New Mass Spectrometry Technique Presents New Questions About MHC II Expression Regulation

By Victoria Osinski, PhD

An exciting paper by Casasola-LaMacchia et al. presents a novel mass spectrometry method for detecting different MHC class II isotypes in multiple cell types.¹ Their results not only present a new useful method for measuring subsets of these molecules, but also intriguing findings regarding regulation of MHC II expression.

The major histocompatibility complex II (MHC II or HLA II) molecules are expressed on antigen-presenting cells (such as dendritic cells (DCs)) and play an important role in adaptive immune responses by presenting immunogenic peptides to CD4+ T cells. Human leukocyte antigens (HLAs) are dimers consisting of alpha and beta chains. Expression of each HLA chain is driven by multiple loci within the larger MHC gene complex. Variants of these different HLA alleles (DR, DQ, DP, DO, DM) have been strongly associated with certain diseases, including type 1 diabetes.² Some of these variants translate to differences in protein structure.² This paper¹ and others³, however, remind us that the regulation of MHC II alleles is more complicated than that: co-factors, variants in non-coding regions of the gene, and post-translational modifications are additional factors.¹

Major findings quantify and introduce new questions about HLA expression

In this study, the authors develop a novel liquid chromatography tandem mass spectrometry (LC MS/MS) method to isolate total HLA II proteins from cell lysates using an anti-panHLA-II antibody (HB-145) and measure the concentrations of alpha chain isotypes HLA-DRA1/DQA2, -DPA1 and -DQA1. They do this by monitoring the proteotypic peptides VEHWGLDEPLLK, VEHWGLDQPLLK, and VEHWGLDKPLLK, respectively. Dendritic-like cell lines MUTZ-3 and KG-1, as well as human monocyte-derived DCs, were stimulated with LPS and HLA-DRA1/DQA2, -DPA1 and -DQA1 isotypes were measured using LC MS/MS.

Their findings demonstrated that the HLA II alpha chain in all samples was predominantly made up of HLA-DRA1, followed by HLA-DPA1, while HLA-DQA1 was not detected in all donors. Interestingly, LPS stimulation of these cells increased total cell surface HLA II expression as detected by flow cytometry in human DC samples. The same treatment, however, did not change the levels of each HLA-II isotype from whole cell lysates when measured by LC MS/MS. These results highlight that HLA-II abundance is also likely regulated by biosynthesis, translocation of the protein to the membrane, and stabilization at the membrane.

This approach will be useful for studies on various autoimmune diseases associated with certain HLA haplotypes and variants including type 1 diabetes and rheumatoid arthritis. It can also be applied to more specific studies investigating the mechanisms driving HLA isotype abundance in DCs and other antigen-presenting cells.