Monitoring antigen cross-presentation by human dendritic cells purified from peripheral blood

Abstract

Tumor immunosurveillance, regression and therapy require most often the action of CD8⁺ T cells. These cells are primed by dendritic cells (DC), which are the only antigen presenting cells able to stimulate naive T cells. Tumor antigen presentation requires cross-presentation of antigens from the tumor cells by DC. Dendritic cells capture antigens from tumor cells into endosomal compartments and process them. Then they expose at their cell surface their own MHC class I molecules complexed with tumor cell epitopes, which are recognized by the T cell receptors of specific CD8⁺ T cells. This allows the activation of anti-tumoral functions of these specific CD8⁺ T cells, mediated by cytokines and by cytotoxic mechanisms. Here, we describe in detail the delicate methods required to (1) prepare antigen donor cells, (2) prepare CD8⁺ T cells specific for these antigens, (3) purify human DC from peripheral blood, (4) preincubate purified DC and antigen donor cells for antigen capture, (5) incubate these DC with antigen-specific CD8⁺ T cells for cross-presentation and (6) assess cross-presentation to specific CD8⁺ T cells. These methods allowed our laboratory to characterize in detail cross-presentation from cells containing viral antigens and can be applied to study cross-presentation from tumor antigens.

Keywords: Antigen processing; Cell sorting; Cross-presentation; Dendritic cells; Fluorescence activated cell sorting; IFN-γ; Immunomagnetic cell sorting; MHC restriction; Tumor antigens.