Immune Inhibitory Molecules Research Articles

HLA-G neo-expression modifies genetic programs governing tumor cell lines

The development of immunotherapies has proved to be clinically encouraging to re-establish the immune function modified by the expression of immune inhibitory molecules in tumors. However, there are still patients with poor survival rates following treatment. The elucidation of molecular mechanisms triggered by the neo-expression of particular IC in tumors would constitute a major step toward better understanding tumor evolution and would help to design future clinical protocols. To this end, we investigate the modifications triggered by the neo-expression of the immune checkpoints HLA-G in ccRCC tumor cells. We demonstrate, for the first time, that HLA-G modifies key genes implicated mainly in tumor development, angiogenesis, calcium flow and mitochondria dynamics. The involvement of HLA-G on the expression of genes belonging to these pathways such as ADAM-12, NCAM1 and NRP1 was confirmed by the CRISPR/Cas9-mediated edition of HLA-G. The data reveal multifaceted roles of HLA-G in tumor cells which are far beyond the well-known function of HLA-G in the immune anti-tumor response. This warrants further investigation of HLA-G and these new partners in tumors of different origin so as to propose future new treatments to improve health patient’s outcome.

Dual CAR-T cell therapy for glioblastoma: strategies to cure tumour diseases based on a mathematical model

AbstractThe CAR-T cell immunotherapy entails the genetic reprogramming of T-lymphocytes, which then engage with cancer cells, triggering an anti-tumour immune response. While this treatment has gained approval for hematological cancers, addressing solid tumours presents new obstacles. Challenges include the heterogeneity of antigen expression within solid tumours, encompassing antigen-positive non-tumoural cells, the presence of immune inhibitory molecules, and the difficulty of CAR-T cell trafficking within the tumour microenvironment. In this article, we analytically study a generalisation of a mathematical model proposed by León-Triana et al. (Cancers 13(4):703, 2021a. https://doi.org/10.3390/cancers13040703, Commun Nonlinear Sci Numer Simul 94:105570). This model focuses on the dynamics of glioblastoma, the most aggressive brain tumour, and its response to CAR-T cell treatment. We study the basic properties of the model, the dynamics of the solutions of the model when the treatment is not sustained during the time, and finally we study analytically the model when the therapy is constant, periodic and/or impulsive. We derive sufficient conditions for global stability of tumour-free equilibrium, as well as necessary and sufficient conditions for local stability of the equilibrium obtaining conditions for an effective treatment. Finally, we perform different numerical simulations to find the strategies to keep the tumour under control. The obtained results are based on a combination of different analytical techniques in differential equations, dynamical systems and numerical simulations.

Altered characteristics of regulatory T cells in target tissues of Sjögren’s syndrome in murine models

Sjӧgren’s syndrome (SS), also known as Sjögren’s disease, is a chronic autoimmune condition predominantly affecting the salivary and lacrimal glands. The disease is driven by autoimmune responses involving the activation and actions of major innate- and adaptive immune cell subsets. However, the specific characteristics and roles of regulatory T cells (Tregs) in SS remain elusive. This study seeks to clarify the main phenotypic and functional attributes of Tregs in the salivary glands and their draining lymph nodes in murine models of SS. Our flow cytometric analysis revealed that Tregs in the salivary gland-draining lymph nodes of female non-obese diabetic (NOD) mice, a spontaneous model of SS, exhibited a greater proportion of activated Tregs and fewer resting Tregs compared to Balb/c mice. Furthermore, Tregs from the salivary gland-draining lymph nodes of female C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec) mice, a model for primary SS, demonstrated significantly lower IL-10 production but markedly higher IFNγ- and IL-17 production than their C57BL/6 counterparts. Additionally, treatment of C57BL/6 Tregs with IL-7, a cytokine critical for SS pathogenesis, resulted in diminished IL-10 production and enhanced IFNγ and IL-17 production in these cells. Notably, the alterations in B6.NOD-Aec Tregs also included an increased expression of the immune-inhibitory molecule CTLA-4 compared to the C57BL/6 Tregs. Intriguingly, in vitro co-cultures of Tregs with conventional CD4 T cells and other key immune populations from lymph nodes indicated that Tregs from salivary gland-draining lymph nodes of both B6.NOD-Aec and C57BL/6 strains exhibited comparable and limited immunosuppressive effects on the proliferation and function of conventional CD4 T cells. The ability of B6.NOD-Aec Tregs to directly inflict damages to salivary gland epithelial tissues and contribute to SS pathologies through IFNγ and IL-17 that they produce warrants further investigations. In addition, enhancing the relatively weak immunosuppressive capacities of these Tregs may also serve as a viable strategy to alleviate the SS phenotype in the mouse models and potentially in patients.

Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.

Immunoprevention is an emerging consideration for solid tumors, including pancreatic ductal adenocarcinoma (PDAC). We and others have shown that Kras mutations in genetic models of spontaneous pancreatic intraepithelial neoplasia (PanIN), which is a precursor to PDAC, results in CD73 expression in the neoplastic epithelium and some populations of infiltrating immune cells, including macrophages and CD8 T cells. CD73 is an ecto-enzyme that converts extracellular adenosine monophosphate to adenosine, a critical immune inhibitory molecule in PDAC. We hypothesized inhibition of CD73 would reduce the incidence of PanIN formation and alter the immune microenvironment. To test our hypothesis, we used the KrasG12D; PdxCre1 (KC) genetically engineered mouse model and tested the utility of AB-680, a small molecule inhibitor targeting CD73, to inhibit PanIN progression. AB-680, or vehicle control, was administered using oral gavage delivery 3 days/week at 10 mg/kg, beginning when the mice were 2 months old and lasting 3 months. We euthanized the mice at 5 months old. In the KC model, we quantified significantly less pancreatitis, early and advanced PanIN, and quantified a significant increase in M1 macrophages in AB-680-treated mice. Single-cell RNA sequencing (scRNA-seq) of pancreata of AB-680-treated mice revealed increased infiltration of CD4+ T cells, CD8+ T cells, and mature B cells. The scRNA-seq analysis showed that CD73 inhibition reduced M2 macrophages, acinar, and PanIN cell populations. CD73 inhibition enhanced immune surveillance and expanded unique clonotypes of TCR and BCR, indicating that inhibition of CD73 augments adaptive immunity early in the neoplastic microenvironment. Prevention Relevance: Previous studies found PanIN lesions in healthy pancreata. Not all progress to PDAC, suggesting a window for enhanced antitumor immunity through immunoprevention therapy. CD73 inhibition in our study prevents PanIN progression, reduces immune-suppressive macrophages and expands TCR and BCR unique clonotypes, highlighting an encouraging therapeutic avenue for high-risk individuals.

CD163+ macrophages in mantle cell lymphoma induce activation of prosurvival pathways and immune suppression

AbstractMantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME) in which infiltration of CD163+ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163+ cells are associated with the biology of MCL, using spatial multiomic investigations of tumor and infiltrating CD163+ and CD3+ cells. A total of 63 proteins were measured using GeoMx digital spatial profiling in tissue microarrays from 100 diagnostic MCL tissues. Regions of interest were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163+ macrophages, CD20+ MCL cells, and CD3+ T-cells was performed. To validate protein profiles, 1811 messenger RNAs were measured in CD20+ cells and 2 subsets of T cells. Image analysis was used to extract the phenotype and position of each targeted cell, thereby allowing the exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163+ cells modulate their immune profile depending on their localization and that the immune inhibitory molecules, V-domain immunoglobulin suppressor of T-cell activation and B7 homolog 3, have higher expression in tumor-sparse than in tumor-rich tissue regions and that targeting should be explored. We showed that MCL tissues with more abundant infiltration of CD163+ cells have a higher proteomic and transcriptional expression of key components of the MAPK pathway. Thus, the MAPK pathway may be a feasible therapeutic target in patients with MCL with CD163+ cell infiltration. We further showed the independent and combined prognostic values of CD11c and CD163 beyond established risk factors.

Abstract LB076: Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer

Abstract Purpose: Despite receiving great attention as a promising treatment, therapeutic efficacies induced by immune checkpoint inhibitors are low in clinical settings, and thus there are high expectations for the development of innovative diagnostic and therapeutic methods. We previously demonstrated that blocking FSTL1 overcomes anti-PD1 resistance in aged mice. In this study, we comprehensively analyzed and compared gene expressions in splenic T cells harvested from young and old mice receiving anti-FSTL1 or anti-PD1 therapy after tumor implantation using the GeneChip microarray in order to identify a key gene in the anti-PD1 resistance. In addition, we evaluated anti-tumor efficacy induced by blocking the identified molecule using aged tumor models and tumor metastasis models (3LL or Colon26), which are resistant to anti-PD1 therapy. Furthermore, to validate the clinical relevancy, we analyzed immune cells isolated from malignant tumor ascites and peripheral blood cells of gastric cancer patients by flow cytometry. Results: Using the GeneChip microarray data of splenic T cells, we identified α-synuclein (SNCA), which is a neuropathological molecule involved in Parkinson's disease and other synucleinopathies, as a gene that was significantly reduced by anti-FSTL1 therapy, but not anti-PD1 therapy, in splenic T cells of aged tumor models. SNCA+ cell subsets significantly increased not only in CD3+ T cells, but also CD11b+ myeloid cells and CD45- mesenchymal stromal/stem cells, within spleen, peripheral blood, and tumor tissues of aged mice and mouse tumor models, but rarely in those of young and naive mice. Increase of the subsets were also observed in malignant tumor ascites and peripheral blood of gastric cancer patients, but not those of healthy donors. Blocking SNCA with the specific mAb significantly induced potent CD8+ T cells with greater cytotoxicity in the in vitro CTL induction setting, and also significantly induced robust anti-tumor immunity in the in vivo therapeutic setting with anti-PD1-resistant tumor models. Combination of anti-SNCA therapy successfully elicited anti-PD1 therapeutic efficacy, providing significantly better prognosis in many tumor models, including severe tumor ascites models. Conclusions: These results suggest that SNCA is a type of immune inhibitory checkpoint molecules and an immunological determinant of anti-PD1 resistance, although the further study is needed to clarify the molecular functions in immune cells. Targeting this molecule may represent a promising strategy to overcome the anti-PD1 resistance and contribute to improved clinical outcomes in cancer treatment. Citation Format: Chie Kudo-Saito, Hiroshi Imazeki, Hirokazu Shoji, Narikazu Boku. Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB076.

Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N-Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor.

The adaptive transfer of T cells redirected to cancer cells via chimeric Ag receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to solid cancer, we screened CARs targeting surface Ags on human lung cancer cells using (to our knowledge) novel expression cloning based on the Ag receptor-induced transcriptional activation of IL-2. Isolated CARs were directed against fragile X mental retardation 1 neighbor (FMR1NB), a cancer-testis Ag that is expressed by malignant cells and adult testicular germ cells. Anti-FMR1NB CAR human T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in mouse xenograft models of lung cancer. Furthermore, to protect CAR T cells from immune-inhibitory molecules, which are present in the tumor microenvironment, we introduced anti-FMR1NB CARs into 2-deoxy-glucose (2DG)-treated human T cells. These cells exhibited reduced binding affinity to immune-inhibitory molecules, and the suppressive effects of these molecules were resisted through blockade of the N-glycosylation of their receptors. Anti-FMR1NB CARs in 2DG-treated human T cells augmented target-specific cytotoxicity invitro and invivo. Thus, our findings demonstrated the feasibility of eradicating lung cancer cells using 2DG-treated human T cells, which are able to direct tumor-specific FMR1NB via CARs and survive in the suppressive tumor microenvironment.

Exosome-Derived Cargos in Immune Microenvironment in Esophageal Carcinoma: A Mini-Review.

Esophageal carcinoma, a lethal malignancy with limited treatment options and poor prognosis, necessitates understanding its underlying mechanisms and identifying novel therapeutic targets. Recent studies have highlighted the critical role of the immune microenvironment in esophageal carcinoma, particularly the interplay between tumor cells and immune cells mediated by exosomes and their cargos. Exosomes, small extracellular vesicles secreted by various cells, including tumor cells, facilitate intercellular communication by transferring bioactive molecules such as proteins, nucleic acids, and lipids to recipient cells. In the context of esophageal carcinoma, tumor-derived exosomes have been shown to play a significant role in shaping the immune microenvironment. In esophageal carcinoma, exosomal cargos have been found to modulate immune cell function and impact tumor progression. These cargos can carry immune inhibitory molecules, such as programmed death-ligand 1 (PD-L1), to suppress T-cell activity and promote immune evasion by tumor cells. Furthermore, exosomal cargos can activate antigen- presenting cells, enhancing their ability to present tumor-specific antigens to T cells and thereby promoting anti-tumor immune responses. Additionally, the cargos of exosomes have been implicated in the induction of immune regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within the esophageal carcinoma microenvironment. These immunosuppressive effectors inhibit the activity of T cells, contributing to tumor immune evasion and resistance to immune therapies. In summary, exosomes and their cargo play a crucial role in the immune microenvironment of esophageal carcinoma. Understanding the mechanisms by which exosomal cargos regulate immune cell function and tumor progression may reveal novel therapeutic targets for this devastating disease.

Comprehensive Immunophenotyping by Polychromatic Cytometry.

The human immune system comprises a diverse array of cells involved in innate and adaptive immunity, and these immune cells coordinate immune responses against pathogens through intricate interactions. Multicolor flow cytometry is a powerful technique for qualitatively and quantitatively measuring the characteristics of immune cells, offering advantages, such as high-dimensional analysis, elucidation of cellular heterogeneity, understanding of pathogenesis, development of therapeutic strategies, and platform flexibility. Here, we demonstrate a new immunophenotyping panel that allows simultaneous evaluation of the characteristics of T and B cells. This panel enables tracking of changes in the immune status due to aging, environmental factors, pathogen infections, and vaccine administration. Additionally, it includes co-stimulatory molecules for assessing the activation state of immune cells and inhibitory checkpoint molecules for evaluating exhaustion status, thereby providing valuable insights into the features of human immune responses. These analyses contribute to understanding the pathophysiology of diseases and developing therapeutic strategies while offering crucial information for assessing the correlation of symptoms with infections and evaluating the efficacy of vaccines.

Induction of local immunosuppression in allogeneic cell transplantation by cell-type-specific expression of PD-L1 and CTLA4Ig

Immune rejection has long hindered allogeneic cell transplantation therapy. Current genetic modification approaches, including direct targeting of major histocompatibility complex or constitutive expression of immune inhibitory molecules, exhibit drawbacks such as severe adverse effects or elevated tumorigenesis risks. To overcome these limitations, we introduce an innovative approach to induce cell-type-specific immune tolerance in differentiated cells. By engineering human embryonic stem cells, we ensure the exclusive production of the immune inhibitory molecules PD-L1/CTLA4Ig in differentiated cells. Using this strategy, we generated hepatocyte-like cells expressing PD-L1 and CTLA4Ig, which effectively induced local immunotolerance. This approach was evaluated in a humanized mouse model that mimics the human immune system dynamics. We thus demonstrate a robust and selective induction of immunotolerance specific to hepatocytes, improving graft survival without observed tumorigenesis. This precise immune tolerance strategy holds great promise for advancing the development of stem cell-based therapeutics in regenerative medicine.

The tumor microenvironment generated by Marek’s disease virus suppresses interferon-gamma-producing gamma delta T cells

The tumor microenvironment (TME) is generated by the cross-talk among tumor cells, immune system cells, and stromal cells. The TME generated by Marek’s disease virus (MDV) is suggested to display an immunosuppressive milieu due to immune inhibitory molecules and cytokines which are possibly induced by MDV-transformed cells and regulatory T cells. Both anti-tumor and pro-tumor gamma delta (γδ) T cells are reported in human cancer. Although anti-tumor like and pro-tumor like γδ T cells are found in MDV-infected chickens at the later phase of infection, how the TME affects circulating and tissue-resident γδ T cells has not been investigated. Here, we demonstrated that the supernatant of the cultured splenocytes derived from MDV-challenegd chickens inhibited interferon (IFN)-γ production and CD25 expression by T cell receptor (TCR)γδ-stimulated tissue-resident γδ T cells, but the supernatant of the cultured MDV-transformed cell line did not affect γδ T cell activation. TCRγδ-stimulated circulating γδ T cells were influenced neither by the supernatant of the cultured splenocytes derived from MDV-challenegd chickens nor by the supernatant of the cultured MDV-transformed cell line. Taken together, activation and IFN-γ production by tissue-resident γδ T cells can be inhibited in the TME generated by MDV while tumor attracted circulating γδ T cells may not be influenced in activation and IFN-γ production by the TME generated by MDV.

Expression of HPV-16 E6 and E7 oncoproteins alters Chlamydia trachomatis developmental cycle and induces increased levels of immune regulatory molecules.

Infection with Human Papillomavirus (HPV) is a recognized risk factor for Chlamydia trachomatis (CT) infection and vice versa. Coinfection of HPV and CT in women is a very common and usually asymptomatic finding that has been linked to increased risk of cervical cancer. It has been demonstrated that CT facilitates the entry of multiple high risk HPV genotypes, leading to damage of the mucosal barrier and interfering with immune responses and viral clearance, which ultimately favours viral persistence and malignant transformation. Although the facilitating effects elicited by CT infection on viral persistence have been reported, little is known about the consequences of HPV infection on CT development. Herein, we took advantage of a genetically modified human cervical cell line co-expressing HPV-16 major oncogenic proteins E6 and E7, as an experimental model allowing to investigate the possible effects that HPV infection would have on CT development. Our results show that CT infection of HPV-16 E6E7 expressing cells induced an upregulation of the expression of E6E7 oncoproteins and host cell inhibitory molecules PD-L1, HVEM and CD160. Additionally, smaller chlamydial inclusions and reduced infectious progeny generation was observed in E6E7 cells. Ultrastructural analysis showed that expression of E6 and E7 did not alter total bacterial counts within inclusions but resulted in increased numbers of reticulate bodies (RB) and decreased production of infectious elementary bodies (EB). Our results indicate that during CT and HPV coinfection, E6 and E7 oncoproteins impair RB to EB transition and infectious progeny generation. On the other hand, higher expression of immune inhibitory molecules and HPV-16 E6E7 are cooperatively enhanced in CT-infected cells, which would favour both oncogenesis and immunosuppression. Our findings pose important implications for clinical management of patients with HPV and CT coinfection, suggesting that screening for the mutual infection could represent an opportunity to intervene and prevent severe reproductive health outcomes, such as cervical cancer and infertility.

Acquired resistance to anti-PD1 therapy in patients with NSCLC associates with immunosuppressive T cell phenotype

Immune checkpoint inhibitor treatment has the potential to prolong survival in non-small cell lung cancer (NSCLC), however, some of the patients develop resistance following initial response. Here, we analyze the immune phenotype of matching tumor samples from a cohort of NSCLC patients showing good initial response to immune checkpoint inhibitors, followed by acquired resistance at later time points. By using imaging mass cytometry and whole exome and RNA sequencing, we detect two patterns of resistance¨: One group of patients is characterized by reduced numbers of tumor-infiltrating CD8+ T cells and reduced expression of PD-L1 after development of resistance, whereas the other group shows high CD8+ T cell infiltration and high expression of PD-L1 in addition to markedly elevated expression of other immune-inhibitory molecules. In two cases, we detect downregulation of type I and II IFN pathways following progression to resistance, which could lead to an impaired anti-tumor immune response. This study thus captures the development of immune checkpoint inhibitor resistance as it progresses and deepens our mechanistic understanding of immunotherapy response in NSCLC.

Immune Inhibitory Molecule PD-1 Homolog (VISTA) Colocalizes with CD11b Myeloid Cells in Melanoma and Is Associated with Poor Outcomes

Tumors evade immunity through the overexpression of immune inhibitory molecules in the tumor microenvironment such as PD-L1/B7-H1. An immune inhibitory molecule named PD-1 homolog (also known as V-domain Ig-containing suppressor of T cell activation [VISTA]) functions to control both T cells and myeloid cells. Current clinical trials using anti-VISTA-blocking agents for treatment of cancer are ongoing. We sought to determine the extent of VISTA expression in primary cutaneous melanomas (n= 190), identify the critical cell types expressing VISTA, and correlate its expression with PD-L1 expression using multiplexed quantitative immunofluorescence. Within the tumor subcompartments, VISTA is most highly expressed on CD11b myeloid cells, and PD-L1 is most highly expressed on CD68 myeloid cells in our melanoma cohort. There is little correlation between VISTA and PD-L1 expression intensity, suggesting that individual tumors have distinct immunosuppressive tumor microenvironments. High levels of VISTA expression on CD11b myeloid cells but not PD-L1 expression were associated with greater melanoma recurrence and greater all-cause mortality. Our findings suggest that cell-specific VISTA expression may be a negative prognostic biomarker for melanoma and a future potential therapeutic target.

Programming Injectable DNA Hydrogels Yields Tumor Microenvironment-Activatable and Immune-Instructive Depots for Augmented Chemo-Immunotherapy.

Injectable hydrogels have attracted increasing attention for promoting systemic antitumor immune response through the co-delivery of chemotherapeutics and immunomodulators. However, the biosafety and bioactivity of conventional hydrogel depots are often impaired by insufficient possibilities for post-gelling injection and means for biofunction integration. Here, an unprecedented injectable stimuli-responsive immunomodulatory depot through programming a super-soft DNAhydrogel adjuvant is reported. This hydrogel system encoded with adenosine triphosphate aptamers can be intratumorally injected in a gel formulation and then undergoes significant molecular conformation change to stimulate the distinct release kinetics of co-encapsulated therapeutics. In this scenario, doxorubicinis first released to induce immunogenic cell death that intimately works together with the polymerized cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) in gel scaffold for effectively recruiting and activating dendritic cells. The polymerized CpG ODN not only enhances tumor immunogenicity but minimizes free CpG-induced splenomegaly. Furthermore, the subsequently released anti-programmed cell death protein ligand 1 (aPDL1) blocks the corresponding immune inhibitory checkpoint molecule on tumor cells to sensitize antitumor T-cell immunity. This work thus contributes to the first proof-of-concept demonstration of a programmable super-soft DNA hydrogel system that perfectly matches the synergistic therapeutic modalities based on chemotherapeutic toxicity, in situ vaccination, and immune checkpoint blockade.

Tryptophan metabolism promotes immune evasion in human pancreatic β cells

To resist the autoimmune attack characteristic of type 1 diabetes, insulin producing pancreatic β cells need to evade T-cell recognition. Such escape mechanisms may be conferred by low HLA class I (HLA-I) expression and upregulation of immune inhibitory molecules such as Programmed cell Death Ligand 1 (PD-L1). The expression of PD-L1, HLA-I and CXCL10 was evaluated in the human β cell line, ECN90, and in primary human and mouse pancreatic islets. Most genes were determined by real-time RT-PCR, flow cytometry and Western blot. Activator and inhibitor of the AKT signaling were used to modulate PD-L1 induction. Key results were validated by monitoring activity of CD8+ Jurkat T cells presenting β cell specific T-cell receptor and transduced with reporter genes in contact culture with the human β cell line, ECN90. In this study, we identify tryptophan (TRP) as an agonist of PD-L1 induction through the AKT signaling pathway. TRP also synergistically enhanced PD-L1 expression on β cells exposed to interferon-γ. Conversely, interferon-γ-mediated induction of HLA-I and CXCL10 genes was down-regulated upon TRP treatment. Finally, TRP and its derivatives inhibited the activation of islet-reactive CD8+ T cells by β cells. Collectively, our findings indicate that TRP could induce immune tolerance to β cells by promoting their immune evasion through HLA-I downregulation and PD-L1 upregulation. Dutch Diabetes Research Foundation, DON Foundation, the Laboratoire d'Excellence consortium Revive (ANR-10-LABX-0073), Agence Nationale de la Recherche (ANR-19-CE15-0014-01), Fondation pour la Recherche Médicale (EQ U201903007793-EQU20193007831), Innovative Medicines InitiativeINNODIA and INNODIA HARVEST, Aides aux Jeunes Diabetiques (AJD) and Juvenile Diabetes Research Foundation Ltd (JDRF).

O-265 Increased CD8+ T-cell exhaustion due to high NKG2A/HLA-E engagement and IL-15 induction in adenomyosis

Abstract Study question Is there dysregulation of immune cells in the local uterine microenvironment of patients with adenomyosis? If any, what’s the underlying mediators and mechanisms? Summary answer Increased CD8+ T-cell exhaustion due to enhanced HLA-E/NKG2A engagement and IL-15 induction in ectopic adenomyotic lesions existed in human patients and mice models with adenomyosis. What is known already The immunological dysfunction has long been considered in the pathogenesis of adenomyosis. However, high heterogeneity in immune cell alterations and contradictory results were reported due to the technique limitation of immunohistochemistry mainly used in previous studies. A comprehensive picture of the dysregulation of immune cells in the local uterine microenvironment of patients with adenomyosis is still lacking. Specially, increased number of CD8+ T cells have been reported in adenomyotic patients, whether functional deficiency of CD8+ T cells may contribute to the abnormal accumulation and residency of ectopic endometrial cells at pathological sites remains unknown. Study design, size, duration The paired ectopic lesions and eutopic endometrium from 80 patients with adenomyosis, and normal endometrium and myometrium from 62 patients with uterine myoma (controls) were collected between 2021 to 2022. Mice models with adenomyosis by neonatal tamoxifen treatment were established for experiments in 2022. Participants/materials, setting, methods The alteration of immune subsets, NKG2A expression, exhausted phenotypes and CD8+ T-cell dysfunction by flow cytometry, the expression/localization of HLA-E and IL-15 by immunohistochemistry were investigated in uterine tissues of patients and mice models with adenomyosis. CD8+ T cells were sorted, and in-vitro cultured with recombinant human transforming growth factor-β (rhTGF-β) or rhIL-15 to determine the NKG2A expression. Correlation analyses were conducted for association between immune disturbances and disease severity (dysmenorrhea, local/diffuse lesion, CA125 level). Main results and the role of chance We found that an increase in CD8+ T cell number was the predominant alteration in ectopic lesions in patients with adenomyosis, and it was significantly associated with the severity of adenomyosis. For CD8+ T cells in adenomyotic lesions, the expression of NKG2A was positively correlated with CD94 expression, and CD94/NKG2A mainly enriched on CD103+ tissue resident CD8+ T subsets. The NKG2A+CD8+ T cells showed exhausted phenotypes characterized by co-expression of multiple immune inhibitory molecules (PD1, LAG3, TIM3, and TOX) and decreased degranulation capability demonstrated by reduced expression of perforin, granzyme B, and CD107. Importantly, the exhausted NKG2A+CD8+ T-cell subset was associated with disease severity and was significantly increased in ectopic uterine tissues of human patients and mice models with adenomyosis. The expression of NKG2A on CD8+ T cells was markedly upregulated by exogenous rhIL-15 but not rhTGF-β treatment. Additionally, we also found increased co-expression of IL-15 and the NKG2A ligand HLA-E on the glandular epithelial cells, where the NKG2A+CD8+ T cells were closely distributed around the gland, in ectopic adenomyotic microenvironment of patients and mice models with adenomyosis. Limitations, reasons for caution The control uterine samples were collected from patients with uterine myoma. Although they were pathologically confirmed as normal, the potential effects are unknown and cannot be excluded. Further studies in larger independent populations and exploration on the functional significance of CD8+ T-cell exhaustion in adenomyosis pathogenesis is warranted. Wider implications of the findings Our study reveals a previously unrecognized role for CD8+ T-cell exhaustion in the pathogenesis of adenomyosis and suggests that therapeutic interventions targeting and reinvigorating exhausted CD8+ T cells by NKG2A blockade may be beneficial for patients with adenomyosis. Trial registration number not applicable

Assessment of tumor microenvironment expression and clinical significance of immune inhibitory molecule CTLA-4, ligand B7-1, and tumor-infiltrating regulatory cells in Hodgkin lymphoma.

Classical Hodgkin lymphoma represents a paradigm of tumor cell-microenvironment interactions as the neoplastic Hodgkin Reed-Sternberg (HRS) cells typically constitute less than 1% of the total tumor volume. CTLA-4, a member of the CD28/B7 immunoglobulin superfamily, and CD28 and their ligands B7-1 and B7-2 are critically important for the initial activation of naive T cells. Strategies aimed at interfering with the crosstalk between tumoral Reed-Sternberg cells and their cellular partners have been taken into account in the development of new immunotherapies that target different cell components of the HL microenvironment. The study included 50 histopathological confirmed cases of Hodgkin lymphoma. IHC staining for CTLA-4 and B7-1 was performed on archival paraffin-embedded biopsy. SPSS version 17 was used for statistical analysis. CTLA-4 IHC expression in HRS cells was negative in all cases, while in immune cells, CTLA-4 expression was observed in 45 (90%) cases. CD80 expression was present in all cases, both in HRS and immune cells. There was a significant association between HRS cell percentage and IPS score (p-value=0.001). Mean survival duration was longer in <50% immune cells compared to >50% groups, with an overall mean survival of 67.633 months. Considering the CTLA4 expression in immune cells within the microenvironment and the availability of targeted drugs like Iplimumab, which act through CTLA4 blockade, it may be appropriate to use this as targeted therapy in HL cases, particularly in those with refractory disease who are unable to achieve cure prior to ASCT.

Tim-3 blockade enhances the clearance of Chlamydia psittaci in the lung by promoting a cell-mediated immune response

Chlamydia psittaci is remarkable at disrupting immunity and thus poses a great risk to the animal industry and public health. Immune inhibitory molecule upregulation and the accumulation of specialized cells play key roles in chlamydial clearance. It is clear that the T-cell immunoglobulin and mucin domain protein 3 receptor (Tim-3) can regulate effector T cells in infectious disease. However, the immunomodulatory effect of Tim-3 in C. psittaci infection remains unknown. Thus, the expression of Tim-3 in effector T cells and its immune regulatory function during C. psittaci infection were investigated. The level of Tim-3 on CD4+ and CD8+ T cells was meaningfully higher in C. psittaci-infected mice. Blockade of Tim-3 signaling by anti-Tim-3 antibody showed accelerated C. psittaci clearance and less pathological changes in the lung than isotype immunoglobulin treatment. Furthermore, treatment with anti-Tim-3 antibody greatly enhanced the levels of IFN-γ and interleukin (IL)-22/IL-17, which were correlated with an improved Th1- and Th17-mediated immune response, and decreased IL-10, which were related with a decreased Treg immune response. In conclusion, Tim-3 expression in effector T cells negatively regulates Th1 and Th17 immune responses against C. psittaci respiratory infection.

Advanced T and Natural Killer Cell Therapy for Glioblastoma.

Although immunotherapy has been broadly successful in the treatment of hematologic malignancies and a subset of solid tumors, its clinical outcomes for glioblastoma are still inadequate. The results could be due to neuroanatomical structures such as the bloodbrain- barrier, antigenic heterogeneity, and the highly immunosuppressive microenvironment of glioblastomas. The antitumor efficacy of endogenously activated effector cells induced by peptide or dendritic cell vaccines in particular has been insufficient to control tumors. Effector cells, such as T cells and natural killer (NK) cells can be expanded rapidly ex vivo and transferred to patients. The identification of neoantigens derived from tumor-specific mutations is expanding the list of tumor-specific antigens for glioblastoma. Moreover, recent advances in gene-editing technologies enable the effector cells to not only have multiple biological functionalities, such as cytokine production, multiple antigen recognition, and increased cell trafficking, but also relieve the immunosuppressive nature of the glioblastoma microenvironment by blocking immune inhibitory molecules, which together improve their cytotoxicity, persistence, and safety. Allogeneic chimeric antigen receptor (CAR) T cells edited to reduce graft-versushost disease and allorejection, or induced pluripotent stem cell-derived NK cells expressing CARs that use NK-specific signaling domain can be a good candidate for off-the-shelf products of glioblastoma immunotherapy. We here discuss current progress and future directions for T cell and NK cell therapy in glioblastoma.