Inducible T-cell Costimulator Research Articles (Page 1)

T-cell receptor (TCR) clonotype patterns across disease stages and histologic subtypes in mycosis fungoides (MF) and Sézary syndrome (SS) remain poorly characterized, limiting their use in risk stratification. To assess the association of TCR β (TCRB) and γ (TCRG) clonotypes with disease stage, folliculotropism, large-cell transformation, and overall survival (OS) as well as clonal abundance (percentage of total reads) with immune checkpoint expression. This retrospective cohort study conducted at City of Hope (Duarte, California) included patients with stage IA to IVB MF/SS who underwent TCR next-generation sequencing on lesional skin biopsy specimens collected between June 2020 to October 2024; duplicate samples were excluded. Analyses were performed from November 2024 to April 2025. Associations between clinical and genetic categorical variables were evaluated using the Fisher exact test. OS was analyzed using Kaplan-Meier estimates, with univariate and multivariable models applied to assess prognostic factors. Of the 125 patients (42 female [33.6%] and 74 male individuals [66.4%]; mean [SD] age, 62.4 [15.9] years) who underwent TCR sequencing, at least 1 clonal TCRB and/or TCRG gene segment was identified in 98 patients (78%). Clonal TCRB and TCRG segments were detected in 72 (57.6%) and 92 patients (73.6%), respectively. The clonal Vb20 segment was significantly associated with folliculotropism and concurrent large-cell transformation compared with classic MF/SS (7 of 17 [41%] vs 0 of 30 [0%]; P < .001), marginally significantly associated with advanced-stage MF/SS compared with early-stage MF (8 of 38 [21%] vs 0 of 34 [0%]; P = .01). Clonal Vg8 was significantly associated with advanced-stage MF/SS compared with early-stage MF (25 of 53 [47%] vs 8 of 39 [21%]; P = .01) and correlated with poorer OS. Additionally, the higher percentage of total reads for TCRG was positively correlated with increased expression of immune checkpoints programmed cell death 1 and inducible T-cell costimulator but not with programmed cell death ligand 1. This cohort study's analysis of TCRB and TCRG repertoires identified specific clonotypes that were associated with more aggressive subtypes and poorer survival in patients with MF/SS. Incorporating TCR sequencing into clinical practice may enhance risk stratification, enabling earlier identification of high-risk patients who could benefit from closer monitoring and timely implementation of more intensive treatment strategies in the disease course to improve clinical outcomes.

Immune evasion in lung cancer is closely associated with the dysregulation of molecular chaperones and immunoregulatory pathways. Heat shock protein 90 (HSP90), which is frequently overexpressed in lung cancer and correlates with poor prognosis, has emerged as a promising therapeutic target. Here, we investigated whether targeting the HSP90–aryl hydrocarbon receptor (AhR) axis with usnic acid (UA) could suppress immune evasion mechanisms in lung cancer. Through target prediction and molecular docking, UA-bead–based proteomic profiling, and in vitro assays, we identified HSP90 as a direct binding of UA. Unlike classical HSP90 inhibitors, UA downregulates HSP90 protein level and disrupts the HSP90–AhR complex, thereby promoting proteasomal degradation of AhR and reducing its half-life. This disruption suppresses AhR-associated gene expression and tryptophan metabolism-related markers under both AhR ligand-bound and ligand-free conditions. Additionally, the immune checkpoint molecules programmed death-ligand 1 (PD-L1) and inducible T-cell costimulator ligand (ICOSL) were markedly downregulated, demonstrating that UA modulates the tumor immune microenvironment via the HSP90–AhR axis. To assess its translational relevance, a water-soluble derivative of UA, potassium usnate (KU), was evaluated in a syngeneic lung cancer mouse model. KU treatment inhibited tumor growth in a dose-dependent manner, reduced tumor-associated macrophages and programmed cell death protein 1 (PD-1⁺) T cells, and increased the infiltration of cytotoxic T lymphocytes (CD8⁺) and helper T cells (CD4⁺). In addition, KU decreased the proliferation marker, antigen Kiel 67 (Ki67⁺), and HSP90⁺ cell populations within tumors. Together, these findings demonstrate that UA/KU targets the HSP90–AhR axis, suppresses immune evasion pathways, and offers a novel immunomodulatory approach for lung cancer therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s43556-025-00309-z.

Gemcitabine-loaded ICOS-Fc decorated nanosponges: A new chemo immunotherapy combination against pancreatic cancer.

Regulatory T cell (Treg) immunome profile and targets in antiprogrammed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) is a subject of extensive research, but there are still complexities in the area due to the nature of tumor microenvironment (TME). TME of solid tumors contains factors exerting a range of effects on Tregs including development, recruitment, expansion, stability and their immunosuppressive activity. Anti-PD-1 secondarily causes replenishment of intratumoral Tregs, which further intensify tumor immunosuppression. Besides, Treg depletion may also compromise immune checkpoint inhibitor (ICI) efficacy in addition to its other adversarial effects. Thus, a desired ICI booster is to use agents preferentially acting on intratumoral Tregs. Modulation of hypoxia, and regulation of Treg-related cytokines, chemokines, receptors and chromatin modifying factors in tumor TME provide supplementary approaches to anti-PD-(L)1. Factors acting on Tregs have diverse or even dual functions in TME. Treg expansion inhibitory and immunosuppressive tumor-associated macrophage (TAM) recruitment stimulatory effects of stimulator of interferon genes (STING), effector suppressor Treg activating and tumor-specific CD8+ T cell stimulatory effects of interleukin type 2 receptor alpha (IL-2Rα, also called CD25), cell type-dependent dual activities of CXCR3 and inducible T cell costimulatory (ICOS), exposure time-dependent dual effects of glucocorticoid-induced TNFR-related protein (GITR) and CD70 on Tregs and T cells, and exposure level-dependent dual activities of IFN-γ on Tregs are examples require consideration in designing Treg-based strategies. The main direction of this review is to provide updated information about targeting modulators of intratumoral Tregs with agents/compounds aiming to expand anti-PD-(L)1 efficacy and durability in solid tumors.

Caveolin-1/PPARα axis suppresses T follicular helper cell response in primary Sjögren disease.

BackgroundAdvancing cancer immunotherapy requires engineering synthetic immunomodulators that integrate precise receptor targeting, tunable activity, and compatibility with modular biologic formats. The Inducible T-cell Co-Stimulator (ICOS) is a clinically validated co-stimulatory receptor whose engagement enhances T-cell function. However, the development of ICOS-targeting biologics has been hindered by limited receptor affinity and format-dependent agonist activity. To address this, we applied a protein engineering framework to optimize the ICOS ligand (ICOS-L) as a high-affinity, modular component for precision immune modulation.ResultsUsing yeast surface display–based directed evolution, we identified an ICOS-L variant (Y8) containing two synergistic mutations (Q51P and N57H) that improved human ICOS (hICOS) binding affinity by ~ 100-fold relative to wild-type. Structural modeling revealed that Q51P enhances backbone rigidity via a proline-induced conformational constraint, while N57H introduces a salt bridge with Asp86 in hICOS. These mutations reconfigure the receptor-binding interface to support high-affinity engagement. Functionally, Y8 induced potent T-cell proliferation and IFN-γ secretion. When genetically fused to pembrolizumab, Y8 further enhanced T-cell activation and tumor cell lysis, demonstrating synthetic synergy between PD-1 blockade and ICOS agonism. Among fusion formats, light-chain conjugation (pembrolizumab-L-Y8) exhibited superior functional output, highlighting the importance of geometric configuration in optimizing fusion-based agonism.ConclusionThis study establishes Y8 as a high-affinity ICOS-L variant with robust co-stimulatory function, capable of potentiating anti–PD-1 immunotherapy through modular fusion design. The integration of Y8 into therapeutic antibody scaffolds provides a versatile engineering framework for the development of next-generation immunomodulatory biologics, offering opportunities to overcome resistance and enhance clinical efficacy in cancer immunotherapy.

Targeting costimulatory signalling pathways, especially inducible T-cell costimulatory (ICOS)-ICOS ligand(ICOSL) co-stimulatory signal, has been widely used as a therapeutic target in autoimmune diseases. Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating and neurodegenerative disease in the central nervous system. At present, few studies are addressing the role of the ICOS-ICOSL co-stimulatory pathway in MS. We aimed to explore the role of anti-ICOS mAb in the immune response of EAE and further reveal the regulatory mechanism. C57BL/6 female mice (6-8 weeks old, 18-20 g) were randomly divided into EAE group, T1 group, T9 group and T30 group. The EAE model was established in the three groups by MOG active immunisation. Symptom scores and weights of mice were recorded every day after modelling. Mice in the T1 and the T9 groups were given intraperitoneal injections of anti-ICOS mAb on Day 1 or 9 after active immunisation, respectively. Anti-ICOS mAb were injected three times total, each injection separated by 72 h. On the 19th day after modelling, the mice were sacrificed and the spinal cord tissues of each group were collected. Mice in the T30 group were given an intraperitoneal injection of anti-ICOS mAb on Day 9 after active immunisation. These anti-ICOS mAb were injected continuously until Day 30. On Day 30 after the establishment of the model, the mice were sacrificed and spinal cord tissues were collected. HE staining was used to observe the inflammatory infiltration of the spinal cord in each group of mice. Transcriptome sequencing was used to detect gene transcription in spinal cord tissues of mice and to explore the signalling pathways that might be involved. Finally, the signalling pathway was verified by Western blot. (1) Compared with the EAE group, there are significantly lower behavioural scores, heavier weight, delayed onset time and fewer inflammatory infiltrations of the spinal cord in the T9 group and T30 group. However, there were no significant differences in behavioural scores, weight, onset time and inflammatory infiltration between the EAE group and the T1 group. (2) The R 2 value of the spinal cord sample in each group was greater than 0.8. There were 6569 differentially expressed genes (DEGs) between T9 group and EAE group, including 2579 up-regulated genes and 3990 down-regulated genes. There were 487 DEGs between T1 and EAE groups, including 131 up-regulated genes and 356 down-regulated genes. There were 7116 DEGs between T9 and T1 groups, including 2921 up-regulated genes and 4195 down-regulated genes. KEGG enrichment showed that DEGs between T9 group and EAE group were mainly enriched in Epstein-Barr virus infection, NF-κB signalling pathway, FcγR-mediated phagocytosis, natural killer cell-mediated cytotoxicity, as well as Th17, Th1 and Th2 cell differentiation pathways. In the most significantly enriched EB pathway, we found that the fold change of key genes SYK, PI3K and AKT was -4.3457, -2.0985 and -0.69373, respectively (p < 0.001). However, in KEGG enrichment analysis between T1 group and EAE group, DEGs were mainly enriched in retrograde endocannabinoid signalling, Parkinson's disease, morphine addiction, Alzheimer's disease, endocrine disorders, Huntington's disease, cholinergic synapses, oxidative phosphorylation and other pathways. In addition, in KEGG enrichment analysis between T9 group and T1 group, DEGs were mainly in Epstein-Barr virus infection, Th17, Th1 and Th2 cell differentiation, NF-κB signalling pathway, B/T cell receptor signalling pathway, TNF signalling pathway, FcγR-mediated phagocytosis and other pathways. (3) Compared with the control group, the relative expression of SYK, AKT and PI3K protein in the spinal cord of the EAE group was significantly increased. The relative expression of SYK, AKT and PI3K protein in the T9 group and T30 group was significantly decreased compared with the EAE group, while there was no significant difference between the EAE group and the T1 group. Anti-ICOS mAb treatment showed a therapeutic effect in EAE mice during the immune response period (Days 9 and 30 after active immunisation), but not in the immune activation period (Day 1 after active immunisation). The therapeutic effect of anti-ICOS mAb through blocking ICOS-ICOSL signalling in the immune response of EAE mice may be mediated by inhibiting the SYK/PI3K/AKT pathway.

ABSTRACTCanine oral squamous cell carcinoma (COSCC) is the second most common oral tumor in dogs and the most relevant for comparative human trials as a spontaneous large animal model of disease. Historical genomic work has focused primarily on bulk sequencing. The present study describes the complete transcriptomic landscape of COSCC with spatial distinction between the surface tumor, deep invasive tumor, peritumoral dysplastic epithelium, and tumor microenvironment compared to matched normal oral samples. Each region demonstrated distinct molecular signatures. Genes related to epithelial growth factor (EGFR) and epithelial‐mesenchymal transformation (EMT) were upregulated in both peritumoral dysplasia and surface cancer. Additionally, the KRAS gene set, KRT17, and SSP1 were enriched in cancer. We identified five genes that represent dysplastic lesion with high potential for malignant transformation (FZD4, GAS1, HACD2, NOG, and SLC39A6). Also, three genes, SFRP4, FZD1, and IL34 represented a specific signature of the invasive portion of the COSCC that should be explored for prognostic value as a biomarker of malignancy. Lastly, we verified the immunomodulatory tumor microenvironment detecting an increase in macrophages and an abundance of IL‐10 secretion. The other predominant leukocytes were T‐cells, with CD4+ T‐cells being the most prevalent. CD4+ T cells expressed transcripts for both stimulatory (Inducible T‐cell Co‐Stimulator (ICOS) and inhibitory molecules (CTLA4). The observed high CTLA4 suggests that this inhibitory signal may be preventing a robust antitumor immune response. Taken together, this study identified multiple targets to be explored for biomarkers of malignancy, prediction of tumor behavior, and potential targets for development of novel therapies.

Interferon-γ and IL-27 positively regulate type 1 regulatory Tcell development during adaptive tolerance.

Objective Zinc finger protein 335 (Zfp335) plays a crucial role in the early development of thymic T cells and the differentiation of peripheral T cell subpopulations. The objective of this study is to investigate the role and underlying mechanisms of Zfp335 in the regulation of regulatory T cell (Treg) within tumor immunity. Methods The Zfp335 gene was specifically knocked out in Treg using tamoxifen (Zfp335fl/fl FOXP3creERT2), and the MC38 tumor model was established. On the 7th day after tumor inoculation, tumor size was observed and measured. Tumor size was monitored and recorded daily starting from day 7 post-inoculation. On day 12, tumors were harvested, and the proportions of CD4+ T cells, CD8+ T cells, and Treg were analyzed by flow cytometry. Additionally, the mitochondrial function of effector regulatory T cell (eTreg) was assessed. Results From day 10 post-tumor inoculation, tumor volume in the Zfp335CKO group was significantly reduced compared to that of the wild-type (WT) group. Furthermore, the infiltration of CD4+ and CD8+ T cells, along with their respective effector cells, was significantly higher in the Zfp335CKO group than in the WT group. The proportions of CD4+ and CD8+ T cells producing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were also significantly increased in the Zfp335CKO group compared to that of the WT group. In addition, the percentage of CD8+ T cells secreting granzyme B (GzmB) was significantly higher in the Zfp335CKO group than that in the WT group. In contrast, the proportion of Treg and inducible T cell co-stimulator (ICOS)+ Treg in the Zfp335CKO group was significantly lower than that in the WT group. Finally, the expression level of Mitotracker Deep Red in eTreg from the Zfp335CKO group was significantly reduced compared to that in the WT group. Conclusion During tumorigenesis, the specific deletion of Zfp335 impairs Treg activation, which is related to decreased mitochondrial function in eTreg. In Zfp335CKO mice. Tumors exhibit increased infiltration of effector T cells, accompanied by elevated levels of cytotoxic cytokines, ultimately enhancing resistance to tumor progression.

Elevated miR-155 levels in B cell malignancies, such as CLL and DLBCL, correlate with increased aggressiveness of the disease. We recently reported that, in two different mouse models of miR-155-driven B cell malignancy, miR-155 targets and down-regulates transcripts encoding ICOSL, the ligand for the Inducible T cell costimulator (ICOS), thereby impairing the capacity of T lymphocytes to recognize and eliminate malignant cells. In this report, we extend our previous findings to Human by showing that miR-155 levels negatively correlate with those of both ICOSL and MHC-I in samples from DLBCL patients. We present evidence of miR-155 reducing the levels of ICOSL transcripts in ABC, but not in GCB primary tumors (PTs) and cell lines (CLs). In contrast, there was no evidence of miR-155 targeting MHC-I transcript levels in both types of DLBCLs. Nevertheless, miR-155 and MHC-I levels inversely correlated in DLBCLs samples, suggesting the existence of indirect regulatory effects of miR-155. There was also evidence of dose-dependent effects at low miR-155 levels. Altogether, our findings indicate that the deficiency of both ICOSL and MHC-I activity, driven by high levels of miR-155, may be causative in the failure of the host immune system to recognize and eliminate malignant B cells.

Peripheral frequency of inducible T cell co-stimulator (ICOS) + CD4 T cells is associated with early response to immunotherapy and prognosis in gastric cancer (GC); however, limited studies have clarified its association with chemotherapy response. This was a prospective, pilot study. A total of 120 participants with newly diagnosed GC were recruited; 50 advanced GC patients (T3-4N1-3M0-1) underwent curative surgery followed by platinum-based chemotherapy, and their prognosis and survival were assessed. The frequency of ICOS + CD4 T cells in the peripheral blood of each patient was examined using flow cytometry. The frequency of ICOS+CD4 T cells in stage III GC patients was significantly higher than that of stages I and II combined (p = 0.0204) and stage IV GC patients (p = 0.0117). In xenograft GC animals, a positive correlation was noted between the peripheral frequency of ICOS + CD4 and the tumor volume in mice (p = 0.0496). Co-culture experiments showed that the presence of GC cells increased the ratio of ICOS + CD4 T cells derived from peripheral blood in a dose-dependent manner. The initial peripheral frequency of ICOS + CD4 T cells in the GC progression-group was significantly lower than that in the stable-group after 3 months of platinum-based chemotherapy (p = 0.0318). High frequency of peripheral ICOS + CD4 was significantly positively correlated with peripheral IFN-γ concentration in tumor-bearing mice. The frequency of ICOS + CD4 serves as an index reflecting the tumor burden of GC. It effectively predicts the short-term progression risk for resectable advanced GC under platinum-based chemotherapy, primarily because it can reflect the basal immune status of the body.

Inducible T-cell co-stimulator (ICOS) and its ligand (ICOSL) form a complex, two-faced immune machinery that can lead to both immune stimulation and inhibition. We explored ICOS transcriptomic expression patterns and their relationship with other checkpoints and with outcomes in patients with advanced/metastatic cancers. This was a retrospective cohort study. RNA expression for ICOS and other immune checkpoints was quantified by RNA sequencing and stratified by rank values into high (75-100 percentiles) and low (0-24 percentiles). Fischer's exact tests were used for univariate analyses to evaluate independent predictors of ICOS high and logistic regression was used for multivariate analyses. Progression-free survival (PFS) and overall survival (OS) for ICOS high versus not high expression were evaluated using the log-rank test (Kaplan-Meier analysis) and Cox proportional hazards. High ICOS (⩾75 percentile RNA rank) was present in 14% of 514 cancers and independently associated with high PD-1 (p = 0.025), PD-L1 (p < 0.0001), and CTLA-4 RNA expression (p < 0.0001) and with patients not having colorectal cancer (p = 0.0009; multivariate analysis). Patterns of ICOS and ICOSL expression varied between and within tumor types. For 217 patients receiving immune checkpoint inhibitors (ICIs), there were no significant differences in PFS or OS between patients with ICOS high versus not-high expression (multivariate analysis). In 272 immunotherapy-naïve patients, OS was also similar between patients with ICOS high versus not-high expression (p = 0.91). High ICOS expression was not a prognostic marker and did not independently predict outcomes after ICIs. Variable expression of ICOS/ICOSL between tumors and association of high ICOS with high PD-1, PD-L1, and CTLA-4 suggest that individual tumor immunomic analysis may be required for optimized patient selection in clinical trials targeting the ICOS/ICOSL system, especially when given in combination with ICIs. UCSD_PREDICT, NCT02478931.

BackgroundCanine cutaneous mast cell tumors (MCTs) are a common, yet clinically challenging tumor type given their variable biological behavior. Although patients with low grade MCTs can often be effectively managed with surgery alone, most dogs with high grade MCTs succumb to their disease despite multimodal therapy. An improved understanding of the immune tumor microenvironment (TME) may help identify novel prognostic and therapeutic targets.MethodsIn this study, we interrogated the immune transcriptional profiles of the TME in low and high grade MCTs, and quantified intratumoral T cells. Twelve client-owned dogs with MCTs (6 Kiupel low grade with clinically benign behavior and 6 Kiupel high grade with clinically aggressive behavior) that underwent curative-intent surgery were selected. Tumor grade was confirmed by a single veterinary pathologist. RNA was extracted from all tumors followed by immune transcriptional profiling utilizing the NanoString Canine IO panel and analysis using the ROSALIND platform. T cell density was determined by immunohistochemical staining for CD3 and quantified using ImageScope software (Leica Biosystems) following digital slide capture. Lymphocytic infiltrate was further characterized in the TME of one high grade MCT using co-immunofluorescence.ResultsImmune transcriptional profiling identified 9 differentially expressed genes between low and high grade MCTs (p-adj < 0.05). Programmed cell death protein 1 (PDCD1) and inducible T-cell costimulator ligand (ICOSLG) gene expression were significantly higher in a subset of high grade MCTs. ICOSLG expression positively correlated with T cell score (rs = 0.6434, p = 0.0278). Although the T cell density was not significantly different between low (mean of 76.42 CD3 + /mm2, SD 12 CD3 + /mm2) and high grade MCTs (mean of 129.1 CD3 + /mm2, SD 96.06 CD3 + /mm2), greater variation of T cell densities was observed across high grade MCTs compared to low grade (p = 0.0059). Immunofluorescence of one high grade MCT with marked T cell infiltration revealed organized aggregates of T and B cells consistent with tertiary lymphoid structures (TLS).ConclusionsOur data revealed significant differences in the immune TME of low and high grade MCTs and provides rationale to further investigate potential prognostic and therapeutic roles of immune checkpoints in canine MCTs.

Immune thrombocytopenia (ITP) is a hemorrhagic autoimmune disease characterized by antibody-mediated platelet injury. ITP has complicated immunopathological mechanisms that need further elucidation. It is well known that the costimulatory molecules OX40 ligand (OX40L) and OX40 play essential roles in the immunological mechanisms of autoimmune diseases. Previously, we discovered that the expression of OX40L and OX40 is significantly increased in the peripheral blood mononuclear cells (PBMCs) of ITP patients. In our present study, OX40L-induced follicular helper T (Tfh) cells exhibited an activated phenotype with elevated expression of inducible T-cell costimulator (ICOS), programmed cell death protein-1 (PD-1), and cluster of differentiation 40 ligand (CD40L) in vitro. Moreover, aberrant OX40L‒OX40 expression might promote the Tfh1-to-Tfh2 shift in vivo, inducing the generation of autoantibodies by enhancing the helper function of Tfh cells for B lymphocytes in a mouse model, which might accelerate the progression of ITP. Additionally, signal transduction through the OX40L‒OX40 axis might be related to the activation of tumor necrosis factor receptor-associated factor (TRAF)‒nuclear factor-κB (NF-κB) and Janus kinase (JAK)‒signal transducer and activator of transcription (STAT) signaling pathways. Overall, OX40L‒OX40 signaling is proposed as a potential novel therapeutic target for ITP.

Abstract INTRODUCTION Regulatory CD8 T cells (CD8 Treg) are responsible for the selective killing of self-reactive and pathogenic T cells to maintain peripheral immune tolerance. In autoimmune disease, CD8 Treg fail to control the expansion of pathogenic T cells that subsequently cause tissue destruction. This CD8 Treg dysfunction is due in part to the expression of inhibitory KIR receptors (KIR2DL1/2/3), which serve as autoimmune checkpoints, and insufficient ICOS (inducible T cell co-stimulator) signaling, which is a primary costimulatory receptor for CD8 Treg. We have demonstrated that in inflammatory bowel disease (IBD) patient peripheral blood mononuclear cells (PBMC), the expression of inhibitory KIR and ICOS on CD8 Treg is increased relative to that in healthy controls, with downstream dysfunctions of their cytolytic capacity and signaling pathways that promote CD8 T cell longevity and memory. Here we present pre-clinical characterization of the CD8 Treg modulator MTX-201, a bispecific antibody that binds to inhibitory KIR and ICOS that are co-expressed by CD8 Treg. MTX-201 binding antagonizes inhibitory KIR signaling and promotes ICOS co-stimulation, resulting in restored CD8 Treg activation and memory, cytolytic capacity, and functions in highly inflamed tissues, including improved metabolic flexibility and deferred exhaustion. APPROACH AND RESULTS Using in vitro testing of Crohn’s and ulcerative colitis (UC) patient derived PBMC, ex vivo tissue organoids, and in vivo testing in a highly inflammatory humanized acute graft vs host disease (GVHD) mouse model, we assessed the binding, specificity, and mechanisms of action of MTX-201. MTX-201 bound to CD8 Treg in mixed PBMC at low nanomolar concentrations, and select single target expressing immune cells, such as activated CD4 T cells. However, MTX-201 increased ICOS downstream effects in inhibitory KIR-expressing CD8 Treg without activating immune cells expressing only ICOS or KIR. In the GVHD model, MTX-201 extended survival and reduced disease score, marked by an increase in CD8 Treg and a reduction of activated CD4 T cells. In antigen stimulated PBMC from patients with either UC or Crohn’s disease, MTX-201 also reduced pathogenic T cell responses without evidence of broad immunosuppression, and reduced antigen induced epithelial cell death in colonic tissue derived organoids containing either tissue resident or engrafted IBD patient-derived T cells. MTX-201 had antibody-like pharmacokinetic parameters with good tolerability, no toxicity, or induction of proinflammatory serum cytokines in repeat dose studies at doses up to 50 mg/kg in CD34+NSG-Tg(Hu-IL15) mice harboring stable engraftments of human lymphocytes, including CD8 Treg cells. CONCLUSION Collectively, our data support the development of MTX-201 as a therapeutic to restore CD8 Treg functions in patients for the treatment of IBD.

BackgroundActivated T cells play a pivotal role in rheumatoid arthritis (RA) pathogenesis, and imaging of activated T cells may provide a non-invasive tool for RA detection. Here, we first developed an optical probe targeting human inducible T cell co-stimulator (ICOS) and tested its capacity in RA diagnosis by capturing ICOS+ activated T cells in vivo in a humanized mouse model.MethodsThe humanized arthritis model, Human peripheral blood mononuclear cells- adjuvant induced arthritis (HuPBMC-AIA) was established, and flow cytometry and immunofluorescence were employed to determine ICOS expression in huPBMC-AIA model. Anti-human ICOS monoclonal antibody (mAb) was conjugated to Cy7 via NHS ester amine reaction. A cell uptake study was used to confirm the specificity of Cy7-ICOS mAb to activated T cells. 4-view near-infrared fluorescence (NIRF) imaging study was performed to test Cy7-ICOS mAb in detecting RA in vivo.FindingsICOS was confirmed as an indicator of RA pathogenesis via RNA-seq, flow cytometry and immunofluorescence data. An in-vitro cellular uptake study validated the specificity of Cy7-ICOS mAb to activated T cells. Cy7-ICOS mAb could detect ICOS+ activated T cells in vivo through 4-view NIRF imaging. The receiver operating characteristic (ROC) curve created based on NIRF imaging quantification could distinguish the huPBMC-AIA group from the control group at all time points imaged.ConclusionIn this study, we first developed an optical imaging probe targeting human ICOS, Cy7-ICOS mAb. The 4-view NIRF imaging with Cy7-ICOS mAb could detect pathogenic ICOS+ activated T cells with high sensitivity and specificity in vivo, which indicated the great potential of this imaging probe in RA early diagnosis.

Type 1 regulatory T (Tr1) cells are critical players in maintaining peripheral tolerance, by producing high IL-10 levels in association with inducible T-cell co-stimulator (ICOS) expression. Whether these cells play a role in naturally acquired baked egg tolerance is unknown. Evaluate frequencies of egg-responsive Tr1 and Th2 cells in egg-allergic children that naturally acquired baked egg tolerance (BET) versus non-egg-allergic (NEA) children. Peripheral blood mononuclear cells from 70 natural BET and 15 NEA children were stimulated for 7 days with ovalbumin and ovomucoid. By flowcytometry, egg-responsive Tr1 cells were identified by co-expression of CD49b and LAG3, and Th2 cells by expression of CD49b but absence of LAG3. Seven-day cultured supernatant was analyzed for Th1, Th2, Tr1, and Th17 cytokines by MSD. Natural BET children had a higher percentage of egg-responsive Th2 cells vs. NEA children (6.75% vs. 10.35%, p = .006). No significant difference was found in frequencies of egg-responsive Tr1 cells between NEA and natural BET children (11.40% vs. 12.55%, p = .42), although Tr1-related IL-10 and IL-21 production was higher in BET children. Interestingly, egg-responsive Tr1 cells from NEA children expressed higher ICOS levels vs. natural BET children (97.90 vs. 88.20, p < .0001). Supernatant from natural BET children showed elevated levels of Th2 cytokines IL-5, IL-9 and IL-13 and Th17 cytokine IL-17A. Natural BET children maintain increased egg-specific Th2 responses, along with comparable proportions of egg-responsive Tr1 cells exhibiting higher IL-10 but lower ICOS expression in comparison with NEA children.

Inducible T-cell costimulator (ICOS, CD278) is a costimulatory receptor primarily expressed by activated T cells. It binds to ICOS ligand (ICOSL, CD275), which is expressed by various immune and non-immune cell types, particularly in inflamed tissues. ICOSL can also bind to osteopontin (OPN), a protein that functions both as a component of the extracellular matrix and as a soluble pro-inflammatory cytokine. Previous studies, including ours, have shown that ICOS and ICOSL play a role in skin wound healing, as mice deficient in either ICOS or ICOSL exhibit delayed healing. The aim of this study was to investigate the involvement of the ICOS/ICOSL/OPN network in skin wound healing by analyzing mice that are single knockouts for ICOS, ICOSL, or OPN, or double knockouts for ICOS/OPN or ICOSL/OPN. Our results showed that wound healing is impaired in all single knockout strains, but not in the two double knockout strains. Cellular and molecular analyses of the wound healing sites revealed that the healing defect in the single knockout strains is associated with reduced neutrophil infiltration and decreased expression of α-SMA (a marker of myofibroblasts), IL-6, TNFα, and VEGF. In contrast, the normalization of wound closure observed in the double knockout strains was primarily linked to increased vessel formation. A local treatment with recombinant ICOS-Fc improved healing in all mouse strains expressing ICOSL, but not in those lacking ICOSL, and led to a local increase in vessel formation and macrophage recruitment, predominantly of the M2 type.

Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound 9 with a promising ICOS/ICOS-L inhibitory profile (IC50=29.38±3.41 μM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound 9 in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.