Genomics of Immune Diseases and New Therapies.

Genomic DNA sequencing technologies have been one of the great advances of the 21st century, having decreased in cost by seven orders of magnitude and opening up new fields of investigation throughout research and clinical medicine. Genomics coupled with biochemical investigation has allowed the molecular definition of a growing number of new genetic diseases that reveal new concepts of immune regulation. Also, defining the genetic pathogenesis of these diseases has led to improved diagnosis, prognosis, genetic counseling, and, most importantly, new therapies. We highlight the investigational journey from patient phenotype to treatment using the newly defined XMEN disease, caused by the genetic loss of the MAGT1 magnesium transporter, as an example. This disease illustrates how genomics yields new fundamental immunoregulatory insights as well as how research genomics is integrated into clinical immunology. At the end, we discuss two other recently described diseases, CHAI/LATAIE (CTLA-4 deficiency) and PASLI (PI3K dysregulation), as additional examples of the journey from unknown immunological diseases to new precision medicine treatments using genomics.

CTLA-4 tail fusion enhances CAR-T antitumor immunity.

Chimeric antigen receptor (CAR)-T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail, we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex or triplex CTLA-4 cytoplasmic tails (CCTs) fused to the C terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of proinflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior antitumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

Cancer-Germline Antigen Expression Discriminates Clinical Outcome to CTLA-4 Blockade.

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

Releasing the brake: CTLA-4 loss turbocharges CAR T cells.

Immune checkpoint receptor-induced T cell dysfunction is a major cause of CAR T cell treatment failure. In this issue, Agarwal et al. report that CRISPR/Cas9 deletion of CTLA4, but not PDCD1 or CTLA4 and PDCD1, enhances CD28 signaling, restoring fitness and antitumor function of CAR T cells, including those derived from patients who failed CAR T cell therapy.

Deletion of the inhibitory co-receptor CTLA-4 enhances and invigorates chimeric antigen receptor T cells.

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has achieved tremendous success treating B cell malignancies; however, some patients fail to respond due to poor autologous T cell fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR T cell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR T cells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the T cell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of T cells from patients with leukemia that previously failed CAR T cell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient T cells, providing a strategy for increasing patient responses to CAR T cell therapy.

Quality of TCR signaling determined by differential affinities of enhancers for the composite BATF-IRF4 transcription factor complex.

Variable strengths of signaling via the T cell antigen receptor (TCR) can produce divergent outcomes, but the mechanism of this remains obscure. The abundance of the transcription factor IRF4 increases with TCR signal strength, but how this would induce distinct types of responses is unclear. We compared the expression of genes in the TH2 subset of helper T cells to enhancer occupancy by the BATF-IRF4 transcription factor complex at varying strengths of TCR stimulation. Genes dependent on BATF-IRF4 clustered into groups with distinct TCR sensitivities. Enhancers exhibited a spectrum of occupancy by the BATF-IRF4 ternary complex that correlated with the sensitivity of gene expression to TCR signal strength. DNA sequences immediately flanking the previously defined AICE motif controlled the affinity of BATF-IRF4 for direct binding to DNA. Analysis by the chromatin immunoprecipitation-exonuclease (ChIP-exo) method allowed the identification of a previously unknown high-affinity AICE2 motif at a human single-nucleotide polymorphism (SNP) of the gene encoding the immunomodulatory receptor CTLA-4 that was associated with resistance to autoimmunity. Thus, the affinity of different enhancers for the BATF-IRF4 complex might underlie divergent signaling outcomes in response to various strengths of TCR signaling.

Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade.

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer.

Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States.

Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire. CTLA-4 imposes major boundaries on CD4+ T cell phenotypes, whereas PD-1 subtly limits CD8+ T cell phenotypes. By computationally reconstructing T cell differentiation paths, we identified protein expression changes that underlied the abnormal phenotypic expansion and pinpointed when lineage choice events occurred during differentiation. Similar alterations in T cell phenotypes were observed after anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate negative co-stimulation as a key regulator and determinant of T cell differentiation and suggest that checkpoint blockade might work in part by altering the limits of T cell phenotypes.

T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity.

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack.

Soluble CTLA-4 attenuates T cell activation and modulates anti-tumor immunity.

CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumor control. Antibodies targeting CTLA-4 have been promising treatments for numerous cancers, but the mechanistic basis of their anti-tumoral immune-boosting effects is poorly understood. Although the ctla4 gene also encodes an alternatively spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in a murine cancer model. We show that expression of sCTLA-4 by tumor cells suppresses CD8+ T cells in vitro and accelerates growth and experimental metastasis of murine tumors in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8+ T cells in a non-cytotoxic state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoral CD8+ T cell activation and cytolytic potential, correlating with therapeutic efficacy and tumor control. This previously unappreciated role of sCTLA-4 suggests that the biology and function of multi-gene products of immune checkpoint receptors need to be fully elucidated for improved mechanistic understanding of cancer immunotherapies.

CTLA-4 on thymic epithelial cells complements Aire for T cell central tolerance.

Medullary thymic epithelial cells (mTECs) are essential for the establishment of T cell central tolerance. The transcription factor Aire plays a key role in this process, but other factors remain understudied. We found that a small population of mTECs expressed the coinhibitory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These CTLA-4+ cells were detectable in perinates, peaked around young adulthood and expanded sixfold in the absence of Aire. Single-cell transcriptomics revealed CTLA-4+ mTECs to express a distinct gene signature encoding molecules associated with antigen presentation and interferon-gamma signaling. Mice conditionally lacking CTLA-4 in thymic epithelial cells had no major immunological deficiencies but displayed a mildly increased inflammatory tone and a partial defect in the generation of Foxp3+CD4+ regulatory T cells. Consequently, these mice developed modest levels of autoantibodies and lymphocytic infiltration of peripheral tissues. Thus, CTLA-4 expression in mTECs complements Aire to establish T cell central tolerance.

Biomarker discovery with quantum neural networks: a case-study in CTLA4-activation pathways.

BACKGROUND: Biomarker discovery is a challenging task due to the massive search space. Quantum computing and quantum Artificial Intelligence (quantum AI) can be used to address the computational problem of biomarker discovery from genetic data. METHOD: We propose a Quantum Neural Networks architecture to discover genetic biomarkers for input activation pathways. The Maximum Relevance-Minimum Redundancy criteria score biomarker candidate sets. Our proposed model is economical since the neural solution can be delivered on constrained hardware. RESULTS: We demonstrate the proof of concept on four activation pathways associated with CTLA4, including (1) CTLA4-activation stand-alone, (2) CTLA4-CD8A-CD8B co-activation, (3) CTLA4-CD2 co-activation, and (4) CTLA4-CD2-CD48-CD53-CD58-CD84 co-activation. CONCLUSION: The model indicates new genetic biomarkers associated with the mutational activation of CLTA4-associated pathways, including 20 genes: CLIC4, CPE, ETS2, FAM107A, GPR116, HYOU1, LCN2, MACF1, MT1G, NAPA, NDUFS5, PAK1, PFN1, PGAP3, PPM1G, PSMD8, RNF213, SLC25A3, UBA1, and WLS. We open source the implementation at: https://github.com/namnguyen0510/Biomarker-Discovery-with-Quantum-Neural-Networks .

A heterogeneous tumor immune microenvironment of uncommon epidermal growth factor receptor mutant non-small cell lung cancer.

Common epidermal growth factor receptor (EGFR) mutations are usually not considered for immunotherapy in non-small cell lung cancer (NSCLC) due to poor efficacy. However, whether uncommon EGFR mutations are suitable for immunotherapy has not been thoroughly studied. Thus, we explored the tumor immune microenvironment (TME) features in uncommon EGFR mutant NSCLC. In this study, a total of 41 patients with EGFR mutations were included, the majority (85.4%) of whom were stage I. Among them, 22 patients harbored common mutations, while 19 patients presented with uncommon mutations. Compared with common mutations, uncommon mutations exhibited more infiltrating T cells and fewer M2 macrophages, upregulated expression of antigen processing and a presentation pathway. Unsupervised clustering based on the mIF profile identified two classes with heterogeneous TME in uncommon mutations. Class 1 featured the absence of PD-1+ cytotoxic T cell infiltration, and class 2 displayed a hotter TME because of the downregulated expression of hypoxia (p < 0.001), oxidative phosphorylation (p = 0.009), and transforming growth factor beta signaling (p = 0.01) pathways as well as increased expression of CTLA4 (p = 0.001) and PDCD1 (p = 0.004). The association of CTLA4 and PDCD1 with TME profiles was validated in a TCGA lung adenocarcinoma cohort with uncommon EGFR mutations. Our study reveals the distinct and heterogeneous TME features in uncommon EGFR mutant NSCLC.

Effect of DNA methylation at the CTLA4 gene on the clinical status of autoimmune thyroid diseases.

The pathogenesis and manifestation of autoimmune thyroid diseases (AITDs), Graves' disease (GD), and Hashimoto's disease (HD) are associated with T cell activation. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) plays a crucial role in the regulation of T cell activation. DNA methylation levels of eight CpG sites in the CTLA4 gene and expression levels of soluble CTLA-4 were examined. Methylation levels of +22 CpG and CT60 CpG-SNPs in patients with GD and HD with the CT60 GG genotype were lower than those in control subjects. Methylation levels of the-15 CpG sites were lower in patients with intractable GD than those in GD patients in remission. These results suggest that demethylation of +22 CpG and CT60 CpG-SNPs may be associated with susceptibility to GD and HD in subjects with the CTLA4 CT60 GG genotype, and that demethylation of -15 CpG may be associated with the intractability of GD.

Combination of transcriptome and Mendelian inheritance reveals novel prognostic biomarker of CTLA-4-related lncRNAs and protective role of nitrogen metabolism pathway in lung adenocarcinoma development.

OBJECTIVE: Since in the cancer setting, tumor cells may use cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to evade the immune system. This study aimed to identify CTLA-4-related long non-coding RNAs (lncRNAs) and assess their roles in lung adenocarcinoma (LUAD) development. METHODS: Clinical and genomic data were obtained from The Cancer Genome Atlas (TCGA), MSigDB and Gene Weaver. CTLA-4-related lncRNA-based gene signatures (CTLA4LncSigs) were identified using Cox regression, establishing a risk score model and an independent prognostic model. Enrichment analysis (GO/KEGG) was performed. Mendelian randomization (MR) analysis investigated the nitrogen metabolism and lung cancer relationship, with Bayesian weighted MR (BWMR) addressing uncertainties. Correlations with tumor microenvironment and drug sensitivity were explored. RESULTS: Nineteen CTLA4LncSigs significantly influenced LUAD prognosis. The risk score demonstrated independence as a prognostic factor. Functional analysis revealed lncRNAs' impact on nitrogen metabolism. MR and BWMR confirmed the protective role of the nitrogen metabolism pathway in lung cancer. CONCLUSION: Our study identifies CTLA-4-related lncRNAs associated with LUAD prognosis and uncovers a previously undiscovered protective role of the nitrogen metabolism pathway in combating LUAD development, providing new insights into potential therapeutic targets and prognostic biomarkers for this aggressive cancer subtype.

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-ß1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

CTLA-4 expression and polymorphisms in Schizophrenia; a systematic review of literature.

Schizophrenia constitutes a severe psychiatric disorder with detrimental impacts on individuals, their support systems, and the broader economy. Extensive research has revealed a notable association between variations in the Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) gene and an increased susceptibility to schizophrenia.This study represents the first systematic review of the literature investigating the impact of CTLA-4 polymorphisms and expression on the development and progression of schizophrenia.Our investigation involved a comprehensive search strategy, using a combination of title, abstract, and MESH terms in four databases, including PubMed, Scopus, Web of Science, and Google Scholar, until August 29th, 2023. The complete texts of the identified records were obtained and rigorously assessed based on predefined exclusion and inclusion criteria. Out of the numerous records, a total of 88 were identified through the databases. 10 studies met the criteria; therefore, their quality was assessed and included in this systematic study. The records were then categorized into polymorphism and expression groups. Our investigation emphasizes an association between rs3087243, rs231779, rs231777, rs16840252, rs5742909, and rs231775 polymorphisms and the development of schizophrenia. The results demonstrate a correlation between CTLA-4 polymorphisms and schizophrenia, compelling the need for further research to thoroughly examine the role of CTLA-4 in schizophrenia and other psychiatric disorders.