Genetically engineered CD80–pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide–MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols

MHC Class II Presentation in Autoimmunity

Antigen presentation by major histocompatibility complex class II (MHC-II) molecules is crucial for eliciting an efficient immune response by CD4+ T cells and maintaining self-antigen tolerance. Some MHC-II alleles are known to be positively or negatively associated with the risk of the development of different autoimmune diseases (ADs), including those characterized by the emergence of autoreactive T cells. Apparently, the MHC-II presentation of self-antigens contributes to the autoimmune T cell response, initiated through a breakdown of central tolerance to self-antigens in the thymus. The appearance of autoreactive T cell might be the result of (i) the unusual interaction between T cell receptors (TCRs) and self-antigens presented on MHC-II; (ii) the posttranslational modifications (PTMs) of self-antigens; (iii) direct loading of the self-antigen to classical MHC-II without additional nonclassical MHC assistance; (iv) the proinflammatory environment effect on MHC-II expression and antigen presentation; and (v) molecular mimicry between foreign and self-antigens. The peculiarities of the processes involved in the MHC-II-mediated presentation may have crucial importance in the elucidation of the mechanisms of triggering and developing ADs as well as for clarification on the protective effect of MHC-II alleles that are negatively associated with ADs

The CIMP-high phenotype is associated with energy metabolism alterations in colon adenocarcinoma

Abstract Background CpG island methylator phenotype (CIMP) is found in 15–20% of malignant colorectal tumors and is characterized by strong CpG hypermethylation over the genome. The molecular mechanisms of this phenomenon are not still fully understood. The development of CIMP is followed by global gene expression alterations and metabolic changes. In particular, CIMP-low colon adenocarcinoma (COAD), predominantly corresponded to consensus molecular subtype 3 (CMS3, “Metabolic”) subgroup according to COAD molecular classification, is associated with elevated expression of genes participating in metabolic pathways. Methods We performed bioinformatics analysis of RNA-Seq data from The Cancer Genome Atlas (TCGA) project for CIMP-high and non-CIMP COAD samples with DESeq2, clusterProfiler, and topGO R packages. Obtained results were validated on a set of fourteen COAD samples with matched morphologically normal tissues using quantitative PCR (qPCR). Results Upregulation of multiple genes involved in glycolysis and related processes (ENO2, PFKP, HK3, PKM, ENO1, HK2, PGAM1, GAPDH, ALDOA, GPI, TPI1, and HK1) was revealed in CIMP-high tumors compared to non-CIMP ones. Most remarkably, the expression of the PKLR gene, encoding for pyruvate kinase participating in gluconeogenesis, was decreased approximately 20-fold. Up to 8-fold decrease in the expression of OGDHL gene involved in tricarboxylic acid (TCA) cycle was observed in CIMP-high tumors. Using qPCR, we confirmed the increase (4-fold) in the ENO2 expression and decrease (2-fold) in the OGDHL mRNA level on a set of COAD samples. Conclusions We demonstrated the association between CIMP-high status and the energy metabolism changes at the transcriptomic level in colorectal adenocarcinoma against the background of immune pathway activation. Differential methylation of at least nine CpG sites in OGDHL promoter region as well as decreased OGDHL mRNA level can potentially serve as an additional biomarker of the CIMP-high status in COAD