Self and parasite-derived peptides selected upon DERAA-bearing HLA-DRB1 alleles activate CD4+ T cells from Chagas cardiomyopathy patients and are associated with ventricular dysfunction

Human infection with the protozoan Trypanosoma cruzi causes Chagas disease, which may lead to a deadly dilated cardiomyopathy resulting from T-cell-mediated inflammation. We found that specific HLA-DRB1 alleles (*0103, *0402, *1301, and *1302) that display the DERAA motif are linked to this severe clinical manifestation of Chagas disease. We employed computational analysis, in vitro functional assays, and single-cell RNA sequencing to determine the response of CD4+ T cells from indeterminate (IND) and cardiac (CCC) Chagas patients to peptides selected on DERAA-bearing alleles. We observed that these alleles display binding affinity towards host-derived peptides with sequence similarity to parasite-derived proteins. These peptides can activate and induce proliferation of CD4+ T-cells from CCC, but not IND. Importantly, the magnitude of this response correlated with the severity of ventricular dysfunction and increased production of soluble factors associated with myocardial fibrosis. Analysis of differentially expressed genes (DEGs) in activated CD4+ T-cells from individuals with the DERAA-DRB1 alleles demonstrated a high expression of cytotoxic, chemotactic and proapoptotic genes, linking these cells with pathogenic functions. Finally, we observed the upregulation of genes that code for the host proteins that contain the potentially pathogenic peptides in the cardiac tissue of CCC, suggesting their involvement in cardiomyopathy. Our findings highlight the ability of CD4+ T-cells from CCC patients to recognize and react to foreign and self-peptides, thereby emphasizing the importance of HLA-DRB1 alleles in the presentation of potentially pathogenic antigens and in the amplification of CCC pathology. Overall design: PBMCs from 24 patients with Chagas disease (Asymptomatic=10, CCC=14) were sequecing. CD45-positive PBMCs were isolated and stained with oligonucleotide-conjugated Sample Tags following the manufacturer's instruction using the BDTM Single-Cell Multiplexing Kit, BD-Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody Express Single-cell analysis system (BD Biosciences), using the BD Rhapsody cDNA (BD Biosciences, cat# 633663) and Target amplification (BD Biosciences, cat# 633664) kits, according to the manufacturer's instructions. Sequencing was performed on Nova SeqTM 6000 (Illumina, San Diego, CA). Data processing was initially conducted through the Seven Bridges Genomics platform, using BD Rhapsody Target Analysis Pipeline (version CWL-v1.2) to identifies and correct artifacts of sequencing, ensuring accurate molecule counting pre cell. DBEC (Distribution-based error correction) files were imported into SeqGeq version 1.6.0 (BD, Ashland, OR). Normalization was performed considering all genes, 10,000 counts per cell as a scaling factor. Quality control of samples was performed considering both cell and gene quality. Quality control for cell removes outlier events or doublets based on number of expressed genes relative to the library size. Gene quality control considered the total expression of each gene versus the cells expressing each gene, as well as the expression dispersion (Supplementary Figure 1D). The Lex BDSMK plugin was employed to identify Sample Tags. Data analyses were performed on sample-normalized and quality-controlled cells. Differentially expressed genes (DEGs) were identified using the FindMarker function, applying a Log2 fold change threshold (= 1.5 or = -1.5) and a Bonferroni-adjusted p value<0.05.