Figure 3. DR1-CII and DR1-HA CAR T cells lyse CD4+ T cells in an antigen specific manner.

CD8+ T cells were transduced with vectors encoding the DR1-CII or DR1-HA CAR and the resulting T cells were incubated with a mixture of CII-specific and HA-specific T cell hybridoma clones at 10:1 effector:target ratio. After 4 hours, the cells were labeled with fluorochrome labeled antibodies specific for CD8, DR1, TCR-BV8, and TCR-BV14, stained with DAPI, and analyzed by flow cytometry. A. Target cells in the absence of CAR T cells, showing roughly an even mix of target cells in the assay. The CII-specific T cells express TCR-BV8, while the HA-specific T-cells express BV14. B. Target cells mixed with non-transduced CD8+ T cells. No lysis of target cells was observed. C. DR1-CII CAR T-cells lyse the T cells specific for CII (0.75% BV8+), but not the HA-specific T cells (98.9% BV14+). D. DR1-HA CAR T cells lyse the HA-specific T cells (1.7% BV14+), but not the CII-specific T cells (97.9% BV8+). E and F. Summary of at least 6 experiments each for the cytolytic efficacy and specificity of the DR1-CII and DR1-HA CAR T cells. Each CAR T cell was highly effective in lysing its appropriate target cell (★ indicates p value <0.00001), whereas NT CD8+ T cells had no effect on the targets. Error bars indicate standard deviation. Data are based on DAPI negative, CD8 negative, and DR negative gates in the flow cytometry data.