Fig 6. In vitro assessment of mutation-reactive T cells for recognition of ID8-G7 tumor cells.

Mice were vaccinated as in Fig 2, and splenocytes were harvested on day 28. OT-I splenocytes were used as positive controls. Splenocytes (5 x 10⁵ per well) were stimulated in duplicate with cognate mutant peptide (20 μg/ml), media alone, or ID8-G7 tumor cells (10⁵ cells per well) and assessed by A. IFN-γ ELISPOT, or B. IL-2 ELISPOT. C. To assess tumor recognition by mutation-reactive CD4 T cells, 4-1BB⁺OX40⁺ CD4 T cells from the above mice were FACS purified and cultured in recombinant murine IL-2 (100 U/ml). ID8-G7 tumor cells were incubated in IFN-γ (100 U/ml) for 72 hours to cause upregulation of MHCI and MHCII. D. Sorted and expanded CD4 T cells (10⁴ per well) were stimulated in duplicate with either 20 μg/ml of cognate mutant peptide, media, ID8-G7 tumor cells or ID8-G7 tumor cells incubated in IFN-γ for 72 hours. OT-I T cells were used as a positive control for tumor cell recognition. Bars and lines represent mean and standard deviation of duplicate wells, respectively.