Adoptive Transfer of Antigen-Specific CD8+ T Cells

Donor mice were immunized with either PLP_178-191 or control OVA_323-339 (ISQAVHAAHAEINEAGR, GenScript, Piscataway, NJ) in CFA. At 15 days post-immunization, splenocytes and inguinal lymphocytes were harvested and as published previously (5 (link), 23 (link)–29 (link)), single cell suspensions were restimulated in vitro with 20μg/mL cognate antigen and 10pg/mL rIL-2 for 72h in complete RPMI (Corning, Tewksbury, MA). CD8 T cells were sorted to above 90% purity using Ly-2 microbeads (Miltenyi Biotech, Auburn, CA), and 5x10⁶ cells were adoptively transferred i.v. into recipient mice at times indicated. For mixed adoptive transfer experiments, CD8 T cells from cultures including equal inputs of bulk day 15-immune IFNγR−/− and IFNγ−/− cells (inguinal lymphocytes and splenocytes) were Ly-2 bead-sorted and transferred at 5x10⁶ per recipient. Unless otherwise specified, “control” groups consist of recipient mice injected i.v. with either PBS alone or OVA-CD8 donor cells, both of which have no effect on disease, as shown in this report and published previously (5 (link), 27 (link), 28 (link)). A 2:1 donor to recipient ratio was used in these experiments.