Memory-like differentiation enhances NK cell responses against colorectal cancer.

Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Spontaneous Akt2 deficiency in a colony of NOD mice exhibiting early diabetes.

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes.

Transient receptor potential vanilloid-1 (TRPV1) channels act as suppressors of the growth of glioma.

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 1 (TRPV1) in glioma. We found that the expression of TRPV1 mRNA and protein were upregulated in glioma compared with normal brain by qPCR and western blot analysis. In order to investigate the function of TRPV1 in glioma, short hairpin RNA (shRNA) and the inhibitor of TRPV1 were used. In vitro, the activation of TRPV1 induced cell apoptosis with decreased migration capability and inhibited proliferation, which was abolished upon TRPV1 pharmacological inhibition and silencing. Mechanistically, TRPV1 modulated glioma proliferation through the protein kinase B (Akt) signaling pathway. More importantly, in immunodeficient (NOD-SCID) mouse xenograft models, tumor size was significantly increased when TRPV1 expression was disrupted by a shRNA knockdown approach in vivo. Altogether, our findings indicate that TRPV1 negatively controls glioma cell proliferation in an Akt-dependent manner, which suggests that targeting TRPV1 may be a potential therapeutic strategy for glioma.

CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition.

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors.

Tumor Cell-Associated IL-1α Affects Breast Cancer Progression and Metastasis in Mice through Manipulation of the Tumor Immune Microenvironment.

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as an Effective Therapy for Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumors (DSRCT) are a type of aggressive, pediatric sarcoma characterized by the EWSR1::WT1 fusion oncogene. Targeted therapies for DSRCT have not been developed, and standard multimodal therapy is insufficient, leading to a 5-year survival rate of only 15% to 25%. Here, we depleted EWSR1::WT1 in DSRCT and established its essentiality in vivo. Transcriptomic analysis revealed that EWSR1::WT1 induces unique transcriptional alterations compared with WT1 and other fusion oncoproteins and that EWSR1::WT1 binding directly mediates gene upregulation. The E-KTS isoform of EWSR1::WT1 played a dominant role in transcription, and it bound to the CCND1 promoter and stimulated DSRCT growth through the cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. As palbociclib has been approved by the FDA for the treatment of breast cancer, these findings demonstrate the sensitivity of DSRCT to palbociclib and support immediate clinical investigation of palbociclib for treating this aggressive pediatric cancer. SIGNIFICANCE: EWSR1::WT1 is essential for desmoplastic small round cell tumors and upregulates the cyclin D-CDK4/6-RB axis that can be targeted with palbociclib, providing a targeted therapeutic strategy for treating this deadly tumor type.

Altering ß Cell Antigen Exposure to Exhausted CD8+ T Cells Prevents Autoimmune Diabetes in Mice.

Chronic destruction of insulin-producing pancreatic ß cells by T cells results in autoimmune diabetes. Similar to other chronic T cell-mediated pathologies, a role for T cell exhaustion has been identified in diabetes in humans and NOD mice. The development and differentiation of exhausted T cells depends on exposure to Ag. In this study, we manipulated ß cell Ag presentation to target exhausted autoreactive T cells by inhibiting IFN-γ-mediated MHC class I upregulation or by ectopically expressing the ß cell Ag IGRP under the MHC class II promotor in the NOD8.3 model. Islet PD-1+TIM3+CD8+ (terminally exhausted [TEX]) cells were primary producers of islet granzyme B and CD107a, suggestive of cells that have entered the exhaustion program yet maintained cytotoxic capacity. Loss of IFN-γ-mediated ß cell MHC class I upregulation correlated with a significant reduction in islet TEX cells and diabetes protection in NOD8.3 mice. In NOD.TII/8.3 mice with IGRP expression induced in APCs, IGRP-reactive T cells remained exposed to high levels of IGRP in the islets and periphery. Consequently, functionally exhausted TEX cells, with reduced granzyme B expression, were significantly increased in these mice and this correlated with diabetes protection. These results indicate that intermediate Ag exposure in wild-type NOD8.3 islets allows T cells to enter the exhaustion program without becoming functionally exhausted. Moreover, Ag exposure can be manipulated to target this key cytotoxic population either by limiting the generation of cytotoxic TIM3+ cells or by driving their functional exhaustion, with both resulting in diabetes protection.

De novo identification of CD4+ T cell epitopes.

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes.

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice.

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

Murine MHC-Deficient Nonobese Diabetic Mice Carrying Human HLA-DQ8 Develop Severe Myocarditis and Myositis in Response to Anti-PD-1 Immune Checkpoint Inhibitor Cancer Therapy.

Myocarditis has emerged as an immune-related adverse event of immune checkpoint inhibitor (ICI) cancer therapy associated with significant mortality. To ensure patients continue to safely benefit from life-saving cancer therapy, an understanding of fundamental immunological phenomena underlying ICI myocarditis is essential. We recently developed the NOD-cMHCI/II-/-.DQ8 mouse model that spontaneously develops myocarditis with lower mortality than observed in previous HLA-DQ8 NOD mouse strains. Our strain was rendered murine MHC class I and II deficient using CRISPR/Cas9 technology, making it a genetically clean platform for dissecting CD4+ T cell-mediated myocarditis in the absence of classically selected CD8+ T cells. These mice are highly susceptible to myocarditis and acute heart failure following anti-PD-1 ICI-induced treatment. Additionally, anti-PD-1 administration accelerates skeletal muscle myositis. Using histology, flow cytometry, adoptive transfers, and RNA sequencing analyses, we performed a thorough characterization of cardiac and skeletal muscle T cells, identifying shared and unique characteristics of both populations. Taken together, this report details a mouse model with features of a rare, but highly lethal clinical presentation of overlapping myocarditis and myositis following ICI therapy. This study sheds light on underlying immunological mechanisms in ICI myocarditis and provides the basis for further detailed analyses of diagnostic and therapeutic strategies.

Extracellular metallothionein as a therapeutic target in the early progression of type 1 diabetes.

Type 1 diabetes (T1D) is characterized by lymphocyte infiltration into the pancreatic islets of Langerhans, leading to the destruction of insulin-producing beta cells and uncontrolled hyperglycemia. In the nonobese diabetic (NOD) murine model of T1D, the onset of this infiltration starts several weeks before glucose dysregulation and overt diabetes. Recruitment of immune cells to the islets is mediated by several chemotactic cytokines, including CXCL10, while other cytokines, including SDF-1α, can confer protective effects. Global gene expression studies of the pancreas from prediabetic NOD mice and single-cell sequence analysis of human islets from prediabetic, autoantibody-positive patients showed an increased expression of metallothionein (MT), a small molecular weight, cysteine-rich metal-binding stress response protein. We have shown that beta cells can release MT into the extracellular environment, which can subsequently enhance the chemotactic response of Th1 cells to CXCL10 and interfere with the chemotactic response of Th2 cells to SDF-1α. These effects can be blocked in vitro with a monoclonal anti-MT antibody, clone UC1MT. When administered to NOD mice before the onset of diabetes, UC1MT significantly reduces the development of T1D. Manipulation of extracellular MT may be an important approach to preserving beta cell function and preventing the development of T1D.

An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes.

Introduction: The antigen-presenting cell function of insulin-reactive B cells promotes type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by stimulating pathogenic T cells leading to destruction of insulin-producing ß-cells of pancreatic islets. Methods/Results: To target insulin-reactive B cells, AKS-107, a human IgG1 Fc molecule fused with human insulin A and B chains, was engineered to retain conformational insulin epitopes that bound mouse and human B cell receptors but prevented binding to the insulin metabolic receptor. AKS-107 Fc-mediated deletion of insulin-reactive B cells was demonstrated via ex vivo and in vivo experiments with insulin-reactive B cell receptor transgenic mouse strains, VH125Tg/NOD and Tg125(H+L)/NOD. As an additional immune tolerance feature, the Y16A mutation of the insulin B(9-23) dominant T cell epitope was engineered into AKS-107 to suppress activation of insulin-specific T cells. In mice and non-human primates, AKS-107 was well-tolerated, non-immunogenic, did not cause hypoglycemia even at high doses, and showed an expectedly protracted pharmacokinetic profile. AKS-107 reproducibly prevented spontaneous diabetes from developing in NOD and VH125Tg/NOD mice that persisted for months after cessation of treatment, demonstrating durable immune tolerance. Discussion: These preclinical outcomes position AKS-107 for clinical development in T1D prevention settings.

Mapping of a hybrid insulin peptide in the inflamed islet ß-cells from NOD mice.

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of ß-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous ß-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within ß-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive ß-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within ß-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.

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

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

Irisin delays the onset of type 1 diabetes in NOD mice by enhancing intestinal barrier.

Type 1 diabetes (T1D), a complex autoimmune disease, is intricately linked to the gut's epithelial barrier function. Emerging evidence emphasizes the role of irisin, an exercise-related hormone, in preserving intestinal integrity. This study investigates whether irisin could delay T1D onset by enhancing the colon intestinal barrier. Impaired colon intestinal barriers were observed in newly diagnosed T1D patients and non-obese diabetic (NOD) mice, worsening with age and accompanied by islet inflammation. Using an LPS-induced colonic inflammation model, a dose-dependent impact of LPS on colon cells irisin expression, secretion, and barrier function was revealed. Exogenous irisin demonstrated remarkable effects, mitigating islet insulitis, enhancing energy expenditure, and alleviating autoimmune symptoms by reducing colon intestinal permeability. Single-cell RNA sequencing (scRNA-seq) highlighted irisin's positive impact on colon epithelial cell clusters, effectively restoring the intestinal barrier. Irisin also selectively modulated bacterial composition, averting potential bacterial translocation. Mechanistically, irisin enhanced colon intestinal barrier tight junction proteins through the AMPK/PI3K/AKT pathway, with FAM120A playing a crucial role. Irisin upregulated MUC3 expression, a protector against damage and inflammation. Harnessing irisin's exercise-mimicking properties suggests therapeutic potential in clinical settings for preventing T1D progression, offering valuable insights into fortifying the colon's intestinal barrier and managing autoimmune conditions associated with T1DM.

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

TLR5-deficiency controls dendritic cell subset development in an autoimmune diabetes-susceptible model.

Introduction: The incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D. Methods: To fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study. Results: We found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice. Discussion: In summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.