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-c ell epitopes) from class I complexes expressed at the cell surface of viable cells. Cells treated at pH 3.3 with citrate-phosphate buffer fo r periods as short as 15 s remained viable and became phenotypically c lass I deficient. Qualitative loss of class I determinants was verifie d both serologically and by the incapacity of acid-treated cells to be lysed by class I-restricted cytolytic T lymphocytes (CTLs) in contras t to non-acid-treated controls. Flow cytometric analysis of acid-treat ed cells suggests that class I heavy chains remain associated with the cell membrane, while the class I light chain (beta2-microglobulin) is absent. Since the physical dissociation of beta2-microglobulin from c lass I heavy chain is correlated with the release of previously class I-bound peptides, we examined acid-eluted cell-free supernatants for t he 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 Ml 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 conta ined a peptide of Mr 968 with a sequence similar to the Flu M1 58-66 s equence GILGFVFTL. The application of this technique to other T-cell-b ased systems may aid in the definition of peptide epitopes relevant to viral, autoimmune or neoplastic disorders.