Identification of T-cell epitopes: rapid isolation of class I-presented peptides from viable cells by mild acid elution.
A novel method was developed to isolate immunogenic peptides (CD8+ T-cell epitopes) from class I complexes expressed at the cell surface of viable cells. Cells treated at pH 3.3 with citrate-phosphate buffer for periods as short as 15 s remained viable and became phenotypically class I deficient. Qualitative loss of class I determinants was verified both serologically and by the incapacity of acid-treated cells to be lysed by class I-restricted cytolytic T lymphocytes (CTLs) in contrast to non-acid-treated controls. Flow cytometric analysis of acid-treated cells suggests that class I heavy chains remain associated with the cell membrane, while the class I light chain (beta 2-microglobulin) is absent. Since the physical dissociation of beta 2-microglobulin from class I heavy chain is correlated with the release of previously class I-bound peptides, we examined acid-eluted cell-free supernatants for the presence of immunogenic peptides. Peptides were acid eluted from an influenza A strain-infected, HLA-A2+ cell line and were subsequently fractionated by reverse-phase high performance liquid chromatography (RP-HPLC). These fractionated peptides were examined for their capacity to sensitize an HLA-A2+ B cell line to lysis mediated by an influenza A matrix peptide- (Flu M1 57-68) specific, HLA-A2-restricted CTL line. A single peak of biologic activity was identified in HPLC fractions 47 and 48 derived from influenza-infected cells. These fractions contained a peptide of M(r) 968 with a sequence similar to the Flu M1 58-66 sequence GILGFVFTL. The application of this technique to other T-cell-based systems may aid in the definition of peptide epitopes relevant to viral, autoimmune, or neoplastic disorders.
Storkus
W J
WJ
Department of Surgery, University of Pittsburgh School of Medicine, PA 15261.
Zeh
H J
HJ
Salter
R D
RD
Lotze
M T
MT
D000143:Acids; D002470:Cell Survival / Q000187:drug effects; D000939:Epitopes / Q000097:blood; D015395:Histocompatibility Antigens Class I / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000143:Acids; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D010455:Peptides