Article Figures & Data
Figures
- FIGURE 1.
Generation of anti-TRP-2 CTL with high and low peptide concentrations. INF-γ release of primary CTL cultures (day 9) generated with either 10−5 M (A) or 10−9 M (B) TRP-2180–188 peptide in response to 293Kb target cells pulsed with peptide and tumor targets. Both naive and immune animals generated low avidity CTL when 10−5 M of in vitro-stimulating peptide was used. Only immune animals were able to yield Ag-specific CTL when the concentration of in vitro-stimulating peptide was reduced to 10−9 M.
- FIGURE 2.
Generation of anti-p15E CTL with high and low peptide concentrations. INF-γ release of primary CTL cultures (day 9) generated with either 10−5 M or 10−9 M p15e604–611 peptide in response to 293Kb cells pulsed peptide and tumor targets. As was true for TRP-2, both naïve and immune animals generated low avidity CTL when 10−5 M of in vitro-stimulating peptide was used A. Only immune animals were able to yield Ag-specific CTL when the concentration of in vitro-stimulating peptide was reduced to 10−9 M (B).
- FIGURE 3.
High concentrations of in vitro-stimulating peptide are detrimental to the generation of high avidity CTL. Splenocytes from B16-immune animals (A) or from animals immune to the p15E-expressing tumor MCA205 (B) were stimulated in vitro with titered concentrations of the appropriate peptide epitope. Primary cultures were tested (day 9) for the ability to release IFN-γ in response to tumor targets or to 293Kb cells pulsed with the appropriate autologous peptide. High concentrations of in vitro-stimulating peptide were detrimental to development of high avidity CTL for both these tumor-associated Ags. Results are representative of at least four independent experiments for each Ag.
- FIGURE 4.
Selective expansion of high or low avidity CTL lines. CTL lines were generated by restimulation of primary cultures as described in Materials and Methods. TRP-2-specific CTL lines expanded over four in vitro stimulations with high concentrations of peptide (10−5 M) demonstrated low avidity for peptide-pulsed targets. CTL expanded in low concentrations (10−9 M) of stimulating peptide demonstrated high avidity for the peptide Ag (A). Similarly, high and low avidity CTL lines were able to be generated for the p15E Ag (B).
- FIGURE 5.
Treatment of day 4 B16 pulmonary metastases by high and low avidity CTL lines. A total of 3 × 106 high and low avidity TRP-2-specific CTL were adoptively transferred into C57BL/6 mice bearing day 4 pulmonary metastases as described in Materials and Methods. Fourteen days later mice were sacrificed an the number of pulmonary metastases were enumerated in a coded, blinded fashion. Two-tailed p values were calculated using the Wilcoxon Rank Sum test. In a separate experiment, 7 × 104 high or low avidity p15E-specific CTL were used to treat 4-day established B16 pulmonary metastases (B). For both Ags, only the high avidity CTL effectively reduced the number of pulmonary metastases. Results are representative of two independent experiments for each Ag.
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