Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells

• Published in: Scientific reports Vol. 11; no. 1; p. 1028
• Main Authors: Casasola-LaMacchia, A., Ritorto, M. S., Seward, R. J., Ahyi-Amendah, N., Ciarla, A., Hickling, T. P., Neubert, H.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13.01.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/250; 631/45/475; 631/45/611; 631/45/612; Antigen presentation; Antigens; Biosynthesis; CD4 antigen; Cell activation; Cell lines; Cell surface; Dendritic cells; DQA1 protein; Flow cytometry; Gene expression; Histocompatibility antigen HLA; Humanities and Social Sciences; Lipopolysaccharides; Lymphocytes T; Major histocompatibility complex; Mass spectrometry; Mass spectroscopy; Monocytes; multidisciplinary; Peptide mapping; Peptides; Protein folding; Science; Science (multidisciplinary); Scientific imaging
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-77024-y

The major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLD K PLLK, VEHWGLD Q PLLK and VEHWGLD E PLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3–7% and ≤ 1%, respectively.