Costimulatory Pathway Research Articles

New Developments and Therapeutic Drug Monitoring Options in Costimulatory Blockade in Solid Organ Transplantation: A Systematic Critical Review.

In this review, the authors summarized the latest developments in costimulatory blockade to prevent rejection after solid organ transplantation (SOT) and discussed possibilities for future research and the need for therapeutic drug monitoring (TDM) of these agents. Studies about costimulatory blockers in SOT in humans or animal transplant models in the past decade (2014-2024) were systematically reviewed in PubMed, European Union clinical trials (EudraCT), and ClinicalTrials.gov. Seventy-five registered clinical trials and 58 published articles were found on costimulation blockade of the CD28-CD80/86, CD40-CD40L, and OX40-OX40L pathways. Belatacept, an antagonist of the CD28-CD80/86 pathway, is the only approved costimulatory agent in SOT, hence accounting for most of the research. Other identified costimulatory blocking agents included abatacept and CD28 antagonists tegoprubart, dazodalibep, and TNX-1500. Although tegoprubart was unsuccessful in pancreas transplantation in nonhuman primates, trials in human kidney transplantation are underway. Dazodalibep trials faced recruitment challenges. TNX-1500 was unsuccessful in animal studies and is currently not pursued in humans. After discontinuation of iscalimab (CD40-CD154 pathway antagonist) in SOT, the alternatives, bleselumab and KPL404, showed promising results in kidney transplantation and cardiac xenotransplantation. Studies on secondary costimulatory pathway antagonists, such as OX40-OX40L, have only used animal models. Despite the low interindividual variability in pharmacokinetics (PK) in all studied agents, TDM could be useful for optimizing dosing in PK/pharmacodynamic (PD) studies. The routine use of costimulation blockade in SOT is hindered by problems in efficacy compared with the standard of care. Costimulatory inhibitors could be combined in a calcineurin inhibitor-free regimen. Future PK/pharmacodynamic studies in costimulatory agents and personalized medicine could warrant TDM of these agents.

225.9: Delayed immune tolerance for cardiac allografts in nonhuman primates by targeting CD154, CD2, and CD28 costimulation pathways.

225.9: Delayed immune tolerance for cardiac allografts in nonhuman primates by targeting CD154, CD2, and CD28 costimulation pathways.

Update on the pathophysiology and treatment of primary Sjögren syndrome.

Sjögren syndrome or Sjögren disease is a chronic form of autoimmune epithelitis characterized by lymphocytic infiltration of the exocrine glands, particularly the salivary and lacrimal glands, leading to progressive glandular dysfunction and subsequent xerostomia and xerophthalmia. Other common manifestations include pain and fatigue, various systemic manifestations and non-Hodgkin's lymphoma. Sjögren syndrome is therefore a complex and disabling disease associated with a reduced quality of life and with considerable long-term damage. Most of the available treatments are merely symptomatic with limited efficacy in both preventing glandular damage and suppressing systemic disease activity. In the past 10 years, great progress has been made in understanding the pathophysiology of Sjögren syndrome, opening new avenues towards a more targeted and individualized therapeutic approach to the disease. Indeed, several randomized controlled trials have just been completed or are poised to commence evaluating the effectiveness of novel drugs targeting both innate and adaptive immune pathways, including pro-inflammatory cytokines, the type I interferon system, B cell activation, B cell and T cell co-stimulation pathway, and ectopic germinal centre formation. Novel clinical trials are also ongoing exploring various targeted approaches (that is, IgG recycling inhibition, nuclease therapy and CAR-T cell therapy) for Sjögren syndrome.

The role of OX40 ligand/OX40 axis signalling in atopic dermatitis.

Atopic dermatitis (AD) is a heterogeneous inflammatory condition involving multiple immune pathways mediated by pathogenic T cells. OX40 ligand (OX40L) and OX40 are costimulatory immune checkpoint molecules that regulate effector and memory T-cell activity and promote sustained immune responses in multiple immunological pathways, including T helper (Th)2, Th1, Th17 and Th22. As such, OX40L/OX40 signalling between antigen-presenting cells (APCs) and activated T cells postantigen recognition promotes pathogenic T-cell proliferation and survival. Under inflammatory conditions, OX40L is upregulated on APCs, enhancing the magnitude of antigen-specific T-cell responses and secretion of proinflammatory cytokines. In AD, OX40L/OX40 signalling contributes to the amplification and chronic persistence of T-cell-mediated inflammation. Recent therapeutic success in clinical trials has highlighted the importance of the OX40L/OX40 axis as a promising target for the treatment of AD. Here, we discuss the many factors that are involved in the expression of OX40L and OX40, including the cytokine milieu, antigen presentation, the inflammatory environment in AD, and the therapeutic direction influenced by this costimulatory pathway.

Generation of a novel agonist of the human CD137 costimulatory pathway for cancer immunotherapy

Abstract The use of immune checkpoint stimulators is an increasingly attractive therapeutic approach against cancer, and costimulatory pathway agonists are being developed for clinical use. Our laboratory has focused on the 4-1BB (CD137) pathway because of its critical role in CD8+ T cell expansion, survival, acquisition of effector function, and establishment of long-term memory. We previously reported the generation of a recombinant oligomeric form of the mouse ligand, muSA-4-1BBL, which, unlike the natural 4-1BB ligand, demonstrated robust costimulatory activity in soluble form. muSA-4-1BBL showed therapeutic efficacy as the immune adjuvant component of subunit vaccines in various mouse transplantable tumor models. In this study, we generated a human version of this ligand, huSA-4-1BBL, and characterized its structure and function. The molecule binds to 4-1BB on LPS-activated human THP-1 monocytes, but not ConA-stimulated mouse T cells expressing the 4-1BB receptor. Conversely, muSA-4-1BBL binds to ConA-stimulated mouse T cells, but not LPS-activated human THP-1 cells, demonstrating species-specific binding for both molecules. To assess the cancer immunotherapeutic efficacy of huSA-4-1BBL, we generated a transgenic mouse model where mouse 4-1BB was replaced with the human ortholog. The assessment of huSA-4-1BBL anti-tumor efficacy in this model will serve as a prelude for further development and translation of this novel agonist to the clinic for cancer immunotherapy.

Monophosphoryl Lipid A-based Adjuvant to Promote the Immunogenicity of Multivalent Meningococcal Polysaccharide Conjugate Vaccines.

Activation of the adaptive immune system requires the engagement of costimulatory pathways in addition to B and T cell Ag receptor signaling, and adjuvants play a central role in this process. Many Gram-negative bacterial polysaccharide vaccines, including the tetravalent meningococcal conjugate vaccines (MCV4) and typhoid Vi polysaccharide vaccines, do not incorporate adjuvants. The immunogenicity of typhoid vaccines is due to the presence of associated TLR4 ligands in these vaccines. Because the immunogenicity of MCV4 is poor and requires boosters, I hypothesized that TLR4 ligands are absent in MCV4 and that incorporation of a TLR4 ligand-based adjuvant would improve their immunogenicity. Consistent with this hypothesis, two Food and Drug Administration-approved MCV4 vaccines, MENVEO and MenQuadfi, lack TLR4 ligands. Admixing monophosphoryl lipid A, a TLR4 ligand-based adjuvant formulation named "Turbo" with MCV4 induced significantly improved IgM and IgG responses to all four meningococcal serogroup polysaccharides in adult and aged mice after a single immunization. Furthermore, in infant mice, a single booster was sufficient to promote a robust IgG response and 100% seroconversion when MCV4 was adjuvanted with Turbo. Turbo upregulated the expression of the costimulatory molecules CD40 and CD86 on B cells, and Turbo-driven adjuvanticity is lost in mice deficient in CD40 and CD86. These data suggest that Turbo induces the required costimulatory molecules for its adjuvant activity and that incorporation of Turbo could make bacterial polysaccharide vaccines more immunogenic, minimize booster requirements, and be cost-effective, particularly for those individuals in low- and middle-income and disease-endemic countries.

Pigs in Transplantation Research and Their Potential as Sources of Organs in Clinical Xenotransplantation.

The pig has long been used as a research animal and has now gained importance as a potential source of organs for clinical xenotransplantation. When an organ from a wild-type (i. e., genetically unmodified) pig is transplanted into an immunosuppressed nonhuman primate, a vigorous host immune response causes hyperacute rejection (within minutes or hours). This response has been largely overcome by 1) extensive gene editing of the organ-source pig and 2) the administration to the recipient of novel immunosuppressive therapy based on blockade of the CD40/CD154 T cell costimulation pathway. Gene editing has consisted of 1) deletion of expression of the 3 known carbohydrate xenoantigens against which humans have natural (preformed) antibodies and 2) the introduction of human 'protective' genes. The combination of gene editing and novel immunosuppressive therapy has extended life-supporting pig kidney graft survival to greater than 1 y and of pig heart survival to up to 9 mo. This review briefly describes the techniques of gene editing, the potential risks of transfer of porcine endogenous retroviruses with the organ, and the need for breeding and housing of donor pigs under biosecure conditions.

Abstract 5306: PTPN22 targeting partners with 41BB/OX40 costimulation to overcome anti-PD1 resistance in pancreatic ductal adenocarcinoma

Abstract Background: The pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) is notable for a scarcity of cytotoxic T cells and an overabundance of immunosuppressive myeloid and regulatory T cells. Checkpoint immunotherapies targeting PD/L1 or CTLA4 have shown efficacy in immunogenic cancers but remain ineffective in PDAC, highlighting a need for novel approaches. Abrogating protein tyrosine phosphatase non-receptor type 22 (PTPN22), a phosphatase in the TCR signaling cascade, has been shown to modulate multiple mechanisms of immune resistance. Furthermore, 41BB costimulatory pathway has recently been investigated as a potential strategy to enhance immunotherapy against PDAC. Using immune-resistant murine models of PDAC, we investigated PTPN22 as an immunotherapeutic target in combination with 41BB co-stimulation and PD1 inhibition against PDAC. Methods: We employed a well-characterized murine PDAC cell line expressing mutant KrasG12D and Trp53R172H under the Pdx1-cre promoter (KPCY 6419c5). This tumor line is resistant to combination immuno- and chemotherapies. To determine treatment efficacy, PTPN22 deficient mice (KO) were inoculated with KPCY6419c5 cells either subcutaneously or orthotopically into the pancreas. To extend the translatability of PTPN22 targeting, we also injected PTPN22 competent mice with the same tumor cells and treated them with our novel PTPN22 inhibitor L1. Harvested tumors were processed for FFPE and mass cytometry (CyTOF) analysis to track changes in the immune compartment. Results: When PTPN22 was abrogated, treatment with anti-PD1 and 41BB agonism led to a 10-day delay in substantial tumor formation and roughly 75% reduction in tumor volume by 21-days post treatment. These tumors showed elevated levels of OX40 and OX40L primarily in the setting of PTPN22 KO/PD1/41BB therapy. In a subsequent in vivo experiment using an OX40L neutralizing antibody, we found that OX40 signaling is essential for the efficacy of PTPN22 KO/PD1/41BB therapy. Furthermore, combining OX40 agonist with anti-PD1 in PTPN22 KO sufficiently mimicked the efficacy seen with the anti-PD-1 and 41BB combination. CyTOF analysis indicated that OX40 expression is enriched in Tregs and OX40L is enriched in myeloid derived suppressor cells (MDSCs). Moreover, combination therapy efficacy was reproduced using the PTPN22 inhibitor L1. Conclusions: PTPN22 abrogation enhances the success of PD-1 inhibition given with either OX-40 or 41BB agonist therapies. In addition, PTPN22 abrogation together with PD1 inhibition and 41BB costimulation reduces MDSC and Treg suppression of effector T cells via the OX40L-OX40 axis. Finally, PTPN22 can be blocked by a small molecule inhibitor strengthening its candidacy as a therapeutic target. Citation Format: Soren Charmsaz, Nicole E. Gross, Jae W. Lee, Jianping Lin, Alexei G. Hernandez, Erin M. Coyne, Sarah M. Shin, Courtney Cannon, Xuan Yuan, Zhong-Yin Zhang, Elizabeth M. Jaffee, Won Jin Ho. PTPN22 targeting partners with 41BB/OX40 costimulation to overcome anti-PD1 resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5306.

Abstract 4034: Lag3 blockade enhances the anti-tumor effect of dual GITR agonism and PD-1 blockade in a preclinical melanoma model

Abstract Immune checkpoint blockade (ICB) therapies, specifically anti PD-1 and anti CTLA-4, have resulted in tremendous success in the clinic. However, a substantial number of patients still relapse due to either inherent or acquired resistance to ICB therapies. In this scenario, activation of T cell co-stimulation pathways along with ICB emerges as a promising strategy to improve anti-tumor responses. Engagement of Glucocorticoid induced TNFR Related protein (GITR) costimulatory molecule, leads to enhanced activation of cytotoxic T cells while destabilizing suppressive T regulatory cells in the tumor microenvironment. Additionally, combining GITR agonism with PD-1 blockade improves the anti-tumor response in a murine model of advanced melanoma resistant to ICB, positioning GITR as an attractive immunotherapeutic target. In this study, we evaluated the therapeutic efficacy of combining blockade of the immune checkpoint LAG3 with GITR agonism for treating ICB-resistant advanced melanoma, using the B16 model. The combination of LAG3 blockade with GITR agonism was well tolerated, leading to better tumor control and improved survival. Our data revealed increased B cell infiltration and activation within tumors and draining lymph nodes (LNs) of treated mice. B cell depletion diminished the anti-tumor effect of the combination therapy, suggesting that B cells play an active role in controlling tumor growth. Spleens from treated mice exhibited increased lymphocytic hyperplasia, along with more and larger germinal centers. B cell receptor sequencing revealed an increased clonality and reduced entropy. Treating B cells with GITR agonism and LAG3 blockade in vitro led to increased activation and proliferation, suggesting a direct effect of these therapies on B cells. Furthermore, adding LAG3 blockade to the dual combination of GITR agonism and PD-1 blockade resulted in superior tumor control and increased B cell activity in the draining LNs. Collectively, our findings suggest that the humoral response significantly contributes to the anti-tumor responses elicited by ICB when combined with GITR agonism. This triple combination therapy (LAG3 blockade + PD1 blockade + GITR agonism) holds promise as a safe and potent therapeutic strategy to overcome ICB resistance. Further investigation into the mechanism of action by which GITR agonism leads to enhanced B cell responses when combined with ICB is warranted in order to design more effective therapies for cancer treatment. Citation Format: Rachana R. Maniyar, Yuval Elhanati, Levi Mangarin, Yacine Marouf, Ashley Ahmed, Benjamin Greenbaum, Jedd Wolchok, Taha Merghoub. Lag3 blockade enhances the anti-tumor effect of dual GITR agonism and PD-1 blockade in a preclinical melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4034.

Synthetic dual co-stimulation increases the potency of HIT and TCR-targeted cell therapies.

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies.

Role of co‑inhibitory molecules in the treatment of psoriasis (Review).

Psoriasis is a common chronic inflammatory skin disease characterized by abnormal activation and infiltration of T-cells and excessive proliferation of keratinocytes (KCs). Its pathogenesis is complex and frequently accompanied by the imbalance of T-cell subpopulations, contributing to its development and further exacerbation. Therefore, the immune system, especially T-cells, is mainly involved in the pathogenesis of psoriasis. While T-cell activation not only requires the first recognition of T-cell receptor and major histocompatibility complex peptide, co-stimulatory and co-inhibitory pathways are reported to promote or dampen T-cell responses through a variety of mechanisms. In recent years, immuno-related agents have been applied in the treatment of numerous clinical diseases, including psoriasis, and are starting to show promising and potential therapy prospects in autoimmune skin diseases. The present review outlined the role of co-inhibitory molecules in the pathogenesis of psoriasis and their application in the treatment of psoriasis.

TIGIT acts as an immune checkpoint upon inhibition of PD1 signaling in autoimmune diabetes.

Chronic activation of self-reactive T cells with beta cell antigens results in the upregulation of immune checkpoint molecules that keep self-reactive T cells under control and delay beta cell destruction in autoimmune diabetes. Inhibiting PD1/PD-L1 signaling results in autoimmune diabetes in mice and humans with pre-existing autoimmunity against beta cells. However, it is not known if other immune checkpoint molecules, such as TIGIT, can also negatively regulate self-reactive T cells. TIGIT negatively regulates the CD226 costimulatory pathway, T-cell receptor (TCR) signaling, and hence T-cell function. The phenotype and function of TIGIT expressing islet infiltrating T cells was studied in non-obese diabetic (NOD) mice using flow cytometry and single cell RNA sequencing. To determine if TIGIT restrains self-reactive T cells, we used a TIGIT blocking antibody alone or in combination with anti-PDL1 antibody. We show that TIGIT is highly expressed on activated islet infiltrating T cells in NOD mice. We identified a subset of stem-like memory CD8+ T cells expressing multiple immune checkpoints including TIGIT, PD1 and the transcription factor EOMES, which is linked to dysfunctional CD8+ T cells. A known ligand for TIGIT, CD155 was expressed on beta cells and islet infiltrating dendritic cells. However, despite TIGIT and its ligand being expressed, islet infiltrating PD1+TIGIT+CD8+ T cells were functional. Inhibiting TIGIT in NOD mice did not result in exacerbated autoimmune diabetes while inhibiting PD1-PDL1 resulted in rapid autoimmune diabetes, indicating that TIGIT does not restrain islet infiltrating T cells in autoimmune diabetes to the same degree as PD1. Partial inhibition of PD1-PDL1 in combination with TIGIT inhibition resulted in rapid diabetes in NOD mice. These results suggest that TIGIT and PD1 act in synergy as immune checkpoints when PD1 signaling is partially impaired. Beta cell specific stem-like memory T cells retain their functionality despite expressing multiple immune checkpoints and TIGIT is below PD1 in the hierarchy of immune checkpoints in autoimmune diabetes.

A brief review of the current status of pig islet xenotransplantation.

An estimated 1.5 million Americans suffer from Type I diabetes mellitus, and its incidence is increasing worldwide. Islet allotransplantation offers a treatment, but the availability of deceased human donor pancreases is limited. The transplantation of islets from gene-edited pigs, if successful, would resolve this problem. Pigs are now available in which the expression of the three known xenoantigens against which humans have natural (preformed) antibodies has been deleted, and in which several human 'protective' genes have been introduced. The transplantation of neonatal pig islets has some advantages over that of adult pig islets. Transplantation into the portal vein of the recipient results in loss of many islets from the instant blood-mediated inflammatory reaction (IBMIR) and so the search for an alternative site continues. The adaptive immune response can be largely suppressed by an immunosuppressive regimen based on blockade of the CD40/CD154 T cell co-stimulation pathway, whereas conventional therapy (e.g., based on tacrolimus) is less successful. We suggest that, despite the need for effective immunosuppressive therapy, the transplantation of 'free' islets will prove more successful than that of encapsulated islets. There are data to suggest that, in the absence of rejection, the function of pig islets, though less efficient than human islets, will be sufficient to maintain normoglycemia in diabetic recipients. Pig islets transplanted into immunosuppressed nonhuman primates have maintained normoglycemia for periods extending more than two years, illustrating the potential of this novel form of therapy.

Co-stimulatory pathway competitive assay development using Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Inhibition of CD40L with Frexalimab in Multiple Sclerosis

BackgroundThe CD40–CD40L costimulatory pathway regulates adaptive and innate immune responses and has been implicated in the pathogenesis of multiple sclerosis. Frexalimab is a second-generation anti-CD40L monoclonal antibody being evaluated for the treatment of multiple sclerosis.MethodsIn this phase 2, double-blind, randomized trial, we assigned, in a 4:4:1:1 ratio, participants with relapsing multiple sclerosis to receive 1200 mg of frexalimab administered intravenously every 4 weeks (with an 1800-mg loading dose), 300 mg of frexalimab administered subcutaneously every 2 weeks (with a 600-mg loading dose), or the matching placebos for each active treatment. The primary end point was the number of new gadolinium-enhancing T1-weighted lesions seen on magnetic resonance imaging at week 12 relative to week 8. Secondary end points included the number of new or enlarging T2-weighted lesions at week 12 relative to week 8, the total number of gadolinium-enhancing T1-weighted lesions at week 12, and safety. After 12 weeks, all the participants could receive open-label frexalimab.ResultsOf 166 participants screened, 129 were assigned to a trial group; 125 participants (97%) completed the 12-week double-blind period. The mean age of the participants was 36.6 years, 66% were women, and 30% had gadolinium-enhancing lesions at baseline. At week 12, the adjusted mean number of new gadolinium-enhancing T1-weighted lesions was 0.2 (95% confidence interval [CI], 0.1 to 0.4) in the group that received 1200 mg of frexalimab intravenously and 0.3 (95% CI, 0.1 to 0.6) in the group that received 300 mg of frexalimab subcutaneously, as compared with 1.4 (95% CI, 0.6 to 3.0) in the pooled placebo group. The rate ratios as compared with placebo were 0.11 (95% CI, 0.03 to 0.38) in the 1200-mg group and 0.21 (95% CI, 0.08 to 0.56) in the 300-mg group. Results for the secondary imaging end points were generally in the same direction as those for the primary analysis. The most common adverse events were coronavirus disease 2019 and headaches.ConclusionsIn a phase 2 trial involving participants with multiple sclerosis, inhibition of CD40L with frexalimab had an effect that generally favored a greater reduction in the number of new gadolinium-enhancing T1-weighted lesions at week 12 as compared with placebo. Larger and longer trials are needed to determine the long-term efficacy and safety of frexalimab in persons with multiple sclerosis. (Funded by Sanofi; ClinicalTrials.gov number, NCT04879628.)

An exosome-derived lncRNA signature identified by machine learning associated with prognosis and biomarkers for immunotherapy in ovarian cancer.

Ovarian cancer (OC) has the highest mortality rate among gynecological malignancies. Current treatment options are limited and ineffective, prompting the discovery of reliable biomarkers. Exosome lncRNAs, carrying genetic information, are promising new markers. Previous studies only focused on exosome-related genes and employed the Lasso algorithm to construct prediction models, which are not robust. 420 OC patients from the TCGA datasets were divided into training and validation datasets. The GSE102037 dataset was used for external validation. LncRNAs associated with exosome-related genes were selected using Pearson analysis. Univariate COX regression analysis was used to filter prognosis-related lncRNAs. The overlapping lncRNAs were identified as candidate lncRNAs for machine learning. Based on 10 machine learning algorithms and 117 algorithm combinations, the optimal predictor combinations were selected according to the C index. The exosome-related LncRNA Signature (ERLS) model was constructed using multivariate COX regression. Based on the median risk score of the training datasets, the patients were divided into high- and low-risk groups. Kaplan-Meier survival analysis, the time-dependent ROC, immune cell infiltration, immunotherapy response, and immune checkpoints were analyzed. 64 lncRNAs were subjected to a machine-learning process. Based on the stepCox (forward) combined Ridge algorithm, 20 lncRNA were selected to construct the ERLS model. Kaplan-Meier survival analysis showed that the high-risk group had a lower survival rate. The area under the curve (AUC) in predicting OS at 1, 3, and 5 years were 0.758, 0.816, and 0.827 in the entire TCGA cohort. xCell and ssGSEA analysis showed that the low-risk group had higher immune cell infiltration, which may contribute to the activation of cytolytic activity, inflammation promotion, and T-cell co-stimulation pathways. The low-risk group had higher expression levels of PDL1, CTLA4, and higher TMB. The ERLS model can predict response to anti-PD1 and anti-CTLA4 therapy. Patients with low expression of PDL1 or high expression of CTLA4 and low ERLS exhibited significantly better survival prospects, whereas patients with high ERLS and low levels of PDL1 or CTLA4 exhibited the poorest outcomes. Our study constructed an ERLS model that can predict prognostic risk and immunotherapy response, optimizing clinical management for OC patients.

520 - Evaluating the expression of OX40 and OX40 ligand in lesional skin versus non-lesional skin in volunteers with atopic dermatitis and in healthy control skin

Abstract Introduction/Background Growing evidence suggests that blocking the OX40 ligand (OX40L)/OX40 costimulatory pathway could have therapeutic effects in inflammatory diseases such as atopic dermatitis (AD). Amlitelimab (SAR445229; KY1005) is a potential first-in-class, fully human, non-depleting anti-OX40L monoclonal antibody that blocks OX40L-OX40 interactions. We report OX40L and OX40 expression from skin biopsies collected in two studies: an amlitelimab Phase 2a (Ph2a) study in patients with moderate-to-severe AD, and a small-scale study of patients with mild AD and healthy volunteers. Objectives Assess OX40L and OX40 expression in AD skin (lesional and non-lesional) and healthy control skin. Methods As part of a Ph2a randomized, double-blind, placebo-controlled study (NCT03754309) of amlitelimab in moderate-to-severe AD, 174 baseline-paired lesional/non-lesional skin biopsies were collected from all patients (n=87). In a separate small-scale study, 24 skin biopsies were collected, including healthy control skin from healthy volunteers (n=6) and paired lesional/non-lesional skin from patients with mild AD (n=9). Each biopsy underwent immunohistochemical (IHC; formalin-fixed paraffin-embedded) analyses using various antibodies (OX40L: rabbit IgG antibody from Cell Signaling Technology for Ph2a biopsies, proprietary mouse IgG2a KY1005 for small-scale study biopsies; OX40: clone Ber-ACT35 from BioLegend for all biopsies). A trained pathologist assessed IHC staining in the dermis and/or epidermis using semi-quantitative scoring (0 [no stained cells] to 10 [all cells stained]) or quantitative methods (cells/mm2, Tissue Studio 4.0, and HALO link image analysis software). Wilcoxon matched-pairs signed-rank test was used to assess IHC staining of Ph2a biopsies. Ph2a biopsies also underwent transcriptomic analyses (stored in RNALater) for OX40L (TNFSF4) and OX40 (TNFRSF4) on sequenced RNA using a linear mixed model with Benjamini-Hochberg adjustment. Results In baseline Ph2a biopsies, IHC analysis revealed elevated OX40L and OX40 staining in lesional versus non-lesional AD skin (P <0.0001 for both markers). For OX40L, the median score was 3 (lesional) versus 1 (non-lesional) and the Wilcoxon matched-pairs signed-rank test found P<0.0001. For OX40+ cells, the median expression was 179.8 cells/mm2 in lesional AD skin versus 54.45 cells/mm2 in non-­lesional AD skin (P<0.0001 using the Wilcoxon matched-pairs signed-rank test). These findings were confirmed by RNA sequencing analysis, with TNFSF4 (OX40L) and TNFRSF4 (OX40) differentially expressed in lesional and non-lesional AD skin (P<0.01 and P<0.0001, respectively). A larger scale study is ongoing that may further support this finding. In the small-scale study including patients with mild AD and healthy volunteers, there was a trend for an increase of mean OX40L+ and OX40+ cells/mm2 in lesional skin versus non-lesional skin and healthy control skin in the dermis and epidermis (OX40L+ dermal lesional, non-lesional, healthy, respectively: 17.6, 11.2, 11.5; OX40L+ epidermal: 5.8, 2.1, 1.9; OX40+ dermal: 5.7, 2.1, 0.4; OX40+ epidermal: 10.9, 9.0, 1.8); the increase was significant for lesional versus healthy control skin for dermal OX40+ cells/mm2 (P<0.001). Conclusions We confirm with IHC and RNA sequencing that OX40L and OX40 expression is elevated in skin lesions of patients with moderate-to-severe AD. We also demonstrate a trend to elevated OX40L and elevated OX40 in mild AD skin versus healthy volunteers. A potential explanation for the lower levels of OX40L and OX40 in patients with mild AD in this study may be their lower disease severity versus Ph2a study patients. These findings support the OX40L/OX40 pathway as a relevant target in T cell immune dysregulation; its blockade may represent a novel treatment approach for moderate-to-severe AD.

The susceptibility of single nucleotide polymorphisms located within co-stimulatory pathways to systemic lupus erythematosus.

Autoimmune diseases result from the loss of immune tolerance, and they exhibit complex pathogenic mechanisms that remain challenging to effectively treat. It has been reported that the altered expression levels of co-stimulatory/inhibitory molecules will affect the level of T/B cell activation and lead to the loss of immune tolerance. In this study, we evaluated the gene polymorphisms of the ligand genes corresponding co-stimulatory system that were expressed on antigen-presenting cells (CD80, CD86, ICOSLG, and PDL1) from 60 systemic lupus erythematosus (SLE) patients and 60 healthy controls. The results showed that rs16829984 and rs57271503 of the CD80 gene and rs4143815 of the PDL1 gene were associated with SLE, in which the G-allele of rs16829984 (p=0.022), the A-allele of rs57271503 (p=0.029), and the GG and GC genotype of rs4143815 (p=0.039) may be risk polymorphisms for SLE. These SNPs are in the promoter and 3'UTR of the genes, so they may affect the transcription and translation activity of the genes, thereby regulating immune function and contributing to the development of SLE.

Comparison of immune-related gene signatures and immune infiltration features in early- and late-onset preeclampsia.

Preeclampsia, a severe pregnancy syndrome, is widely accepted divided into early- and late-onset preeclampsia (EOPE and LOPE) based on the onset time of preeclampsia, with distinct pathophysiological origins. However, the molecular mechanism especially immune-related mechanisms for EOPE and LOPE is currently obscure. In the present study, we focused on placental immune alterations between EOPE and LOPE and search for immune-related biomarkers that could potentially serve as potential therapeutic targets through bioinformatic analysis. The gene expression profiling data was obtained from the Gene Expression Omnibus database. ESTIMATE algorithm and Gene Set Enrichment Analysis were employed to evaluate the immune status. The intersection of differentially expressed genes in GSE74341 series and immune-related genes set screened differentially expressed immune-related genes. Protein-protein interaction network and random forest were used to identify hub genes with a validation by a quantitative real-time PCR. Kyoto Encyclopedia of Genes and Genomes pathways, Gene Ontology and gene set variation analysis were utilized to conduct biological function and pathway enrichment analyses. Single-sample gene set enrichment analysis and CIBERSORTx tools were employed to calculate the immune cell infiltration score. Correlation analyses were evaluated by Pearson correlation analysis. Hub genes-miRNA network was performed by the NetworkAnalyst online tool. Immune score and stromal score were all lower in EOPE samples. The immune system-related gene set was significantly downregulated in EOPE compared to LOPE samples. Four hub differentially expressed immune-related genes (IL15, GZMB, IL1B and CXCL12) were identified based on a protein-protein interaction network and random forest. Quantitative real-time polymerase chain reaction validated the lower expression levels of four hub genes in EOPE compared to LOPE samples. Immune cell infiltration analysis found that innate and adaptive immune cells were apparent lacking in EOPE samples compared to LOPE samples. Cytokine-cytokine receptor, para-inflammation, major histocompatibility complex class I and T cell co-stimulation pathways were significantly deficient and highly correlated with hub genes. We constructed a hub genes-miRNA regulatory network, revealing the correlation between hub genes and hsa-miR-374a-5p, hsa-miR-203a-3p, hsa-miR-128-3p, hsa-miR-155-3p, hsa-miR-129-2-3p and hsa-miR-7-5p. The innate and adaptive immune systems were severely impaired in placentas of EOPE. Four immune-related genes (IL15, GZMB, IL1B and CXCL12) were closely correlated with immune-related pathogenesis of EOPE. The result of our study may provide a new basis for discriminating between EOPE and LOPE and acknowledging the role of the immune landscape in the eventual interference and tailored treatment of EOPE.

TLR4 Ligands in Typhoid Vi Polysaccharide Subunit Vaccines Contribute to Immunogenicity.

Activation of B cells and T cells requires the engagement of costimulatory signaling pathways in addition to Ag receptor signaling for efficient immune responses. None of the typhoid Vi polysaccharide (ViPS) subunit vaccines contains adjuvants that could activate costimulatory signaling pathways, yet these vaccines are very immunogenic. I hypothesized that residual TLR ligands present in the ViPS preparation used for making typhoid subunit vaccines account for the robust immune response generated by these vaccines. I show the presence of endotoxin, a potent agonist of TLR4, in ViPS preparations and ViPS vaccines. Furthermore, I found that ViPS obtained from various sources induces the production of proinflammatory cytokines such as IL-6 from mouse peritoneal exudate cells. Unconjugated and tetanus toxoid-conjugated ViPS vaccines activate human and mouse TLR4. Mice deficient in TLR4 or the signaling adaptors MyD88 and Trif (Toll/IL-1R domain-containing adapter inducing IFN-β) are severely impaired in generating anti-ViPS responses to these vaccines. Elimination of the TLR4 agonist in ViPS preparation resulted in the loss of immunogenicity, and addition of lipid A, a known TLR4 agonist, restored the immunogenicity. These data highlight the importance of associated TLR ligands in the immunogenicity of ViPS subunit vaccines.