title: Generation of human alloantigen-specific T cells from peripheral blood.
abstract: The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.
authors.affiliation: Department of Pathology, University of California, San Diego.
authors.forename: Burhan P
authors.initials: BP
authors.lastname: Jama
authors.affiliation: Department of Pathology, University of California, San Diego; gpmorris@ucsd.edu.
authors.forename: Gerald P
authors.initials: GP
authors.lastname: Morris
mesh_terms: D017951:Antigen Presentation; D002999:Clone Cells; D000939:Epitopes; D005434:Flow Cytometry / Q000379:methods; D006086:Graft vs Host Disease / Q000276:immunology; D006801:Humans; D007519:Isoantigens / Q000097:blood / Q000276:immunology*; D008297:Male; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
chemical_list: D000939:Epitopes; D007519:Isoantigens; D011948:Receptors, Antigen, T-Cell