Figure 4

Polyfunctional CD8 ⁺ T effector memory cells specific to apoptotic epitopes in multiple sclerosis patients. (A) Representative flow cytometry analysis of peripheral blood mononuclear cells from a multiple sclerosis (MS) patient. Cells were incubated with or without the relevant peptides plus anti-CD28 mAb for 18 h at 37°C. Then cells were stained with dextramers complexed to corresponding peptides, phycoerythrin-cyanine-labeled mAb to CD8 and the dump channel reagents and processed for the detection of IL-17 and IFN-γ, by intracellular staining (ICS) assay with the relevant mAbs. Dot plot analyses are gated on CD8⁺dextramer⁺ cells and show percentages of cytokine-producing cells. The percentage of cells is reported in each quadrant. (B) Percentages of CD8⁺dextramer⁺ cells specific to the corresponding peptides producing IL-17, IFN-γ, or both within 18 h of contact with the relevant peptides in the 12 patients analyzed. (C) Representative flow cytometry analysis of an antigen-specific T cell line obtained upon 15 days stimulation with the non-muscle myosin heavy chain 9 (MYH9)_478-486 epitope and IL-2. Cells were stained with mAb to CD8, the indicated dextramers, and were then stimulated or not with the relevant soluble peptide plus anti-CD28 mAb for detecting IL-17 and IFN-γ production by ICS assay, as described above. Contourplot analyses are gated on CD8⁺dextramer⁺ cells and show percentages of cytokine-producing cells. Similar results were obtained in six patients tested with different apoptotic epitopes.