Table 1.
C57/BL6 mice used in each different experimental group.
Figure 1.
Inhibition of B7-1 expressing RMA-S tumor growth in Lass5-antigen immunized mice.
A: a) B7-1 expression in the transfectants. B7-1 expression was determined by FACS assay using FITC-conjugated anti-mouse CD80 mAb; b, c and d) NK1.1 population in mouse splenocytes were detected by anti-NK1.1 mAb. b) Normal mouse splenocytes, c) and d) the splenocytes from tumor-immunized and anti-NK1.1 mAb treated mouse (c: on the tumor cell challenge time and d: end of experiment). B and C: In vivo tumor growth assays. B: e) mice immunized with PBS (0), Lass5-peptide-pulsed and mitomycin-c-treated RMA-S/pUB (1) or RMA-S/B7-1 (2) cells. After immunization, the mice were challenged s.c with RMA-S/pUB or RMA-S/B7-1 cells. The insert indicates tumor growth during the time point of the initial tumor cell injection through two weeks. f) Mice immunized with Lass5-peptide-pulsed and mitomycin-c-treated RMA-S/pUB cells and followed by anti-NK1.1 mAb treatment. Afterwards, the mice were challenged s.c with RMA-S/pUB or RMA-S/B7-1 cells. Statistical analysis of tumor sizes indicated significant differences between RMA-S/pUB ‘’ and RMA-S/B7-1 ‘*’ cell challenge groups at relevant time points (P value≤0.05 or 0.01). C: Tumor sizes at the endpoint were shown in the mice immunized with Lass5-peptide-pulsed and mitomycin-c-treated RMA-S/pUB or RMA-S/B7-1 cells and followed by challenge with live RMA-S/pUB or RMA-S/B7-1 cells.
Table 2.
Tumor formation in the mouse groups during the initial time points.
Figure 2.
Efficient killing of B7-1 expressing tumor cells by bulk culture T cells.
In vitro 51Cr-release assays were conducted. (A): Bulk-culture T effectors were generated by immunizing mice with Lass5 peptide-pulsed mitomycin-c-treated RMA-S/pUB cells. (B): Bulk-culture T effectors were generated by immunizing mice with Lass5 peptide-pulsed mitomycin-c-treated RMA-S/B7-1 cells. One out of three experiments with similar results was shown. * indicated that P-values were less than 0.05.
Figure 3.
Effects of anti-CD80 and CD28 antibodies on reducing killing activities of bulk culture T effectors against RMA-S/B7-1 cells.
Lift-panel (A and C): The cytolytic T effectors were generated by immunization of mice with mitomycin-c-treated RMA-S/pUB cells pulsed with Lass5 peptide. Right-panel (B and D): The cytolytic T effectors were generated by immunization of mice with mitomycin-c-treated RMA-S/B7-1 cells pulsed with Lass5 peptide. Up-panel (A and B): 51Cr-labeled RMA-S/B7-1 and RMA-S/pUB target cells were incubated with either anti-mouse B7-1 mAb or relevant IgG-control. After incubation, the cells were then incubated with antigen-specific bulk culture T effectors for in vitro 51Cr-release assays. Bottom-panel (C and D): Cytolytic bulk culture T effectors were incubated with either anti-mouse CD28 mAb or relevant IgG-control. After incubation, the T-cells were then incubated with 51Cr-labeled RMA-S/B7-1 and RMA-S/pUB target cells for in vitro 51Cr-release assays. ** indicated that P-values were less than 0.05 among ‘RMA-S/B7-1+ Isotype’ and other targets at each ‘Target: Effector’ ratio.
Figure 4.
Importance of B7-1:CD28 axis in enhancing a Lass5 specific LnB5 T-cell clone activation.
The RMA-S/pUB and RMA-S/B7-1 transfectants were used as targets recognized by a Lass5 specific LnB5 T-cell clone. Lass5 specific T-cell clone activation detected by the intracellular IFN-gamma release assays were conducted with stimulators RMA-S/pUB and RMA-S/B7-1 cells in (A) to (D). (A): 8×103 T-cells were incubated with indicated amounts of RMA-S/pUB and RMA-S/B7-1 cells. (B): 8×103 T-cells were incubated with 1×105 stimulators that previously incubated with either anti-B7-1 mAb or isotype control (for RMA-S/B7-1). (C): 8×103 T cells were incubated with either anti-CD28 mAb or isotype control before co-culture with 1×105 stimulators (RMA-S/pUB or RMA-S/B7-1). (D): Before co-culture of the T-cells and stimulators, 8×103 T-cells were incubated with either anti-CD28 mAb or Isotype control and 1×105 RMA-S/B7-1 stimulator cells were incubated with either anti-B7-1 mAb or Isotype control. One out of at least two experiments with similar results was shown. * and ** indicated that P-values were less than 0.05.
Table 3.
Lass5 mRNA expression in RMA-S transfectants.
Figure 5.
Increase in Lass5 expression Bypasses B7-1/CD28 requirement for T effectors’ response.
Lass5 specific LnB5 T-cell clone (A) and T-cell bulk culture (B) were used to determine B7-1/CD28 requirement. (A): Lass5 high expressing RMA-S/pUB.Trh4 and RMA-S/B7-1.Trh4 cells were used as targets that were recognized by LnB5 T-cell clone. The antibodies against CD80 (B7-1) or CD28 molecules were used to block B7-1/CD28 axis. The isotype Ig was used as a control. (B): Lass5-peptide (50 micromole) pulsed RMA-S/pUB and RMA-S/B7-1 cells were used as targets that were recognized by T-cell bulk culture for 51Cr-release assays. Pep means Lass5 peptide. One out of two experiments with similar results for each assay was shown. * * and *** indicated no statistical significance.