Abstract
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 VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, 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.
Highlights
Specialized antigen presenting cells, such as dendritic cells (DCs), play a fundamental role in the adaptive immune response by presenting antigen-derived peptides to T-cells
Consistent with a previous published results[34,35,36,37,38,39,40,41], we confirmed an increase of cell surface-associated HLA-DR (csHLADR) following TNF and LPS treatment for the monocytic-like cell line MUTZ-3, where both stimulants elicited a response of similar magnitude
Our assessment of total HLA-II abundance by mass spectrometry showed a larger effect in stimulated MUTZ-3 cells, suggesting that an accumulation of HLA- α chain may occur at the intracellular compartments
Summary
Specialized antigen presenting cells, such as dendritic cells (DCs), play a fundamental role in the adaptive immune response by presenting antigen-derived peptides to T-cells. According to the linear model of DC maturation, immature DCs are functionally poor activators of T cells but have a high capacity for internalizing antigenic compounds through active endocytosis[1,2,3,4] This development state correlates with low levels of HLA-II at the cell surface. We demonstrated the quantification of HLA-II from the dendritic-like cell model MUTZ-334–41, as well as a population of myeloid DCs (CD11c+) derived from human blood, both of which can be differentiated and activated by proinflammatory stimuli. These CD14+ monocyte-derived dendritic cells were activated with LPS to examine HLA-DR at the cell surface as well as total HLAII alpha chains
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