Clonal expansion of freshly isolated CD4T cells by randomized peptides andidentification of peptide ligands using combinatorial peptide libraries

We synthesized Xn (n = 9-19) peptides that consist of 9 to 19 residues with random sequences. X19 is considered to deliver antigenic Stimuli to CD4 T ells, because: (a) X19 induces proliferation of peripheral blood mononuclea r cells (PBMC), in the presence of IL-2, which is abrogated by monoclonal a ntibodies to class II HLA; (b) X19 + IL-2 induces proliferation of CD4 T ce ll clones of distinct specificities; and (c) T cell clones recognizing the same TCR ligands with distinct VP usage are equally stimulated by X19 + IL- 2. We next co-cultured single peripheral CD4 T cells with X19 and mitomycin -treated autologous PBMC. Indeed, single T cells of CD45RA(-) memory phenot ype exhibited clonal expansion, with variable rates of proliferation, when IL-4, IL-7, IL-9, IL-15 and agonistic antibody to CD29 were included in the culture. These T cell clones showed heterogeneous proliferation patterns a gainst KGXXXXXXXXX and KGXXXXXXXXXGKGKK-based combinatorial peptides librar ies, in the presence of IL-2. Pattern-match search on a T cell clone result ed in peptide ligand candidates, one of which induced proliferation, as did protein molecules carrying the corresponding sequence. These results indic ate that X19 can induce proliferation of peripheral memory T cells, the pep tide ligands of which can be determined using combinatorial peptide librari es.