T-cell Signaling Research Articles

Predicting protein interactions of the kinase Lck critical to T cell modulation

Protein-protein interactions (PPIs) play pivotal roles in directing Tcell fate. One key player is the non-receptor tyrosine protein kinase Lck that helps to transduce Tcell activation signals. Lck is mediated by other proteins via interactions that are inadequately understood. Here, we use the deep learning method AF2Complex to predict PPIs involving Lck, by screening it against ∼1,000 proteins implicated in immune responses, followed by extensive structural modeling for selected interactions. Remarkably, we describe how Lck may be specifically targeted by a palmitoyltransferase using a phosphotyrosine motif. We uncover "hotspot" interactions between Lck and the tyrosine phosphatase CD45, leading to a significant conformational shift of Lck for activation. Lastly, we present intriguing interactions between the phosphotyrosine-binding domain of Lck and the cytoplasmic tail of the immune checkpoint LAG3 and propose a molecular mechanism for its inhibitory role. Together, this multifaceted study provides valuable insights into Tcell regulation and signaling.

Unveiling the Innate and Adaptive Immunity Interplay: Global Transcriptomic Profiling of the Host Immune Response in Candida albicans Endophthalmitis in a Murine Model.

Intraocular fungal infection poses a significant clinical challenge characterized by chronic inflammation along with vision impairment. Understanding the host defense pathways involved in fungal endophthalmitis will play a pivotal role in identifying adjuvant immunotherapy. Clinical isolates of Candida albicans (15,000 CFU/μL) were intravitreally injected in C57BL/6 mice followed by enucleation at 24 and 72 h postinfection. Histopathological analysis was performed to evaluate the retinal changes and the disease severity. RNA-seq analysis was conducted on homogenized eyeballs to assess the relevant gene profiles and their differentially expressed genes (DEGs). Pathway enrichment analysis was performed to further annotate the functions of the DEGs. Histopathological analysis demonstrated a higher disease severity with increased inflammatory cells at 72 hpi and transcriptome analysis revealed 27,717 DEGs, of which 1493 were significant (adj p value ≤0.05, FC ≥ 1.5). Among these, 924 were upregulated, and 569 were downregulated. Majority of the upregulated genes were associated with the inflammatory/host immune response and signal transduction and enriched in the T-cell signaling pathway, natural killer cell-mediated cytotoxicity, C-type receptor signaling pathway, and NOD-like receptor signaling pathway. Furthermore, inflammation-associated genes such as T-cell surface glycoprotein CD3, cathelicidin antimicrobial peptide, and lymphocyte cell-specific protein tyrosine kinase were enriched, while pathways such as MAPK, cAMP, and metabolic pathways were downregulated. Regulating the T-cell influx could be a potential strategy to modulate excessive inflammation in the retina and could potentially aid in better vision recovery in fungal endophthalmitis.

Development of a FUT8 Inhibitor with Cellular Inhibitory Properties.

Core fucosylation is catalyzed by α-1,6-fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N-glycans. Given the association of FUT8 with various diseases including cancer, selective FUT8 inhibitors applicable to in vivo or cell-based systems are highly sought-after. Here, we report the discovery of a compound that selectively inhibits FUT8 in cell-based assays. High-throughput screening revealed a FUT8-inhibiting pharmacophore, and further structural optimization yielded an inhibitor with a KD of 49 nM. Notably, this binding occurs only in the presence of GDP (a product of the enzymatic reaction catalyzed by FUT8). Mechanistic studies suggested that this inhibitor generates a highly reactive naphthoquinone methide derivative at the binding site in FUT8, which subsequently reacts with FUT8. Furthermore, prodrug derivatization of this inhibitor improved its stability, enabling suppression of core fucose expression and subsequent EGFR and T-cell signaling in cell-based assays, paving the way for the development of drugs targeting core fucosylation.

Intron retention as an excellent marker for diagnosing depression and for discovering new potential pathways for drug intervention.

Peripheral inflammation is often associated with depressive disorders, and immunological biomarkers of depression remain a focus of investigation. We performed RNA-seq analysis of RNA transcripts of human peripheral blood mononuclear cells from a case-control study including subjects with self-reported depression in the pre-symptomatic state of major depressive disorder and analyzed differentially expressed genes (DEGs) and the frequency of intron retention (IR) using rMATS. Among the statistically significant DEGs identified, the 651 upregulated DEGs were particularly enriched in the term "bacterial infection and phagocytosis", whereas the 820 downregulated DEGs were enriched in the terms "antigen presentation" and "T-cell proliferation and maturation". We also analyzed 158 genes for which the IR was increased (IncIR) and 211 genes for which the IR was decreased (DecIR) in the depressed subjects. Although the Gene Ontology terms associated with IncIR and DecIR were very similar to those of the up- and downregulated genes, respectively, IR genes appeared to be particularly enriched in genes with sensor functions, with a preponderance of the term "ciliary assembly and function". The observation that IR genes specifically interact with innate immunity genes suggests that immune-related genes, as well as cilia-related genes, may be excellent markers of depression. Re-analysis of previously published RNA-seq data from patients with MDD showed that common IR genes, particularly our predicted immune- and cilia-related genes, are commonly detected in populations with different levels of depression, providing validity for using IR to detect depression. Depression was found to be associated with activation of the innate immune response and relative inactivation of T-cell signaling. The DEGs we identified reflect physiological demands that are controlled at the transcriptional level, whereas the IR results reflect a more direct mechanism for monitoring protein homeostasis. Accordingly, an alteration in IR, namely IncIR or DecIR, is a stress response, and intron-retained transcripts are sensors of the physiological state of the cytoplasm. The results demonstrate the potential of relative IR as a biomarker for the immunological stratification of depressed patients and the utility of IR for the discovery of novel pathways involved in recovery from depression.

Discovery of GNE-6893, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of HPK1.

Hematopoietic progenitor kinase 1 (HPK1) serves a key immunosuppressive role as a negative regulator of T-cell receptor (TCR) signaling. HPK1 loss-of-function is associated with augmentation of immune function and has demonstrated synergy with immune checkpoint inhibitors in syngeneic mouse cancer models. These data offer compelling evidence for the use of selective small molecule inhibitors of HPK1 in cancer immunotherapy. We identified a novel series of isoquinoline HPK1 inhibitors through fragment-based screening that displayed promising levels of biochemical potency and activity in functional cell-based assays. We used structure-based drug design to introduce key selectivity elements while simultaneously addressing pharmacokinetic liabilities. These efforts culminated in a molecule demonstrating subnanomolar biochemical inhibition of HPK1 and strong in vitro augmentation of TCR signaling in primary human T-cells. Further profiling of this molecule revealed excellent kinase selectivity (347/356 kinases <50% inhibition @ 0.1 μM), a favorable in vitro safety profile, and good projected human pharmacokinetics.

Abstract We077: Mechanisms of T-lymphocyte Dysfunction in Dilated Cardiomyopathy Patients

Preclinical studies have shown that T-cells infiltrate the heart during heart failure (HF), promoting remodeling. However, it remains unclear whether activated T-cells infiltrate the human failing hearts also. Importantly, cellular mechanisms that are involved in T-cell activation during human HF are unknown. To address this question, we analyzed published single-cell RNA sequencing data from patients with dilated cardiomyopathy (DCM) and compared it with control hearts. Our analysis focused on characterizing the gene expression of cardiac T-cells to uncover potential mechanisms underlying their role in human HF. We found that patients with DCM had significantly more cardiac CD3+ T-cells (%CD45+ cells; 35.5±5.9 vs 22.0±0.8 p &lt; 0.01), with gene signatures indicative of higher TCR dependent antigenic activation, proliferation, and exhaustion. Although there were no differences in the proportion of CD4+ helper and CD8+ cytotoxic T-cells between groups, patients with DCM had a higher proportion of activated (%CD45+ cells, 6.6±0.8 vs 3.1±0.3 p = 0.03) and proliferative CD4+ T-cells (%CD45+ cells, 8.0±0.8 vs 3.1±0.3 p &lt; 0.01). Additionally, genes involved in TNF and TGFβ signaling were significantly enriched in cardiac T-cells derived from patients with DCM (normalized enrichment scores = 2.35, p adj &lt; 0.01, and 1.72, p adj &lt; 0.01, respectively),consistent with preclinical studies published by us and others. Recently, we also showed that dysfunctional T-cells exhibit increased estrogenic signaling with activation of Estrogen Receptor (ER)α in mice. Thus, we also looked at estrogen signaling in T-cells infiltrated into the DCM hearts and found that, indeed, cardiac T-cells in DCM patients exhibit significant upregulation of genes involved in estrogen signaling (normalized enrichment score = 1.69, p adj &lt; 0.01). This comprehensive analysis, for the first time, shows that T-cells are antigenically activated, actively infiltrate the failing human hearts and exhibit increased estrogenic signaling during DCM, providing novel insights and potential avenues for targeted immunotherapies.

Epigenetic disease markers in primary sclerosing cholangitis and primary biliary cholangitis-methylomics of cholestatic liver disease.

The epigenome, the set of modifications to DNA and associated molecules that control gene expression, cellular identity, and function, plays a major role in mediating cellular responses to outside factors. Thus, evaluation of the epigenetic state can provide insights into cellular adaptions occurring over the course of disease. We performed epigenome-wide association studies of primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) using the Illumina MethylationEPIC Bead Chip. We found evidence of increased epigenetic age acceleration and differences in predicted immune cell composition in patients with PSC and PBC. Epigenetic profiles demonstrated differences in predicted protein levels including increased levels of tumor necrosis factor receptor superfamily member 1B in patients with cirrhotic compared to noncirrhotic PSC and PBC. Epigenome-wide association studies of PSC discovered strongly associated 5'-C-phosphate-G-3' sites in genes including vacuole membrane protein 1 and SOCS3, and epigenome-wide association studies of PBC found strong 5'-C-phosphate-G-3' associations in genes including NOD-like receptor family CARD domain containing 5, human leukocyte antigen-E, and PSMB8. Analyses identified disease-associated canonical pathways and upstream regulators involved with immune signaling and activation of macrophages and T-cells. A comparison of PSC and PBC data found relatively little overlap at the 5'-C-phosphate-G-3' and gene levels with slightly more overlap at the level of pathways and upstream regulators. This study provides insights into methylation profiles of patients that support current concepts of disease mechanisms and provide novel data to inspire future research. Studies to corroborate our findings and expand into other -omics layers will be invaluable to further our understanding of these rare diseases with the goal to improve and individualize prognosis and treatment.

Bioinformatics analysis of high-intensity intermittent exercise for prevention of myocardial infarction.

The mechanism of target interaction involving high-intensity interval training (HIIT) in improving prognosis of myocardial infarction (MI) remains unclear. This study aimed to establish a visual network of "HIIT-target-disease" by referring to the methods of pharmacological disease and drug bioinformatic analysis, to explore the potential targets, and key targets and predict the potential biological mechanism of high-intensity intermittent exercise in preventing and treating myocardial infarction. Public data resources such as OMIM, NCBI and GeneCards were used to find potential targets of high-intensity intermittent exercise and myocardial infarction. Key targets of overlap between exercise and disease were determined according to the Relevance score values analyzed by GeneCards. The visual network diagram of "HIIT - Multi-target-disease" was constructed by Cytoscape. A total of 4820 disease targets and 528 high-intensity intermittent exercise targets were screened out, and 444 overlapped targets were obtained, including 425 protein targets. Five core protein targets were selected: IL10, PPARA, TNF, IL6, and STAT3. It may pass PI3K-AKT signaling pathway, Insulin resistance pathway, T-cell signaling pathway, TNF signaling pathway, and JAX-STAT signaling pathway and other pathways play a role. Our study comprehensively elucidated the potential targets, key targets and molecular mechanisms of high-intensity intermittent exercise in improving the prognosis of myocardial infarction, and proved that high-intensity intermittent exercise can act on multiple targets and multiple pathways to play a good preventive and therapeutic effect on myocardial infarction, providing scientific theoretical basis for revealing the mechanism of high-intensity intermittent exercise in the prevention and treatment of cardiovascular disease.

EFCAB4B (CRACR2A/Rab46) Genetic Variants Associated with COVID-19 Fatality

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 692 million cases worldwide and nearly 7 million deaths (August 2023). Severe COVID-19 is characterised in part by vascular thrombosis and a cytokine storm due to increased plasma concentrations of pro-thrombotic proteins such as von Willebrand factor and cytokines secreted from endothelial and T-cells. EFCAB4B is a gene that encodes for two proteins (CRACR2A and Rab46) that play important roles in endothelial and T-cell secretion. In this study, using patient data recorded in the UK Biobank, we demonstrate the importance of variants in the EFCAB4B genetic sequence with COVID-19 fatality. Using logistic regression analysis, we determined that three single-nucleotide polymorphisms (SNPs) in the gene cause missense variations in CRACR2A and Rab46, which are associated with COVID-19 fatality (rs9788233: p = 0.004, odds ratio = 1.511; rs17836273: p = 0.012, odds ratio = 1.433; rs36030417: p = 0.013, odds ratio = 1.393). All three SNPs cause changes in amino acid residues that are highly conserved across species, indicating their importance in protein structure and function. Two SNPs, rs17836273 (A98T) and rs36030417 (H212Q), cause amino acid substitutions in important functional domains: the EF-hand and coiled-coil domain, respectively. Molecular modelling shows minimal impact by the substitution of threonine at position 98 on the structure of the EF-hand. Since Rab46 is a GTPase that regulates both endothelial cell secretion and T-cell signalling, these missense variants may play a role in the molecular mechanisms underlying the thrombotic and inflammatory characteristics observed in patients with severe COVID-19 outcomes.

An open-label, randomized controlled trial to assess a ketogenic diet in critically ill patients with sepsis.

Patients with sepsis experience metabolic and immunologic dysfunction that may be amplified by standard carbohydrate-based nutrition. A ketogenic diet (KD) may offer an immunologically advantageous alternative, although clinical evidence is limited. We conducted a single-center, open-label, randomized controlled trial to assess whether a KD could induce stable ketosis in critically ill patients with sepsis. Secondary outcomes included assessment of feasibility and safety of KD, as well as explorative analysis of clinical and immunological characteristics. Forty critically ill adults were randomized to either a ketogenic or standard high-carbohydrate diet. Stable ketosis was achieved in all KD patients, with significant increases in β-hydroxybutyrate levels compared with controls [mean difference 1.4 milimoles per liter; 95% confidence interval (CI): 1.0 to 1.8; P < 0.001). No major adverse events or harmful metabolic side effects (acidosis, dysglycemia, or dyslipidemia) were observed. After day 4, none of the patients in the KD group required insulin treatment, whereas in the control group, insulin dependency ranged between 35% and 60% (P = 0.009). There were no differences in 30-day survival, but ventilation-free [incidence rate ratio (IRR) 1.7; 95% CI: 1.5 to 2.1; P < 0.001], vasopressor-free (IRR 1.7; 95% CI: 1.5 to 2.0; P < 0.001), dialysis-free (IRR 1.5; 95% CI: 1.3 to 1.8; P < 0.001), and intensive care unit-free days (IRR 1.7; 95% CI: 1.4 to 2.1; P < 0.001) were higher in the ketogenic group. Next-generation sequencing of CD4+/CD8+ T cells and protein analyses showed reduced immune dysregulation, with decreased gene expression of T-cell activation and signaling markers and lower pro-inflammatory cytokine secretion. This trial demonstrated the safe induction of a stable ketogenic state in sepsis, warranting larger trials to investigate potential benefits in sepsis-related organ dysfunction.

RhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors

BackgroundChimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy...

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Development of an in vitro assay for screening programmed death receptor-1/programmed cell death ligand 1 monoclonal antibody therapy in dogs

Immunomodulatory antibody drugs that modulate the function of immune checkpoint molecules, such as programmed death receptor-1 (PD-1) and programmed cell death ligand 1 (PD-L1), have been established as new cancer treatments in human medicine. In recent years, there have also been reports on antibodies that inhibit immune checkpoint molecules in dogs, and clinical trials using such antibodies for canine cancer have been gradually increasing in number. Because inhibitory antibodies restore T-cell function by inhibiting the binding of PD-1 on T cells and its ligand PD-L1, the quality of antibody function has been evaluated using activated T cells or peripheral blood mononuclear cells isolated from healthy dogs; however, the assays and dogs used significantly vary. Therefore, in the present study, we developed a reporter gene assay using reporter cells (Jurkat/NFATluc/cPD1) and effector cells (CTAC/OKT3/cPDL1). Jurkat/NFATluc/cPD1 were generated by introducing both of the NFAT-responsive luciferase gene as a marker of T-cell signaling and canine PD-1, into a human T lymphoid cell line, Jurkat. CTAC/OKT3/cPDL1 were generated by introducing single-chain FV (scFV) of anti-human CD3 antibody (OKT3) and canine PD-L1 into a canine thyroid carcinoma cell line, CTAC. Ligation of PD-1 on Jurkat/NFATluc/cPD1 via binding of PD-L1 on CTAC/OKT3/cPDL1 suppressed NFAT luciferase activity induced by CD3 ligation by scFV of OKT3. The addition of anti-canine PD-1 and PD-L1 antibodies, both of which were previously developed in our laboratory, restored this suppression with high sensitivity, although the anti-human PD-L1 antibody atezolizumab induced a very weak restoration. This assay is an useful method for functionally evaluating the inhibition of canine PD-1 and PD-L1 binding.

Hypericin Alleviates Chronic Kidney Disease-induced Left Ventricular Hypertrophy by Regulation of FGF23-FGFR4 Signaling Pathway.

Chronic kidney disease (CKD) is a significant global health threat that imposes a substantial burden on both individuals and societies. CKD frequently correlates with cardiovascular events, particularly left ventricular hypertrophy (LVH), which contributes to the high mortality rate associated with CKD. Fibroblast growth factor 23 (FGF23), a hormone primarily involved in regulating calcium and phosphorus metabolism, has been identified as a major risk factor for LVH in CKD patients. Elevated serum FGF23 levels are known to induce LVH and myocardial fibrosis by activating the fibroblast growth factor receptor 4 (FGFR4) signal pathway. Therefore, targeting FGFR4 and its downstream signaling pathways holds potential as a treatment strategy for cardiac dysfunction in CKD. In our current study, we have discovered that Hypericin, a key component derived from Hypericum perforatum , has the ability to alleviate CKD-related LVH by targeting the FGFR4/phospholipase C gamma 1 (PLCγ1) signaling pathway. Through in vitro experiments using rat cardiac myocyte H9c2 cells, we observed that Hypericin effectively inhibits FGF23-induced hypertrophy and fibrosis by suppressing the FGFR4/PLCγ1/calcineurin/nuclear factor of activated T-cell (NFAT3) signaling pathway. In addition, our in vivo studies using mice on a high-phosphate diet and rat models of 5/6 nephrectomy demonstrated that Hypericin has therapeutic effects against CKD-induced LVH by modulating the FGFR4/PLCγ1/calcineurin/NFAT3 signaling pathway. In conclusion, our research highlights the potential of Hypericin as a candidate for the treatment of CKD-induced cardiomyopathy. By suppressing the FGFR4/PLCγ1 signaling pathway, Hypericin shows promise in attenuating LVH and myocardial fibrosis associated with CKD.

Dysregulated Wnt and NFAT signaling in a Parkinson’s disease LRRK2 G2019S knock-in model

Parkinson’s disease (PD) is a progressive late-onset neurodegenerative disease leading to physical and cognitive decline. Mutations of leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. LRRK2 is a complex scaffolding protein with known regulatory roles in multiple molecular pathways. Two prominent examples of LRRK2-modulated pathways are Wingless/Int (Wnt) and nuclear factor of activated T-cells (NFAT) signaling. Both are well described key regulators of immune and nervous system development as well as maturation. The aim of this study was to establish the physiological and pathogenic role of LRRK2 in Wnt and NFAT signaling in the brain, as well as the potential contribution of the non-canonical Wnt/Calcium pathway. In vivo cerebral Wnt and NFATc1 signaling activity was quantified in LRRK2 G2019S mutant knock-in (KI) and LRRK2 knockout (KO) male and female mice with repeated measures over 28 weeks, employing lentiviral luciferase biosensors, and analyzed using a mixed-effect model. To establish spatial resolution, we investigated tissues, and primary neuronal cell cultures from different brain regions combining luciferase signaling activity, immunohistochemistry, qPCR and western blot assays. Results were analyzed by unpaired t-test with Welch’s correction or 2-way ANOVA with post hoc corrections. In vivo Wnt signaling activity in LRRK2 KO and LRRK2 G2019S KI mice was increased significantly ~ threefold, with a more pronounced effect in males (~ fourfold) than females (~ twofold). NFATc1 signaling was reduced ~ 0.5-fold in LRRK2 G2019S KI mice. Brain tissue analysis showed region-specific expression changes in Wnt and NFAT signaling components. These effects were predominantly observed at the protein level in the striatum and cerebral cortex of LRRK2 KI mice. Primary neuronal cell culture analysis showed significant genotype-dependent alterations in Wnt and NFATc1 signaling under basal and stimulated conditions. Wnt and NFATc1 signaling was primarily dysregulated in cortical and hippocampal neurons respectively. Our study further built on knowledge of LRRK2 as a Wnt and NFAT signaling protein. We identified complex changes in neuronal models of LRRK2 PD, suggesting a role for mutant LRRK2 in the dysregulation of NFAT, and canonical and non-canonical Wnt signaling.

Abstract 2114: Differences In The Circulating Transcriptome Between Peripheral Artery Disease And Coronary Artery Disease

Introduction: Despite common risk factors, peripheral artery disease (PAD) and coronary artery disease (CAD) exhibit distinct pathological presentations. Previous gene expression profiling of blood from CAD and PAD patients shows alterations in inflammatory and immune related transcripts relative to controls. Studies have not directly compared gene expression in PAD versus CAD. Here, we aimed to use whole-blood RNA sequencing to investigate transcriptomic differences between PAD and CAD. Methods: Whole blood from subjects age 40-65 years with symptomatic PAD (n=20, mean age 59, 75% male, 35% white) and CAD (n=51, mean age 52, 82% male, 78% white) was collected into PAXgene tubes. Patients with concomitant CAD and PAD were excluded. RNA was isolated, sequenced and analyzed to investigate differential circulating gene expression between PAD and CAD subjects adjusting for age, sex, and race/ethnicity. Cell type deconvolution method dtangle was applied to characterize cell type proportions in the whole blood transcriptome. Results: Between PAD and CAD subjects, 106 transcripts were differentially expressed (adjP &lt; 0.1), of which 38 were enriched in PAD and 68 were enriched in CAD. Gene set enrichment analysis (GSEA) revealed 29 pathways differentially expressed (FDR 0.05) between PAD and CAD, of which 8 were enriched in PAD and 21 enriched in CAD. The top enriched pathways in PAD were “specific granule” (Normalized enrichment score [NES] = 2.1, q value = 2.5 x 10 -5 ) and “secretory granule membrane” (NES = 1.9, q = 1.0 x 10 -4 ). Top enriched pathways in CAD were “T cell receptor complex” (NES = 3.1, q = 2.2 x 10 -7 ) and “response to interferon-alpha” (NES = 3.1, q = 2.2 x 10 -9 ). Cellular deconvolution using dtangle showed increased CD8 T-cells (p=0.01) and CD4 Naïve T-cells (p=0.007) in subjects with CAD versus PAD. Conclusion: Whole blood transcriptomics revealed significant differences in the circulating transcriptome between patients presenting with PAD and CAD. Our data suggest upregulation of pathways related to secretory granules in PAD, and enrichment of CD8 and CD4 Naïve T-cells and pathways related to T-cell signaling in CAD. Differences in the circulating transcriptome may help explain pathological and clinical differences between PAD and CAD.

IGSF8 is an innate immune checkpoint and cancer immunotherapy target

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival invitro or invivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells invitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and Tcell signaling invivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Assembled/Disassembled Modular Scaffolds for Multicellular Tissue Engineering.

The behavior of tissue resident cells can be influenced by the spatial arrangement of cellular interactions. Therefore, it is of significance to precisely control the spatial organization of various cells within multicellular constructs. It remains challenging to construct a versatile multicellular scaffold with ordered spatial organization of multiple cell types. Herein, a modular multicellular tissue engineering scaffold with ordered spatial distribution of different cell types is constructed by assembling varying cell-laden modules. Interestingly, the modular scaffolds can be disassembled into individual modules to evaluate the specific contribution of each cell type in the system. Through assembling cell-laden modules, the macrophage-mesenchymal stem cell (MSC), endothelial cell-MSC, and chondrocyte-MSC co-culture models are successfully established. The in vitro results indicate that the intercellular cross-talk can promote the proliferation and differentiation of each cell type in the system. Moreover, MSCs in the modular scaffolds may regulate the behavior of chondrocytes through the nuclear factor of activated T-Cells (NFAT) signaling pathway. Furthermore, the modular scaffolds loaded with co-cultured chondrocyte-MSC exhibit enhanced regeneration ability of osteochondral tissue, compared with other groups. Overall, this work offers a promising strategy to construct a multicellular tissue engineering scaffold for the systematic investigation of intercellular cross-talk and complex tissue engineering.

Tetracyclines enhance antitumor T-cell immunity via the Zap70 signaling pathway

BackgroundCancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we...

RNA-sequencing of Human Kidney Allografts and Delineation of T-Cell Genes, Gene Sets, and Pathways Associated With Acute T Cell-mediated Rejection.

Delineation of T-cell genes, gene sets, pathways, and T-cell subtypes associated with acute T cell-mediated rejection (TCMR) may improve its management. We performed bulk RNA-sequencing of 34 kidney allograft biopsies (16 Banff TCMR and 18 no rejection [NR] biopsies) from 34 adult recipients of human kidneys. Computational analysis was performed to determine the differential intragraft expression of T-cell genes at the level of single-gene, gene set, and pathways. T-cell signaling pathway gene sets for plenary T-cell activation were overrepresented in TCMR biopsies compared with NR biopsies. Heightened expression of T-cell signaling genes was validated using external TCMR biopsies. Pro- and anti-inflammatory immune gene sets were enriched, and metabolism gene sets were depleted in TCMR biopsies compared with NR biopsies. Gene signatures of regulatory T cells, Th1 cells, Th2 cells, Th17 cells, T follicular helper cells, CD4 tissue-resident memory T cells, and CD8 tissue-resident memory T cells were enriched in TCMR biopsies compared with NR biopsies. T-cell exhaustion and anergy were also molecular attributes of TCMR. Gene sets associated with antigen processing and presentation, and leukocyte transendothelial migration were overexpressed in TCMR biopsies compared with NR biopsies. Cellular deconvolution of graft infiltrating cells by gene expression patterns identified CD8 T cell to be the most abundant T-cell subtype infiltrating the allograft during TCMR. Our delineation of intragraft T-cell gene expression patterns, in addition to yielding new biological insights, may help prioritize T-cell genes and T-cell subtypes for therapeutic targeting.