Inducible Costimulator Ligand Research Articles

Association between Increased Inducible Costimulator/Inducible Costimulator Ligand Expression with Bone Destruction in Apical Periodontitis

IntroductionThe aim was to assess the association of inducible costimulator (ICOS) and ICOS ligand with bone destruction in apical periodontitis (AP). MethodsSpecimens from patients presenting with AP were obtained during apicoectomy and subjected to histopathologic analysis and molecular assessment of ICOS/ICOS ligand. In addition, the experimental AP was induced by exposing the pulp of first mandibular molars of rats. Histologic and radiographic examinations were performed to validate the periapical lesions. The immunolocalization and messenger RNA expression of ICOS/ICOS ligand were evaluated by immunofluorescence staining and quantitative real-time polymerase chain reaction. The osteoclastic activities in periapical lesions, including the lesion size and the expression of tartrate-resistant acid phosphatase and the receptor activator of nuclear factor kappa B ligand, were recorded and followed by correlation analysis with ICOS/ICOS ligand expression. ResultsIn excisional specimens from AP patients, a significantly increased expression of ICOS/ICOS ligand was found compared with the healthy control. In the experimental AP samples, the expression of ICOS/ICOS ligand, tartrate-resistant acid phosphatase, and receptor activator of nuclear factor kappa B ligand was significantly elevated in inflamed periapical tissues (AP group) when compared with the healthy control. The number of ICOS+/ICOS ligand+ cells was highly correlated with the periapical lesion size (r = 0.892, P < .01 and r = 0.930, P < .01, respectively). ConclusionsThe increased expression of ICOS/ICOS ligand in periapical lesions was associated with the inflammatory infiltration and alveolar bone destruction of AP.

Expression of ICOSL is associated with decreased survival in invasive breast cancer

BackgroundInducible co-stimulator (ICOS) is a CD28-related molecule exclusively expressed on activated T cells and plays a critical role in modulating the immune response in breast cancer. The blockage of ICOS pathway has been shown to inhibit the activity of Type 2 T helper cells, thus potentially protecting against cancer growth. The current study aims to investigate the correlation between inducible co-stimulator ligand (ICOSL) expression in tumor tissues and the prognoses of patients with invasive breast cancer.MethodsTumor samples from 562 Chinese patients with invasive breast carcinomas were collected between 2003 and 2010. The expression of ICOSL on breast tumor and adjacent non-cancerous tissue was determined via immunohistochemistry. The overall survival (OS) of patients with positive and negative ICOSL expression were described using Kaplan–Meier curves, respectively. Parametric correlation method was used to analyze the correlation between ICOSL expression and other clinicopathological parameters. ICOSL was selected as a dependent variable for multivariate analysis.ResultsPositive ICOSL expression was identified on the plasma membrane in both cytoplasm and the nucleus of breast cancer cells. Membrane-expressed ICOSL is determined as an independent prognostic factor for OS in breast cancer but without significantly correlating with other clinicopathologic parameters such as age, menopausal status, depth of invasion, lymph node metastasis status, histologic classification, etc.ConclusionOur study suggests that the up-regulated expression of ICOSL protein in breast tumor cells can be associated with poor prognoses in invasive breast carcinomas.

Synergy between T-dependent antibodies and IL-10 limits susceptibility to inflammatory bowel disease

Abstract Inflammatory bowel diseases (IBD) are chronic autoinflammatory conditions in which the immune system loses tolerance to gastrointestinal microbes. The role of humoral immunity in IBD is largely undefined. We examined the potential of T-dependent antibodies to prevent intestinal inflammation using mouse models of IBD. For this, we used mice without inducible T cell costimulator ligand (ICOSL). Icosl−/− mice produce significantly fewer T-dependent antibodies. When tested, we found Icosl−/− mice had similar IgA binding but significantly reduced IgG binding to mucosally-associated microbes. Additionally, Icosl−/− mice have more CD4 T cells producing IL-10, an immunosuppressive cytokine, in the colon. We hypothesized that T-dependent antibodies and IL-10 cooperatively promote intestinal homeostasis. To test this, we temporarily eliminated IL-10 producing cells from adult mice. Icosl−/−, but not wild type or IgA−/− mice, rapidly developed colitis under these conditions. This colitis could be prevented if mice were first depleted of Gram-positive or anaerobic bacterial flora. We further investigated the synergy between these T-dependent antibodies and IL-10 utilizing mice with a codeletion of CD4 T cell-derived IL-10 (Il10cKO) and ICOSL. This codeletion resulted in colitis before 28 days of age. However, fostering Icosl−/− Il10cKO mice on wild-type mothers delayed this early-onset inflammation. This suggests T-dependent antibodies conveyed through breastmilk are protective against early-onset colitis. Collectively, our results suggest that IL-10 and T-dependent antibodies toward mucosa-associated Gram-positive anaerobes continually cooperate to maintain intestinal homeostasis throughout the lifespan.

The costimulatory molecule ICOS promotes establishment of resident memory CD8+ T cells

Abstract Resident memory T cells (TRM) are critical for localized protective immunity in non-lymphoid tissues, but the signals involved in generating TRM are incompletely defined. Inducible T-cell costimulator (ICOS) is a costimulatory molecule expressed on activated T cells, and is highly expressed on TRM compared to recirculating memory CD8+ T cell subsets. While the role of ICOS has been well elaborated in T follicular helper cells, its function in CD8+ T cells is largely unexplored. Using both mixed bone-marrow chimeras and adoptive co-transfer of antigen-specific CD8+ T cells with acute infection models, we found ICOS deficient CD8+ T cells showed minimal defects in the initial effector expansion and the generation of recirculating memory population. However, consistent with its expression pattern, ICOS deficiency significantly compromised the establishment of CD8+ TRM cells in diverse non-lymphoid tissues. We further confirmed that the interaction between ICOS ligand (ICOSL) and ICOS is essential for CD8+ T cells tissue residency, and the defect of Icos−/− CD8+ T cells tissue residency can be detected even at early time points post infection, indicating that ICOS signaling affects initial retention of activated CD8+ T cells in non-lymphoid tissues. To gain a better understanding of the ICOS signaling, we used shRNA to knockdown KLF2, a transcription factor that we showed previously inhibits generation of CD8+ TRM and which is negatively regulated by ICOS stimulation in CD4+ Tfh. Our data support the model that ICOS-mediated down-regulation of KLF2 is important for the establishment of CD8+ TRM. Further investigations into the mechanistic basis by which ICOS signaling promotes CD8+ T cells tissue residency are ongoing.

Dysregulation of humoral immunity in Foxp3 conditional-knockout mice

Foxp3+ regulatory T cells (Tregs) are crucial for maintaining tolerance to self-antigens and preventing autoimmune diseases. Loss of Foxp3 expression leads to autoimmunity and disrupts humoral immune responses, including hyperproduction of immunoglobulin E (IgE). Elucidation of how Tregs control antibody production can lead to the development of new therapies for autoimmune and allergic diseases. However, premature death of Foxp3-deficient mice makes it difficult to analyze the roles of Tregs in humoral immunity of adult mice. In this study, we developed Foxp3 conditional-knockout mice (Foxp3floxR26CreERT2) in which the Foxp3 gene was inducibly deleted by tamoxifen administration. After oral administration of tamoxifen, titers of immunoglobulins, particularly IgG2c and IgE, were increased in Foxp3floxR26CreERT2 mice compared with that in controls. Under these conditions, CD4+ T cells from Foxp3floxR26CreERT2 mice had increased expression of several activation markers, including inducible costimulator and CD40 ligand, as well as the cytokines interleukin 4 and interferon gamma. In addition, the proportions of T follicular helper (Tfh) cells and germinal center (GC) B cells were increased in Foxp3floxR26CreERT2 mice compared with those in controls. These results indicated that Tregs controlled excessive or pathogenic antibody production by suppressing Tfh cell differentiation and GC formation. Furthermore, these data suggested that Foxp3floxR26CreERT2 mice could be a useful tool for screening therapeutic agents.

Nonimmune cell-derived ICOS ligand functions as a renoprotective αvβ3 integrin-selective antagonist.

Soluble urokinase receptor (suPAR) is a circulatory molecule that activates αvβ3 integrin on podocytes, causes foot process effacement, and contributes to proteinuric kidney disease. While active integrin can be targeted by antibodies and small molecules, endogenous inhibitors haven't been discovered yet. Here we report what we believe is a novel renoprotective role for the inducible costimulator ligand (ICOSL) in early kidney disease through its selective binding to podocyte αvβ3 integrin. Contrary to ICOSL's immune-regulatory role, ICOSL in nonhematopoietic cells limited the activation of αvβ3 integrin. Specifically, ICOSL contains the arginine-glycine-aspartate (RGD) motif, which allowed for a high-affinity and selective binding to αvβ3 and modulation of podocyte adhesion. This binding was largely inhibited either by a synthetic RGD peptide or by a disrupted RGD sequence in ICOSL. ICOSL binding favored the active αvβ3 rather than the inactive form and showed little affinity for other integrins. Consistent with the rapid induction of podocyte ICOSL by inflammatory stimuli, glomerular ICOSL expression was increased in biopsies of early-stage human proteinuric kidney diseases. Icosl deficiency in mice resulted in an increased susceptibility to proteinuria that was rescued by recombinant ICOSL. Our work identified a potentially novel role for ICOSL, which serves as an endogenous αvβ3-selective antagonist to maintain glomerular filtration.

IL-10 and ICOS Differentially Regulate T Cell Responses in the Brain during Chronic Toxoplasma gondii Infection.

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

Therapeutic Candidate Alpn-101, a Dual ICOS/CD28 Antagonist, Potently Suppresses Human/NSG Mouse Xenograft Graft Vs. Host Disease (GvHD) in a Dose Ranging Study and Reduces Disease Activity in a Mouse Model of Hemophagocytic Lymphohistiocytosis (HLH)

Background/Purpose: ALPN-101 is a potent dual inhibitor of the ICOS and CD28 T cell costimulatory pathways designed for therapeutic application in inflammatory diseases. CD28 and ICOS bind CD80/CD86 and ICOS ligand (ICOSL), respectively, and play critical roles in T cell activation and adaptive immunity. ALPN-101 has previously been demonstrated to have potent efficacy - superior to wild type ICOSL-Fc - in models of graft versus host disease (GvHD), a disease reflecting immune-mediated attack of recipient tissue by donor T cells. Here, we examined the efficacy of a single dose of ALPN-101 or repeat dosing with different dose levels in GvHD. We also explored the potential therapeutic benefit of ALPN-101 in another T cell-driven inflammatory disease, hemophagocytic lymphohistiocytosis (HLH), a spectrum of disorders of the immune system characterized by the excessive production of cytokines by activated T cells and macrophages accumulating in organs such as the liver, spleen, bone marrow, and brain, which mediate significant tissue damage.Methods: ALPN-101 was generated using our proprietary variant Ig domain (vIgD™) platform and is an effector-function negative Fc-fusion protein with an engineered variant Ig ICOSL domain capable of binding both ICOS and CD28 with high affinity. ALPN-101 blocks the interaction of these T cell costimulatory molecules with their respective receptors, downregulating T cell activation. The dose ranging GvHD study was executed with ALPN-101 (3x weekly/4 weeks, 20 ug - 500 ug) treatment of NSGTM mice engrafted with human peripheral blood mononuclear cells (PBMC) in comparison to belatacept, a CTLA-4-Fc fusion protein CD28 pathway inhibitor. Mice were monitored daily for clinical signs of GvHD. In a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), we evaluated both prophylactic (days 0, 3, and 6 post LCMV infection) and delayed (days 3, 5, and 7) treatment with ALPN-101 (400ug/dose).Results: ALPN-101 significantly attenuated T cell activation in the human PBMC-NSG GvHD model at a single 100ug dose and at all multiple doses tested, protecting mice from the effects of xenogeneic T cell activation in vivo. Treated animals exhibited enhanced survival and reduced disease scores compared to control mice treated with saline or belatacept. Flow cytometric analyses of blood collected at 1-2 weeks post cell transfer demonstrated ALPN-101 reduced both the number and activation state of the transferred human CD4+ and CD8+ T cells. In the HLH model, ALPN-101 lessened several of the clinical and laboratory manifestations of HLH, including organomegaly, anemia, CD8+ T cell expansion, and liver inflammation.Conclusion: ALPN-101 is a potent T cell inhibitor capable, even with a single dose, of preventing T cell activation, such as that observed in the huPBMC-NSGTM GvHD and the LCMV-induced HLH models, and thus is a promising novel therapeutic candidate for GvHD and other inflammatory diseases. Preclinical development is underway to support clinical studies of this potentially first-in-class dual ICOS and CD28 inhibitor. DisclosuresDillon:Alpine Immune Sciences: Employment, Equity Ownership. Lewis:Alpine Immune Sciences: Employment, Equity Ownership. Swanson:Alpine Immune Sciences: Employment, Equity Ownership. Evans:Alpine Immune Sciences: Employment, Equity Ownership. Levin:Alpine Immune Sciences: Employment, Equity Ownership. Rixon:Alpine Immune Sciences: Employment, Equity Ownership. Peng:Alpine Immune Sciences: Employment, Equity Ownership. Nichols:Incyte: Research Funding; Alpine Immune Sciences: Research Funding. Swiderek:Alpine Immune Sciences: Employment, Equity Ownership.

TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation.

The delay in parasite-specific B cell development leaves people in malaria endemic areas vulnerable to repeated Plasmodium infections. Here, we investigated the role of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a molecule involved in the generation of antigen-specific antibody secreting cells, in host response to non-lethal Plasmodium yoelii infection. We found that TACI deficiency not only resulted in higher peak parasitemia levels in P. yoelii challenged mice, but also led to a delay in parasite clearance and anti-P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-119]) protein and anti-rMSP-119 and anti-P. yoelii IgG antibody development. There was also a delay in the generation of splenic high affinity antibody secreting cells that recognize rMSP-119 protein as compared to wild-type mice. Interestingly, coinciding with the delay in parasite clearance there was a delay in the resolution of T follicular helper (TFH) cell and germinal center (GC) B cell responses in TACI -/- mice. The persistence of TFH and GC B cells is likely a result of enhanced interaction between TFH and GC B cells because inducible costimulator ligand (ICOSL) expression was significantly higher on TACI -/- GC B cells than wild-type cells. The difference in the kinetics of GC reaction appeared to also impact the emergence of plasma cells (PC) because there was a delay in the generation of TACI -/- mice PC. Nevertheless, following the recovery from P. yoelii infection, TACI -/- and wild-type mice were both protected from a rechallenge infection. Establishment of protective B cell response was responsible for the resolution of parasitemia because B cells purified from recovered TACI -/- or wild-type mice were equally protective when introduced to naïve wild-type mice prior to P. yoelii challenge. Thus, despite the increased susceptibility of TACI -/- mice to P. yoelii infection and a delay in the development of protective antibody levels, TACI -/- mice are able to clear the infection and resist rechallenge infection.

Bone marrow endothelial cells sustain a tumor-specific CD8+ T cell subset with suppressive function in myeloma patients

ABSTRACT Endothelial cells (EC) line the bone marrow microvasculature and are in close contact with CD8+ T cells that come and go across the permeable capillaries. Because of these intimate interactions, we investigated the capacity of EC to act as antigen-presenting cells (APC) and modulate CD8+ T cell activation and proliferation in bone marrow of patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance. We found that EC from MM patients show a phenotype of semi-professional APC given that they express low levels of the co-stimulatory molecules CD40, CD80 and CD86, and of the inducible co-stimulator ligand (ICOSL). In addition, they do not undergo the strong switch from immunoproteasome to standard proteasome subunit expression which is typical of mature professional APC such as dendritic cells. EC can trap and present antigen to CD8+ T cells, stimulating a central memory CD8+ T cell population that expresses Foxp3 and produces high amounts of IL-10 and TGF-β. Another CD8+ T cell population is stimulated by professional APC, produces IFN-γ, and exerts antitumor activity. Thus, two distinct CD8+ T cell populations coexist in the bone marrow of MM patients: the first population is sustained by EC, expresses Foxp3, produces IL-10 and TGF-β, and exerts pro-tumor activity by negatively regulating the second population. This study adds new insight into the role that EC play in MM biology and describes an additional immune regulatory mechanism that inhibits the development of antitumor immunity and may impair the success of cancer immunotherapy.

Acute Myeloid Leukemia Cells Express ICOS Ligand to Promote the Expansion of Regulatory T Cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) accumulate in bone marrow microenvironment in acute myeloid leukemia (AML). However, little is known about how the tumor environment including tumor cells themselves affects this process. Here we demonstrated that AML cells expressed inducible T-cell costimulator ligand (ICOSL) that can provide costimulation through ICOS for the conversion and expansion of Tregs sustaining high Foxp3 and CD25 expression as well as a suppressive function. TNF-a stimulation up-regulated the expression of ICOSL. Furthermore, both the conversion and expansion of CD4+CD25+Foxp3+ T cells and CD4+ICOS+Foxp3+ T cells were induced by co-culture with AML cells overexpressed ICOSL. CD4+CD25+ICOS+ T cells possessed stronger ability to secrete IL-10 than CD4+CD25+ICOS− T cells. The mechanism by which IL-10 promoted the proliferation of AML cells was dependent on the activation of the Akt, Erk1/2, p38, and Stat3 signaling pathways. Blockade of ICOS signaling using anti-ICOSL antibody impaired the generation of Tregs and retarded the progression of an AML mice model injected with C1498 cells. The expression of ICOSL of patient AML cells and ICOS+ Tregs were found to be predictors for overall survival and disease-free survival in patients with AML, with ICOS+ Treg cell subset being a stronger predictor than total Tregs. These results suggest that ICOSL expression by AML cells may directly drive Treg expansion as a mechanism of immune evasion and ICOS+ Treg cell frequency is a better prognostic predictor in patients with AML.

ICOSL-augmented adenoviral-based vaccination induces a bipolar Th17/Th1 T cell response against unglycosylated MUC1 antigen

Cellular immunity established via immunotherapy holds the potential to eliminate solid tumors. Yet, cancer vaccines have failed to induce tumor-reactive T cells of sufficient quality to control disease. The inducible T cell costimulator (ICOS) pathway has been implicated in both the selective induction of immunity over tolerance as well as licensing of IL-17-polarized cellular immunity. Herein, we evaluated the ability of ICOS ligand (ICOSL) to augment the immunogenicity of adenoviral-based vaccination targeting the unglycosylated MUC1 peptide antigen. Vaccination disrupted immunotolerance in a transgenic mouse model recognizing human MUC1 as a self-antigen, inducing robust MUC1-specific immunity. Augmenting vaccination with ICOSL induced a bipolar Th17/Th1 effector profile, marked by increased MUC1-specific IL-17A production and RORγt expression in CD4+ but not CD8+ T cells which predominantly expressed IFNγ/IL-2 and T-bet. The polarization and maintenance of Th17 cells established following ICOSL augmented vaccination was highly durable, with elevated IL-17A and RORγt levels detected in CD4+ T cells up to 10 months after initial immunization. Furthermore, provision of ICOSL significantly enhanced MUC1-specific IgG antibody in response to immunization. ICOSL signaling dramatically influenced CD4+ T cell phenotype, altering gene expression of transcription factors and regulators of effector function following immunization. Interestingly, ICOSL augmentation failed to alter the transcriptional profile of CD8+ T cells following immunization, affecting the magnitude, but not distribution, of gene expression. Collectively, ICOSL supports the induction of durable, antigen-specific Th17/Th1-mediated immunity in vivo, establishing a vaccination platform to enhance CD4+ T cell-mediated antitumor immunity and providing a crucial component of an effective cancer vaccine.

ICOS-L as a Potential Therapeutic Target for Cancer Immunotherapy.

The co-stimulatory B7 family members are cell-surface protein ligands, binding to receptors on lymphocytes to regulate immune responses. One of them is the inducible co-stimulatory molecule ligand (ICOS-L). This protein is expressed on professional antigen-presenting cells (APCs), including B cells, macrophages, and dendritic cells (DCs), but it can also be expressed by endothelial cells, lung epithelium and in tumour microenvironment cells. ICOS-L is important for memory and effector T cells during the specific humoral immune responses, but its role in cancer is not yet understood. To discuss the role of ICOS/ICOS-L in cancer, given importance of identifying selective targets for cancer treatment, and knowing the mechanism of immune evasion by tumour. ICOS/ICOS-L signal has opposite effects on the T-cell response. ICOS-L is activated in several types of cancers to maintain immunosuppressive CD4+ T cell subsets, such as regulatory T cells (Tregs). ICOS-L over-expression is associated with tumour progression and poor overall survival. In colon cancer, activation of this co-stimulatory signal is associated with improved survival suggesting a dualistic effect of the ICOS/ICOs-L signal pathway. Interestingly, following anti-cancer vaccine or anti- CTLA-4 treatment, ICOS+ T cells increased significantly in both the CD4+ and CD8+ population and the ratio Teff/Treg increased in tumour microenvironment. This suggests a potential role of ICOS/ICOS-L in improving effectiveness of cancer therapy. ICOS/ICOS-L signal pathway has the potential to improve cancer treatment. However, studies in other models are needed to understand whether inhibition of ICOS expression or the blockage of its co-stimulation could be a potential therapeutic target or adjuvant treatment for immunotherapy.

Enhanced CD40 and ICOSL expression on dendritic cells surface improve anti-tumor immune responses; effectiveness of mRNA/chitosan nanoparticles

Objective: To improve dendritic cells (DCs) function, we targeted DCs to over express CD40 and inducible costimulator ligand (ICOSL) costimulatory molecules along with total messenger RNA (mRNA) of tumor cells to achieve a safe and effective system for treatment of tumor.Materials and methods: We generated CD40 and ICOSL mRNA in vitro and manipulated DCs using chitosan nanoparticles and also lipofectamine transfection system then examined in vitro and in vivo.Results: Mice bone marrow derived DCs pulsed with total tumor mRNA/CD40 mRNA or ICOSL mRNA showed higher expression of DCs maturation markers (CD40, ICOSL, CD86, and MHC-II) and accelerated secretion of pro-inflammatory cytokines. Co-culture of DCs with T cells enhanced proliferation of T cells and shift toward stronger Th1 cytokine responses especially in presence of CD40 over expressed DCs. Intra-tumor administration of manipulated DCs to 4T1 tumor mice model showed delay in growth of tumor volume, trend to increase in mice survival, and stronger anti-tumor cytokines production in splenocytes of mice model (with higher efficacy of mRNA/chitosan nanoparticle system).Conclusions: Hence, we suggest that targeting intra-tumor DCs to elicit expression of CD40 and ICOSL and present broad range of tumor antigens could yield effective anti-tumor responses. In this regard, CD40 molecule manipulation trigger stronger functions, while mRNA/chitosan nanoparticles system could provide a high potent tool for targeting strategies.

Abstract P1-09-10: ICOSL anti-HER2 V-mAbs: Localizing engineered ICOSL costimulatory agonists to HER2+ tumors through trastuzumab

Abstract Background: The presence of tumor infiltrating lymphocytes (TILs) has been associated with improved prognosis in HER2+ breast cancer patients. Antigen specific TCR and costimulatory receptor signaling drive increases in TIL number, effector function, and tumor cytotoxicity. Improving the number and effector phenotype of tumor localized TILs has curative potential by enhancing the adaptive and memory immune response. Targeting HER2 with the monoclonal anti-HER2 antibody trastuzumabhas improved survival in HER2+ breast cancer patients and is known to increase peripheral type I immunity, which may be reflected by increased TILs. The Immunoglobulin Superfamily (IgSF) includes a large, diverse family of immunotherapy targets expressed on immune cells and tumors. Transmembrane IgSF receptors,CD28 and inducible T cell co-stimulator (ICOS), related costimulatory molecules expressed on T cells, interact with CD80/CD86 and ICOS ligand (ICOSL), respectively, and play critical roles in T cell activation and adaptive immunity.The Alpine Immune Science's VIgDTM platform uses directed evolution to derive novel, therapeutically-applicable IgSF extracellular domains with tailored specificity and affinity. The vIgD platform has generated human ICOSL vIgDs capable of binding both ICOS and CD28, activating both pathways. To promote anti-tumor activity of TILs in HER2+ tumors, we developed trastuzumab-ICOSL “V-mAbs” consisting of trastuzumab fused to activating ICOSL vIgDs. These V-mAbs are designed to localize to HER2+ tumors and activate antigen-specific, resident T-cells through costimulatory receptor agonism. Methods: V-mAbs were generated by fusing ICOSL vIgDs to either the N- or C- termini of the heavy and/or light chains of trastuzumab. V-mAb binding to CD28, ICOS or HER2 was measured by flow cytometric analysis of transfected cells or ForteBio analysis. V-mAb costimulatory activity was confirmed by immobilization in the presence of anti-CD3 in a primary human T cell assay. Finally, V-mAbs were co-cultured with HER2+ target cells and human T cells; T-cell activity was measured by proliferation, cytokine production, and target lysis. Results: V-mAbs were successfully produced and bound to CD28, ICOS and HER2. In a plate bound costimulation assay, the V-mAbs increased the amount of IFN-gamma produced by T-cells stimulated with anti-CD3. When incubated with HER2+ target cells, V-mAbs promoted T-cell proliferation, cytokine secretion, and target cell lysis. Data from in vivo studies, to determine the impact of trastuzumab V-mAbs on HER2+ cancers, will be presented when available. Conclusions: Trastuzumab-ICOSL V-mAbs are novel ICOS- and CD28-activating immunotherapies for HER2-positive tumors, promoting T-cell proliferation, cytokine secretion, and target cell lysis in a HER2 dependent fashion. The V-mAb platform has broad potential to enable tumor-localized immune modulation via the diverse array of IgSF members. Preclinical development of trastuzumab-ICOSL clinical therapeutics is in progress. Citation Format: Rickel E, Evans L, Swanson R, Levin SS, Rixon M, Wolfson M, Bhandari J, MacNeil S, Hoover J, Kornacker M, Capuano I, Peng SL. ICOSL anti-HER2 V-mAbs: Localizing engineered ICOSL costimulatory agonists to HER2+ tumors through trastuzumab [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-10.

ADAM10-Mediated ICOS Ligand Shedding on B Cells Is Necessary for Proper T Cell ICOS Regulation and T Follicular Helper Responses.

The proper regulation of ICOS and ICOS ligand (ICOSL) has been shown to be essential for maintaining proper immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant Ab production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that a disintegrin and metalloproteinase (ADAM)10 is the primary physiological sheddase of ICOSL in mice and humans. Using an in vivo system in which ADAM10 is deleted only on B cells, elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wild-type mice, interaction of ICOS/ICOSL results in ADAM10-induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper development and TH2 polarization, as seen in a house dust mite exposure model. In addition, enhanced TH1 and TH17 immune responses are seen in experimental autoimmune encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and rescues, at least in part, T follicular helper numbers and the abnormal Ab production previously reported in these mice. Overall, we propose a novel regulation of the ICOS/ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL, as well as indirectly regulating ICOS, thus controlling ICOS/ICOSL-dependent responses.

TFH-derived dopamine accelerates productive synapses in germinal centres.

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contained dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and released it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection.

Effect of follicular helper T cells on the pathogenesis of asthma.

Follicular helper T (TFH) cells are considered to be a separate T helper cell subset, specifically to help memory B cell participate in humoral immunity. It has been reported that there is an association between the imbalance of TFH function and certain autoimmune diseases. However, to the best of our knowledge, the effect of TFH cells on the process of bronchial asthma has not been investigated. The aim of the present study was to investigate the associated markers of TFH cells in bronchial asthma-induced mice. In the current study, sensitized and long-term challenges induced a mouse asthmatic model and were used to investigate the associated markers of TFH cells in the pathogenesis of asthma. The results demonstrated that B cell lymphoma 6, inducible T-cell costimulator (ICOS), ICOS ligand, C-X-C chemokine receptor type 5 (CXCR5) and interleukin (IL)-21 protein and mRNA expression levels were higher in the asthma group, as compared with the control group. Furthermore, the ratio of cluster of differentiation (CD) 4+CXCR5+/CD4+ and CD4+CXCR5+ICOS+/CD4+CXCR5+ was significantly increased in the asthma group. The results of the current study suggest that TFH cells and associated markers may have a role in the pathogenesis of chronic bronchial asthma.

ADAM10 is a novel sheddase of Inducible Costimulatory Ligand (ICOSL).

Abstract ICOSL is an important co-stimulatory molecule involved in the development of TH2 responses through germinal center reactions. Although not well defined, the regulation of ICOSL is known to occur through its interaction with ICOS. Evidence suggests surface expression levels are regulated by a catabolic event, and not through internalization. Here we confirm that regulation of ICOSL is mediated by the metalloproteinase ADAM10. Cleavage of recombinant ICOSL results in an amino-terminal fragment of approximately 3kDa, which is blocked by an ADAM10-specific inhibitor. Recombinant and cell-based cleavage assays suggest that ADAM17 can also cleave ICOSL. In vivo, we observed a ten-fold increase in ICOSL expression in ADAM10B−/− mice compared to wildtype, a phenomenon that was absent in ADAM17B−/− mice. This suggests that ADAM10 is the primary physiological sheddase of ICOSL, while ADAM17 may serve as a secondary sheddase. Additionally, ADAM10B−/− mice develop a significantly diminished T follicular helper (TFH) cell population in response to NP-KLH/alum immunization. Together, these data provide insight into the regulatory mechanism between ICOS/ICOSL and suggest ADAM10 may be a potential therapeutic target for attenuating antibody driven diseases.

Smoking reduces circulating CD26hiCD161hi MAIT cells in healthy individuals and patients with multiple sclerosis.

Upon chronic cigarette smoke exposure, inhaled antigens and irritants cause altered lung immune homeostasis. Circulating immune cells are affected, and smoking is associated with an increased risk of developing various disorders, including multiple sclerosis (MS). This study was conducted to determine the impact of smoking on circulating immune cell subsets. Furthermore, we determined whether any smoking-associated changes were related to MS. With the use of flow cytometry, CFSE assays, and ELISpot assays, we analyzed circulating immune cell phenotypes and quantified antigen-induced proliferation and cytokine secretion in smokers and nonsmokers in a cohort of 100 healthy individuals (HI). In addition, we analyzed immune cell subsets associated with smoking in 2 independent cohorts of patients with MS. In HI smokers compared with nonsmokers, we found increased blood cell counts of granulocytes, monocytes, and lymphocytes. These cells were not more proinflammatory, autoreactive, or EBV reactive compared with cells from nonsmokers. Phenotypic differences were seen in plasmacytoid dendritic cells (pDCs) and CD8+ T cells as higher percentages of ICOS ligand (ICOSL)+ pDCs and lower percentages of CD26hiCD161hi CD8+ T cells and CCR6+ CD8+ T cells in smokers compared with nonsmokers. In supplemental analyses, we showed that CD26hiCD161hi CD8+ T cells were mainly mucosal-associated invariant T cells (MAITs). Comparable frequencies of ICOSL+ pDCs, CCR6+ CD8+ T cells, and CD26hiCD161hi CD8+ T cells were found between HI and MS patients who were nonsmokers. Our findings suggest general proinflammatory effects from smoking combined with skewing of specific cell populations in HI and MS patients. The function of these cell populations needs further investigation.