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Naturally processed and presented MHC-II peptides isolated from HHV-6B (Z29)-infected cells

ABSTRACT: Identification of MHC-II naturally processed and presented peptides derived from HHV-6B (strain Z29). MHC-II-peptide complexes were immuno-affinity purified from the total membrane fraction obtained from a T lymphoblastoid cell line infected with HHV-6B (Z29). Peptides were eluted off the purified complexes by acid treatment and separated from protein subunits using C4 columns. LC-MS/MS was used for analysis of the peptide mix and identification of eluted peptides sequences

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Homo Sapiens (ncbitaxon:9606) Human Herpesvirus 6b (strain Z29) Ncbitaxon:36351

SUBMITTER: Lawrence J. Stern

PROVIDER: MSV000083546 | MassIVE | Fri Mar 08 07:02:00 GMT 2019

SECONDARY ACCESSION(S): PXD013015

REPOSITORIES: MassIVE

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Project description:Purpose: Production of broad-spectrum antibodies against T cell-dependent antigens requires marginal zone (MZ) B cells to present antigenic peptides complexed to MHC II. Here we describe the trogocytic acquisition of these complexes from dendritic cells (DCs). Complement C3 (C3) binds to MHC II on murine and human DCs. Recognition of C3 by MZ B cell complement receptor 2 triggers trogocytosis of DC plasma membrane and its receptors, which become exposed on the MZ B cells. Excessive trogocytosis is deleterious but prevented by membrane ubiquitin ligase MARCH1, which limits the number of MHC II–C3 complexes displayed on the DC surface. MHC II molecules are thus receptors for constitutively activated and potentially harmful C3, an activity that enables acquisition of DC-like functions by trogocytic MZ B cells. Methods: mRNA profiles of splenic cDC1s, B cells and CD11cintCD24+CD8int cells of wild type (WT) and March1-/- mice mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. All 100-base-pair single-end reads were mapped to the reference mouse genome (GRCm38/mm10) using STAR aligner, and gene-wise counts were obtained using Subread package. Differential expression analysis of RNA sequencingdata was carried out using the edgeR and limma-voom packages in Bioconductor. Results: Using an optimized data analysis workflow, we mapped about 14 million sequence reads per sample to the mouse genome (build mm10) and identified 16,182 genes in the different cell types from WT and March1−/− mice.Transcriptomic profiling revealed over 6500 genes differentially expressed between cDC1s and B cells. Principle component analyses revealed very few differences between WT and March1−/− cDC1s, and between WT and March1−/− B cells. Notably, the transcriptome of CD11cintCD24+CD8int cells was similar to those of WT or March1−/− B cells, indicating they belonged to the B cell lineage. Conclusions: In our study we have described that MHC II ubiquitination by MARCH1 plays two important roles: first, to enhance the removal of surface MHC II–C3 complexes on all APCs; and second, to limit MZ B cell trogocytosis and elimination of cDCs. It is conceivable these two functions, more than the regulation of MHC II antigen presentation, have been the major drivers for the conservation of MHC II ubiquitination through evolution.

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Naturally processed and presented MHC-II peptides isolated from HHV-6B (Z29)-infected cells

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