natural experiments disclose a critical role of the corresponding proteins, ICOSL and ICOS, in human immunity at mucocutaneous barriers. Here, we review the syndromes of ICOSL and ICOS deficiency and explore the mechanisms by which the ICOSL:ICOS axis mediates epithelial host defenses.

Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

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 TH 2 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.

Role of endocytosis and trans-endocytosis in ICOS costimulator-induced downmodulation of the ICOS Ligand.

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.

IL-3 is essential for ICOS-L stabilization on mast cells, and sustains the IL-33-induced RORγt+ Treg generation via enhanced IL-6 induction.

IL-33 is a member of the IL-1 family. By binding to its receptor ST2 (IL-33R) on mast cells, IL-33 induces the MyD88-dependent activation of the TAK1-IKK2 signalling module resulting in activation of the MAP kinases p38, JNK1/2 and ERK1/2, and of NFκB. Depending on the kinases activated in these pathways, the IL-33-induced signalling is essential for production of IL-6 or IL-2. This was shown to control the dichotomy between RORγt+ and Helios+ Tregs , respectively. SCF, the ligand of c-Kit (CD117), can enhance these effects. Here, we show that IL-3, another growth factor for mast cells, is essential for the expression of ICOS-L on BMMCs, and costimulation with IL-3 potentiated the IL-33-induced IL-6 production similar to SCF. In contrast to the enhanced IL-2 production by SCF-induced modulation of the IL-33 signalling, IL-3 blocked the production of IL-2. Consequently, IL-3 shifted the IL-33-induced Treg dichotomy towards RORγt+ Tregs at the expense of RORγt- Helios+ Tregs . However, ICOS-L expression was downregulated by IL-33. In line with that, ICOS-L did not play any important role in the Treg modulation by IL-3/IL-33-activated mast cells. These findings demonstrate that different from the mast cell growth factor SCF, IL-3 can alter the IL-33-induced and mast cell-dependent regulation of Treg subpopulations by modulating mast cell-derived cytokine profiles.

Structural characterization of the ICOS/ICOS-L immune complex reveals high molecular mimicry by therapeutic antibodies.

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.