A) Human T cells were activated using anti-CD3/CD28 microbeads and transduced with high-titer lentivirus encoding Ly95 TCR. After expansion in vitro, FACS analysis was performed using anti-CD4 and anti-TCR (TCRVβ13.1) antibodies. Results show greater than 50% of cultured T cells expressing the engineered TCR with the majority of the lymphocyte population being CD8 T cells.
B) Ly95 T cells alone or Ly95 T cells co-cultured with A549-A2 or A549-A2-ESO cells at an E:T ratio of 10:1 for 24 hours, were evaluated for upregulation of activation markers CD25, IFNγ, Granzyme B, and CD107a. Only A549-A2-ESO cells stimulated the T cells.
C) Ly95 T cells were co-cultured with different human tumor cell lines including the target A549-A2-ESO cells for 18 hours at E:T ratios of 1:1, 5:1, and 10:1 for 18hrs. Ly95 T cells demonstrated (A) efficient killing of and (B) high secretion of IFNγ in response to A549-A2-ESO tumor cells in a dose dependent manner. (* p<0.05, ** p<0.01).