Elispot analysis of PLP responses in MS

It has been postulated that Th1-like T cells are involved in the pathogenesis of multiple sclerosis (MS). Pro-inflammatory cytokines are thought to be crucial for the initiation and amplification of inflammatory brain lesions and direct myelin damage. The role of Th2 cytokines is unclear, as is the role of CD4⁺ versus CD8⁺ T-cells in MS. We used the cytokine ELISPOT assay to study proteolipid protein (PLP) peptide reactivity in freshly isolated PBMC from MS patients and controls. We used 9-amino-acid length overlapping PLP peptides to study the fine specificity of lymphocytes as measured by their IFNγ and IL-5 profiles. We tested IFNγ responses in peripheral blood from 14 MS patients and 11 controls. When the IFNγ-positive wells were subdivided into single-well positives (putative CD8 responses) versus multiple-adjacent-well positives (putative CD4 responses), the MS patients demonstrated 12 times as many multiple-well positives compared to controls. Comparing the total number of IFNγ-positive spots between MS patients and controls, spots in multiple-adjacent-well positives were 43 times as high in MS patients as controls. The average number of IL-5 positive single wells was almost identical between MS patients and controls and no IL-5 multiple-adjacent positive wells were observed, suggesting that PLP-specific IL-5 responses do not differ between MS patients and controls. Our data suggest that MS patients give a much stronger MHC Class II-restricted immune response to PLP peptides compared to controls, which is characterized by secretion of IFNγ, an inflammatory cytokine that has been strongly implicated in the disease process.