MHC Class I Stability is Modulated by Cell Surface Sialylation in Human Dendritic Cells.

Zélia Silva,José Alexandre Ferreira,Paula A Videira,Sebastian Springer,Helena Soares,Danielle Almeida,Paul J Hensbergen,Fanny M Deschepper,Sandra J Van Vliet,Martina Pirro,Tiago Ferro

doi:10.3390/pharmaceutics12030249

Abstract

Maturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to identify sialylated proteins at DC surface and comprehend their role and modulation. Mass spectrometry analysis of DC’s proteins, pulled down by a sialic acid binding lectin, identified molecules of the major human histocompatibility complex class I (MHC-I), known as human leucocyte antigen (HLA). After desialylation, DCs showed significantly higher reactivity with antibodies specific for properly folded MHC-I-β2-microglobulin complex and for β2-microglobulin but showed significant lower reactivity with an antibody specific for free MHC-I heavy chain. Similar results for antibody reactivities were observed for TAP2-deficient lymphoblastoid T2 cells, which express HLA-A*02:01. Using fluorescent peptide specifically fitting the groove of HLA-A*02:01, instead of antibody staining, also showed higher peptide binding on desialylated cells, confirming higher surface expression of MHC-I complex. A decay assay showed that desialylation doubled the half-life of MHC-I molecules at cell surface in both DCs and T2 cells. The biological impact of DC´s desialylation was evaluated in co-cultures with autologous T cells, showing higher number and earlier immunological synapses, and consequent significantly increased production of IFN-γ by T cells. In summary, sialic acid content modulates the expression and stability of complex MHC-I, which may account for the improved DC-T synapses.

Highlights

Dendritic cells (DCs) are the most potent antigen-presenting cells, bridging the innate and adaptive immune responses [1,2]
Our results showed that the expression level of major human histocompatibility complex class I (MHC-I) at the cell surface is sensitive to the enzymatic removal of sialic acids, and Sambucus nigra (SNA) lectin reactivity against MHC-I molecules immunoprecipitated using a pan-human leucocyte antigen (HLA)-ABC class I antibody (W6/32) suggests that sialylation is a common feature of different class I alleles
Considering that in DCs there is evidence that approximately 50–60% of MHC-I molecules reside in recycling vesicles [35,49], our results suggest that desialylated MHC-I molecules, originating from recycling vesicles, are the main contributors to the increased surface expression of HLA-A2 molecules after sialidase treatment

Summary

Introduction

Dendritic cells (DCs) are the most potent antigen-presenting cells, bridging the innate and adaptive immune responses [1,2]. Cytotoxic CD8+ T cell engagement starts by DCs delivering the first signal through MHC-I This early signaling step is considered to be of low affinity, as T cells “touch and go” DCs, unless a specific peptide presented via MHC molecules is recognized by the T cell receptor (TCR) [7,8]. MHC-I presents peptides generated by the degradation of endogenous proteins by the proteasome These peptides are transported into the endoplasmic reticulum, where they are assembled with MHC-I heavy chain· complexed with β2 microglobulin (β2m) and the resulting peptide:MHC-I (pMHC-I) transit to the cell surface. The peptide or the β2m may dissociate from the heterotrimer complex resulting in the appearance of free MHC-I heavy chain at the cell surface [9]. Free MHC-I heavy chains have reduced cell surface half-life, they do not activate T cells and they are typically internalized to enable the assembly with new peptides. Effector T cell activation is only triggered by the assembled heterotrimer and it can be evaluated by the secretion of interferon (IFN)-γ, a hallmark of T cell-mediated immune responses [10,11]

Objectives

Methods

Results

Discussion

Conclusion