Figure 1.

H3.3K27M peptide is recognized by a high avidity human TCR isolated from TCR transgenic mice. (A) Representation of a reactive CD8⁺ cell population secreting IFNγ on H3.3K27M_26-35 peptide (RMSAPSTGGV) restimulation of splenocytes isolated from immunized ABabDII mice, measured by intracellular staining 7 days after the last immunization. Stimulation without peptide (Ø) was used as a negative control. (B) Transgenic TCR expression levels in human PBMCs after retroviral transduction with H3.3K27M-specific TCRs 27633 and 1H5 (8), as well as CDK4-specific control TCR14/35 (11) and untransduced control, were measured by flow cytometry staining for the mouse TCRβ constant region. Percentage of positive transduced CD8⁺ T cells are shown in the top right quadrant in (A, B), cells were gated on CD3⁺. (C, D) Levels of IFNγ secretion of H3.3K27M-specific TCRs 27633 and 1H5-transduced PBMCs, cocultured with T2 cells loaded with decreasing concentrations of either H3.3K27M mutant (C) or H3.3 wild-type (WT) (D) peptide. A total of 10⁴ CD8⁺ transduced-TCR⁺ cells were cocultured in a 1:1 E:T ratio with T2 cells. P/I represents maximum IFNγ secretion from PBMCs stimulated with PMA/Ionomycin. The experiment was performed three times with similar results and graphs represent means of quadruplicate cultures±SD. TCR, T cell receptor; PBMC, peripheral blood mononuclear cells; E:T ratio, effector to target ratio.