Fig. 5.
Fig. 5. Effect of B7-H1 costimulation in T-cell responses to allogeneic and tumor antigens. / (A) The expression of mB7-H1 by transfected P815 tumor cells. P815 tumor cells were transfected with pcDNA3 vector (Mock; left panel) or B7-H1.pcDNA3 (B7-H1+ P815; right panel). Cells were stained by rabbit anti–B7-H1 antibodies (bold line) or control rabbit immunoglobulin (dashed line) followed by FITC-conjugated goat antirabbit IgG. In addition, B7-H1+ P815 cells were stained by anti–B7-H1 antibodies in the presence of the peptide of B7-H1, which had been used for immunization to make the antibodies (solid line). (B) The effect of mB7-H1 expression by P815 on T-cell proliferative responses to allogeneic antigen. NW-purified T cells obtained from B6 mice (H-2b) were cultured with irradiated mock, mB7-1+, or mB7-H1+ P815 cells (H-2d) at the indicated responder/stimulator (R/S) ratio. After 3-day culture, the proliferative responses of T cells were determined by 3H-TdR incorporation assay. (C) The effect of mB7-H1 expression by P815 on the generation of alloreactive CTL in vitro. NW-purified T cells (2.5 × 106/mL) obtained from B6 spleen cells were cultured with irradiated mock, mB7-1+, or mB7-H1+ P815 cells at 2.5 × 105/mL for 5 days. The cells were harvested, and their cytolytic activity against allogeneic P815 (left panel) or syngeneic EL4 cells (right panel) was determined in a standard 51Cr-release assay. (D) The effect of mB7-H1Ig on the proliferative responses to allogeneic antigen. NW-purified T cells of BALB/c mice (H-2d) (1 × 106/mL) were cocultured with irradiated B6 spleen cells (H-2b) in the presence of immobilized control immunoglobulin or mB7-H1Ig at the indicated responder/stimulator (R/S) ratio. Anti-CD4 (GK1.5) or control rat immunoglobulin was added at 10 μg/mL. After 3-day culture, 3H-TdR incorporation was measured. (E) The effect of mB7-H1Ig on the generation of alloreactive CTL in vitro. NW-T cells (H-2d) (3 × 106) were cultured with 6 × 106 irradiated B6 spleen cells (H-2b) in the presence of immobilized control immunoglobulin or mB7-H1Ig in 2mL of 24-well plates for 5 days. The CTL activity against allogeneic EL4 (left panel) or syngeneic P815 (right panel) was determined in a standard 51Cr-release assay. (F) The effect of B7-H1 expression by P815 cells on the induction of P815-specific CTL in vivo. DBA/2 mice were injected subcutaneously with live cells of mock.P815, mB7-1+ P815, or mB7-H1+ P815 at 1 × 106/mouse. The draining lymph nodes were removed 7 to 10 days after tumor injection, and prepared suspension cells were restimulated with irradiated wild-type P815 cells for 5 days. The cells were harvested, and their cytolytic activity against P815 (left panel) and L1210 cells (right side) was determined in a standard 51Cr release assay. Data represent one experiment of 3 or more. Results are expressed as the mean ± SD of triplicate wells.

Effect of B7-H1 costimulation in T-cell responses to allogeneic and tumor antigens.

(A) The expression of mB7-H1 by transfected P815 tumor cells. P815 tumor cells were transfected with pcDNA3 vector (Mock; left panel) or B7-H1.pcDNA3 (B7-H1+ P815; right panel). Cells were stained by rabbit anti–B7-H1 antibodies (bold line) or control rabbit immunoglobulin (dashed line) followed by FITC-conjugated goat antirabbit IgG. In addition, B7-H1+ P815 cells were stained by anti–B7-H1 antibodies in the presence of the peptide of B7-H1, which had been used for immunization to make the antibodies (solid line). (B) The effect of mB7-H1 expression by P815 on T-cell proliferative responses to allogeneic antigen. NW-purified T cells obtained from B6 mice (H-2b) were cultured with irradiated mock, mB7-1+, or mB7-H1+ P815 cells (H-2d) at the indicated responder/stimulator (R/S) ratio. After 3-day culture, the proliferative responses of T cells were determined by 3H-TdR incorporation assay. (C) The effect of mB7-H1 expression by P815 on the generation of alloreactive CTL in vitro. NW-purified T cells (2.5 × 106/mL) obtained from B6 spleen cells were cultured with irradiated mock, mB7-1+, or mB7-H1+ P815 cells at 2.5 × 105/mL for 5 days. The cells were harvested, and their cytolytic activity against allogeneic P815 (left panel) or syngeneic EL4 cells (right panel) was determined in a standard 51Cr-release assay. (D) The effect of mB7-H1Ig on the proliferative responses to allogeneic antigen. NW-purified T cells of BALB/c mice (H-2d) (1 × 106/mL) were cocultured with irradiated B6 spleen cells (H-2b) in the presence of immobilized control immunoglobulin or mB7-H1Ig at the indicated responder/stimulator (R/S) ratio. Anti-CD4 (GK1.5) or control rat immunoglobulin was added at 10 μg/mL. After 3-day culture, 3H-TdR incorporation was measured. (E) The effect of mB7-H1Ig on the generation of alloreactive CTL in vitro. NW-T cells (H-2d) (3 × 106) were cultured with 6 × 106 irradiated B6 spleen cells (H-2b) in the presence of immobilized control immunoglobulin or mB7-H1Ig in 2mL of 24-well plates for 5 days. The CTL activity against allogeneic EL4 (left panel) or syngeneic P815 (right panel) was determined in a standard 51Cr-release assay. (F) The effect of B7-H1 expression by P815 cells on the induction of P815-specific CTL in vivo. DBA/2 mice were injected subcutaneously with live cells of mock.P815, mB7-1+ P815, or mB7-H1+ P815 at 1 × 106/mouse. The draining lymph nodes were removed 7 to 10 days after tumor injection, and prepared suspension cells were restimulated with irradiated wild-type P815 cells for 5 days. The cells were harvested, and their cytolytic activity against P815 (left panel) and L1210 cells (right side) was determined in a standard 51Cr release assay. Data represent one experiment of 3 or more. Results are expressed as the mean ± SD of triplicate wells.

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