Fig. 5.

Susceptibility of B7-deficient cells to suppression is restored by lentiviral-based expression of full-length, but not truncated, B7 lacking a transmembrane/cytoplasmic domain. (a) Examination of the costimulatory ability of cells infected with full-length or GPI-anchored B7-1 or B7-2. CD28 wt (black bars) or CD28^–/– (gray bars) CD4CD25^– T cells (n = 5 × 10⁴) were incubated with 1 × 10⁴ irradiated B7-deficient lentivirally infected dendritic cells in the presence of anti-CD3 (10 μg/ml). The ability of full-length or GPI-anchored B7-1 or B7-2 to provide costimulatory activity was determined from the CD28-dependent increase in proliferation. Proliferation was determined by measuring incorporation of [³H]thymidine (1 μCi per well) during the last 18 h of a 66-h culture by scintillation counting. Purity of all cell isolations was ≥98%. Results shown are representative of two independent experiments. (b) Lentiviral expression of B7-1 reconstitutes susceptibility of B7-deficient Th cells to T-reg-mediated suppression. Th (CD4CD25^–) mock- or lentivirally infected B7-deficient or B7 wt T cells (n = 10⁶) were transferred intravenously into B7 wt RAG-2-deficient recipients (n = 4 per group) in the presence (black bars) or absence (gray bars) of 0.5 × 10⁶ CD4⁺CD25⁺ T cells. Recipients were killed 6 weeks after transfer, and the mandibular, cervical, axillary, inguinal, and mesenteric lymph nodes were then isolated. Total cell numbers were determined by trypan blue exclusion, and the percentage of CD4⁺ T cells was determined by FACS analysis. Transferred Th cells were ≥97% pure, and expression of lentivirally expressed B7 was ≥95% in all groups as confirmed by FACS analysis (Fig. 6). Data shown for each experimental group is the average of four pooled mice.