Fig 2: Immunohistochemical assays (a) and relative expression (b) of CD27+ cells in the spinal cord and brain tissues of EAE mice (× 200). A and G. control at day 7; B and H, EAE mice at day 7; C and I, control at day 18; D and J, EAE mice at day 18, E and K. control at day 30 and F and L. EAE mice at day 30, respectively. A-F, spinal cord tissue; G-L, brain tissue. Arrows denote CD27++ cells. * denotes P < 0.05 vs control

Fig 3: Expansion and characterization of ?d[T2] cells(A) Healthy donor PBMCs were activated with zoledronic acid (ZOL, n = 27) or immobilized anti-?d TCR antibody (n = 15) and then cultured for 14–17 days in SFM containing IL-2 alone (?d[2] cells) or IL-2 + TGF-ß (?d[T2] cells). The percentage of ?d T cells was measured in PBMCs (day 1) and after expansion in the indicated cytokines.(B) Absolute number of ?d T cells on day 1 and post-expansion. Median fold expansion is indicated for ?d[2] and ?d[T2] cells.(C–I) Post-?d T cell expansion, the following markers were assessed in ?d[2] and ?d[T2] cells by flow cytometry: (C) % CLA; (D) % CXCR4; (E) binding to E-selectin-Fc fusion protein; (F) state of differentiation: naive (CD45RA+ CD27+), central memory (CM; CD45RA- CD27+), effector memory (CD45RA- CD27-), and terminally differentiated with CD45RA re-expression (TEMRA; CD45RA+ CD27-; (G) viability, apoptosis, and necrosis; (H) % CD103; and (I) % KLRG1.(J) ?d[T2] T cells and ?d T cells expanded from the same donor in human serum + IL-2 (?d[2S]) were engineered to co-express firefly luciferase (ffLuc) and red fluorescent protein (RFP) and then analyzed by flow cytometry. GFP-expressing Jurkat cells were injected i.v. in NSG mice. After 4 days, 6 mice each were treated with 10 million RFP/ffLuc-expressing ?d[2S] or ?d[T2] T cells. Untrans., untransduced.(K) Mice were analyzed by BLI after 24 and 48 h to determine persistence of ?d T cells in the whole body and in a region of interest drawn around the femora (means ± SDs; n = 6 at 24 h; n = 3 at 48 h; 2-way ANOVA).(L) After each imaging session, 3 mice per group were culled. The % RFP+ (?d) T cells present in spleen and bone marrow were determined (means ± SDs).(M) The % GFP+ (leukemic) cells present in bone marrow was also determined (means ± SDs). NS, not significant.(C)–(E) and (G)–(I) show means ± SDs and (A), (B), and (F) show medians ± interquartile ranges, in which data were or were not normally distributed, respectively. Accordingly, statistical analysis was performed using a Student’s t test or Wilcoxon signed-rank test, respectively.