Figure 2. High avidity CTL lysis of tumor cells in vitro and suppression of tumor growth in vivo is inhibited by low avidity CTLs in an antigen-specific manner.

Assays were performed as described in legend for Figure 1. Gp100-specific (A) or MART-specific (B) high avidity CTL were combined with Gp100-specific or MART-specific low avidity CTL, or FLU-specific CTL. Percent inhibition of melanoma cell lysis was calculated as in legend for Fig 1. Bars represent standard deviation between 3 separate assays, each performed in triplicate. (C) Suppression of tumor growth in vivo by high avidity CTLs was inhibited by low avidity CTLs, but not by a CTL clone of irrelevant specificity (flu peptide specific CTL). C57BL SCID (common gamma/RAG2 double knockout) mice were injected subcutaneously with mel526 human tumor cells. Results indicate tumor size 40 days post injection of G209n-specific high avidity CTL (tu hi), G209n-specific high avidity CTL combined with G209n-specific low avidity CTL (tu hi lo), or G209n-specific high avidity CTL combined with flu-specific CTL (tu hi flu). (D) Reduction of MFI of iTAg G209 specific MHC Class I Human Tetramer-SA-PE (normalized to initial MFI at 0 minutes) from CD8 labeled low (○) and high (□) avidity G209 specific CTL clones were plotted versus time elapsed post incubation with blocking HLA-A2 fab fragment. Also provided are control plots of low (Δ) and high (◇) avidity CTL clones that are tetramer/CD8 stained and collected at same time points without addition of HLA-A2 fab fragment. Results are representative of three separate experiments. Data were fitted using a linear mixed effects model to test intercept and slope over time by group effects. Calculations were based on the nlme package in R version 2.12.1.