Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen -specific T cells, the high number of putative determinants on the autoanti gens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to s tudy, directly ex vivo, proteolipid protein (PLP)-specific memory cell reac tivity from MS patients and controls. Overlapping 9-aa-long peptides, spann ing the entire PLP molecule in single amino acid steps, mere used to determ ine the frequency and fine specificity of PLP-specific lymphocytes as measu red by their IFN-gamma and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22), The epito pes recognized in individual patients, up to 22 peptides, were scattered th roughout the PLP molecule, showing considerable heterogeneity among MS pati ents. Frequency measurements showed that the number of PLP peptide-specific IFN-gamma-producing cells averaged II times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low f requencies in both MS patients and controls. This first comprehensive asses sment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-gamma-polarized , IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells, The essential absence of uncommitted T h0 cells producing both cytokines may explain why therapeutic strategies th at aim at the induction of immune deviation show little efficacy in the est ablished disease.