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关键词： CAR-T   Cellular senescence   Exhausted T cells   scRNA-seq   Epigenetic changes   Metabolic reprogramming  

摘要： Background: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive. Methods: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion. Results: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells. Conclusions: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture.

关键词： Drosophila   hematopoietic cell tumor   innate immune system   cytokines   JNK pathway   TNF family   JAK-STAT pathway  

摘要： Turandot (Tot) family proteins, which are induced via the JAK/STAT pathway after infection, also suppress lymph gland tumors in Drosophila mxcmbn1 mutant larvae. We investigated the potential role of hemocytes in Tot induction in tumor-bearing mutants via immunostaining and RNAi experiments. Normal hemocytes transplanted into mutant larvae were recruited to the tumor and fat body (FB), suggesting that these cells transmit tumor-related information. The transplanted hemocytes ectopically expressed Unpaired3 (Upd3), which is necessary for the activation of JAK/STAT. Eiger, a Drosophila tumor necrosis factor (TNF) ortholog, was highly expressed in tumors. Depletion of the Eiger receptor in hemocytes reduced Tot levels and eventually enhanced tumor growth. The c-Jun N-terminal kinase (JNK) pathway, acting downstream of the receptor, was also activated in the hemocytes of mutants. Downregulation of the JNK pathway in hemocytes inhibited Tot induction, leading to enhanced tumor growth. These results suggest that upd3 expression in hemocytes depends on the Eiger-JNK pathway. We propose that after Eiger activates the JNK pathway in hemocytes present on the tumor, cells expressing Upd3 are recruited to the FB. Upd3 then activates JAK/STAT to induce the expression of antitumor proteins. This study highlights the intricate communication between tissues via blood cells during tumor suppression.

关键词： Interleukin-1   Interleukin-1 receptor antagonist   Etiology   Drug delivery  

摘要： ObjectiveThis article is aims to provide an overview of studies reported in the literature to investigate the etiological role of IL-1/IL-1ra in various disease conditions and the different drug delivery systems developed to achieve IL-1ra as a possible therapeutic *** reported in PubMed, Google scholar, and other open-source literature related to etiological involvement of IL-1ra in pathophysiological conditions and various drug delivery schemes developed for IL-1ra for its efficacy evaluation as a possible treatment for different disease conditions were *** and conclusionsThe pathophysiological conditions involving IL-1/IL-1 ra spanned CNS-related disorders, Diabetes, Cardiac disorders, Ocular disease conditions, Gastrointestinal conditions, Tumor growth & metastasis, and miscellaneous conditions. The drug delivery systems developed for IL-1ra included a commercial drug product, Gene therapy, Antibody fusions, Extended-release delivery systems, and Pegylated-IL-1ra systems.

关键词： HLA   HLA-DPA1*02:143   Korean   next-generation sequencing   novel allele  

摘要： HLA-DPA1*02:143 is identical to HLA-DPA1*02:02:02:01 except for a single nucleotide substitution in exon 1.

关键词： IL32   Association study   Tuberculosis   Regulatory variant  

摘要： Aim: IL32 is a pleiotropic intracellular cytokine with an emergent role in tuberculosis. The different isoforms of IL32: alpha, beta, gamma and delta have varying pro and anti-inflammatory potentials. We studied the role of genetic variants of IL32 and its isoforms in susceptibility to tuberculosis using a case-household contact association study. Methodology: Using a targeted sequencing approach, IL32 (+1kb) gene was sequenced in 64 pairs of culture positive TB cases and their culture negative household contacts. Subsequently the identified variants were validated in an independent cohort of cases and household contacts using TaqMan genotyping assay. Regulatory role of the associated variants was assessed using GTExPortal, RegulomeDB score, HaploReg and ENCODE histone ChIP-seq data. Expression of IL32 and its isoforms was evaluated by RT-PCR in PBMC from unexposed healthy controls (N = 25) with different genotype background and stimulated with TB antigens ESAT6 and CFP10. similar to 200 bp around the associated variant was cloned into pGL3 promoter vector to assess enhancer activity by dual luciferase assay in cell lines. Results: Intronic variant rs9927163(G/T) was found associated with pulmonary TB, T being the risk allele (OR = 2.3(1.40-3.83, p = 0.03)), while G is the protective allele. This finding was validated in independent set of TB cases and household contacts (p = 0.0435). rs9927163 is an eQTL for the genes IL32 (p = 4.1e-10) and BICDL2 (p = 2.1e-7) in whole blood and interrupts an AP-1 binding site. ENCODE histone ChIP-seq data shows rs9927163 residing within T cell specific H3K4me3 peak. The G allele is associated with greater enhancer activity in a T cell line (2.12 fold, p = 0.0059). The TT genotype showed greater normalized expression of IL32 delta, a less proinflammatory isoform compared to the GT and GG genotypes together following ESAT6 (p = 0.02288) and CFP10 (p = 0.04595) treatment. This indicates that greater expression of a potentially less p

关键词： DRB3*02:185   HLA   new allele   next-generation sequencing  

摘要： The novel allele HLA-DRB3*02:185 differs from HLA-DRB3*02:02:01:02 by one nucleotide substitution in codon 191 of exon 4.

关键词： iRhom2   ADAM17   Secretome   Macrophages   MHC class I molecules  

摘要： Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor alpha (TNF alpha). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

关键词： Microbial-Immune balance   Germ-related autoimmune disorders   Micorbial agents in multifactorial disease   Immune cell trafficking   Environment-related balance of health/disease  

摘要： Here we critically discuss data supporting the view that microbial agents (pathogens, pathobionts or commensals alike) play a relevant role in the pathogenesis of multifactorial diseases, but their role is concealed by the rules presiding over T cell antigen recognition and trafficking. These rules make it difficult to associate univocally infectious agents to diseases' pathogenesis using the paradigm developed for canonical infectious diseases. (Cross-)recognition of a variable repertoire of epitopes leads to the possibility that distinct infectious agents can determine the same disease(s). There can be the need for sequential infection/colonization by two or more microorganisms to develop a given disease. Altered spreading of infectious agents can determine an unwanted activation of T cells towards a pro-inflammatory and trafficking phenotype, due to differences in the local microenvironment. Finally, trans-regulation of T cell trafficking allows infectious agents unrelated to the specificity of T cell to modify their homing to target organs, thereby driving flares of disease. The relevant role of microbial agents in largely prevalent diseases provides a conceptual basis for the evaluation of more specific therapeutic approaches, targeted to prevent (vaccine) or cure (antibiotics and/or Biologic Response Modifiers) multifactorial diseases.

关键词： Probes  

摘要： Human leukocyte antigen (HLA) genes play pivotal roles in numerous immunological applications. Given the immense number of polymorphisms, achieving accurate high-throughput HLA typing remains challenging. This study aimed to harness the human pan-genome reference consortium (HPRC) resources as a potential benchmark for HLA reference materials. We meticulously annotated specific four field-resolution alleles for 11 HLA genes (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5) from 44 high-quality HPRC personal genome assemblies. For sequencing, we crafted HLA-specific probes and conducted capture-based targeted sequencing of the genomic DNA of the HPRC cohort, ensuring focused and comprehensive coverage of the HLA region of interest. We used publicly available short-read whole-genome sequencing (WGS) data from identical samples to offer a comparative perspective. To decipher the vast amount of sequencing data, we employed seven distinct software tools: OptiType, HLA-VBseq, HISAT genotype, SpecHLA, T1K, QzType, and DRAGEN. Each tool offers unique capabilities and algorithms for HLA genotyping, allowing comprehensive analysis and validation of the results. We then compared these results with benchmarks derived from personal genome assemblies. Our findings present a comprehensive four-field-resolution HLA allele annotation for 44 HPRC samples. Significantly, our innovative targeted next-generation sequencing (NGS) approach for HLA genes showed superior accuracy compared with conventional short-read WGS. An integrated analysis involving QzType, T1K, and DRAGEN was developed, achieving 100% accuracy for all 11 HLA genes. In conclusion, our study highlighted the combination of targeted short-read sequencing and astute computational analysis as a robust approach for HLA genotyping. Furthermore, the HPRC cohort has emerged as a valuable assembly-based reference in this realm. © 2024 The Authors

关键词： beta 5-integrin   non-small cell lung cancer   pericytes   tyrosine kinase inhibitor sensitivity  

摘要： EGFR-mutated lung cancer patients sometimes display restricted responses to third-generation tyrosine kinase inhibitors (TKIs), potentially attributable to undervalued input from stromal cells, notably pericytes (PCs). The study shows that PCs isolated from EGFR-mutated patients have a unique secretome profile, notably secreting IL32 and affecting signaling pathways and biological processes linked to TKI sensitivity. Clinical evidence, supported by single-cell RNA sequencing and multiplex immunostaining of tumor tissues, confirms the presence of IL32-expressing pericytes closely interacting with beta 5-integrin-expressing cancer cells in EGFR-mutated patients, impacting therapeutic response and prognosis. Co-culture and conditioned medium experiments demonstrate that PCs reduce TKI effectiveness in EGFR-mutated cancer cells, a reversible phenomenon through silencing IL32 expression in PCs or depleting the IL32 receptor beta 5-integrin on cancer cells, thereby restoring cancer cell sensitivity. Mechanistically, it is shown that YY1 signaling upregulates IL32 secretion in PCs, subsequently activating the beta 5-integrin-Src-Akt pathway in EGFR-mutated cancer cells, contributing to their TKI sensitivity. In animal studies, co-injection of cancer cells with PCs compromises TKI effectiveness, independently of blood vessel functions, while inhibition of beta 5-integrin restores tumor cell sensitivity. Overall, the findings highlight direct crosstalk between cancer cells and pericytes, impacting TKI sensitivity via IL32-beta 5-integrin paracrine signaling, proposing an enhanced therapeutic approach for EGFR-mutated patients.