Soluble HLA-G and HLA-G Bearing Extracellular Vesicles Affect ILT-2 Positive and ILT-2 Negative CD8 T Cells Complementary.

Esther Schwich,Vera Rebmann,Christina Bade-Döding,Gia-Gia T Hò,Edgardo D Carosella,Peter A Horn,Joel Lemaoult

doi:10.3389/fimmu.2020.02046

Abstract

Tumor immune escape is associated with both, the expression of immune checkpoint molecules on peripheral immune cells and soluble forms of the human leukocyte antigen-G (HLA-G) in the blood, which are consequently discussed as clinical biomarker for disease status and outcome of cancer patients. HLA-G preferentially interacts with the inhibitory receptor immunoglobulin-like transcript (ILT) receptor-2 in the blood and can be secreted as free soluble molecules (sHLA-G) or via extracellular vesicles (EV). To investigate the contribution of these two forms to the expression of checkpoint molecules in peripheral blood, we primed peripheral blood mononuclear cells with purified soluble sHLA-G1 protein, or EV preparations derived from SUM149 cells transfected with membrane-bound HLA-G1 or control vector prior to anti-CD3/CD28 T cell activation. Our study demonstrated that priming of PBMC with sHLA-G1 protein prior to 48 h activation resulted in enhanced frequencies of ILT-2 expressing CD8+ T cells, and in an upregulation of immune checkpoint molecules CTLA-4, PD-1, TIM-3, and CD95 exclusively on ILT-2 positive CD8+ T cells. In contrast, when PBMC were primed with EV (containing HLA-G1 or not) upregulation of CTLA-4, PD-1, TIM-3, and CD95 occurred exclusively on ILT-2 negative CD8+ T cells. Taken together, our data suggest that priming with sHLA-G forms induces a pronounced immunosuppressive/exhausted phenotype and that priming with sHLA-G1 protein or EV derived from HLA-G1 positive or negative SUM149 cells affects CD8+ T cells complementary by targeting either the ILT-2 positive or negative subpopulation, respectively, after T cell activation.

Highlights

The human leukocyte antigen-G (HLA-G) belongs to the non-classical class I HLA molecules and can exist in different isoforms expressed either as membrane-anchored structures or as secreted molecules [1,2,3,4]
To mimic whether the expression of immune checkpoint (IC) on T cells can be modulated by the presence of sHLA-G1 in the peripheral blood, peripheral blood mononuclear cells (PBMC) (n = 6) of healthy individuals were primed with sHLA-G1 overnight prior to stimulation with anti-CD3/CD28
Analysis revealed that the fold change (FC) of CTLA-4, PD-1, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and CD95 (Figures 1E–H) was significantly elevated on immunoglobulin-like transcript (ILT)-2 positive CD8+ T cells compared to ILT-2 negative ones

Summary

Introduction

The human leukocyte antigen-G (HLA-G) belongs to the non-classical class I HLA molecules and can exist in different isoforms expressed either as membrane-anchored structures or as secreted molecules [1,2,3,4]. HLA-G can be released as membrane-anchored molecules from various cell types via extracellular vesicles (EV) [5]. Of EV depends on their cell of origin and differs remarkably encompassing a broad spectrum of antigens, cell surface-expressed receptors and/or ligands, metabolites, and nucleic acids [7]. EV can either directly fuse with a target cell enabling the transfer of bioactive molecules to both, adjacent and distant sites, or be internalized via phagocytosis, endocytosis or micropinocytosis, thereby contributing to an intracellular signaling mechanism [10]. Fusion depends on an acidic micro-environment which naturally occurs inside tumors [11,12,13,14], while uptake and internalization of EV are primarily receptor-mediated via adhesion molecules [15]. Thereby, tumor-derived EV (TEV) may represent an alternative mechanism of immunosurveillance deficiency impairing diverse immune cell lineages [6]

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