Figure 7. IL-12 enhances suppressive ability of neuroantigen-specific CD8+ Tregs.

(A) Neuroantigen-specific CD8+ T cells were stimulated by culturing 30 × 10⁶ cells bulk PBMCs from healthy subjects at 10 × 10⁶ /mL for 7 days in 6 well plates. Culture medium was either left untreated or supplemented with 25ng/mL of IL-12 or IL-23. All cultures were supplemented with 1 mg/mL of one of two different neuroantigen peptides (MBP and PLP). 7 days post in vitro PBMC stimulation, CD8+ T cells were isolated by magnetic bead sorting and used in suppression assays with freshly thawed autologous APCs and CD4+CD25− T cells. (B) CD8+ T cells from IL-12-treated cultures expressed significantly higher levels of granzyme B. Enriched CD8+ T cells from cytokine-treated co-cultures were stimulated for 5 hrs with LAF on day 7, followed by Intracellular staining for granzyme B. (C) Suppressive ability of exacerbation derived CD8+ T cells was restored through IL-12 treatment. Exacerbation PBMCs from five RRMS patients were cultured 10 × 10⁶ /mL for 7 days in 6 well plates. Culture medium was either left untreated or supplemented with 25ng/mL of IL-12. All cultures were supplemented with 1 μg/mL of one of two different neuroantigen peptides. 7 days post in vitro stimulation, exacerbation-derived CD8+ T cells were isolated by magnetic bead sorting and used in suppression assays with autologous exacerbation-derived freshly thawed APCs and CD4+CD25− T cells. *p < 0.05; Five independent experiments were performed with PBMCs from 5 healthy control donors for (A) and (B). Five independent experiments were performed with PBMCs from 5 RRMS donors during disease exacerbation for (C).