Sensitivity of undifferentiated, high-TCR density CD8+ cells to methylene groups appended to tumor antigen determines their differentiation or death

CD8(+) cells expressing high numbers of TCR per cell (TCRhi) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CDS+ cells expressing high levels of E75-TCR (TCRhi) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-gamma(+) Perf(-) and to TCRhi IFN-gamma(+) Perf(+) cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCR (hi) Perf(hi) cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCR hi Perf+ CTL required an agonist of optimal CH2 side chain length, which in this study was equal to two CH2 groups appended to E75 at the Gly(4) position. Side chains one CH2 shorter or longer than optimal were either less stimulatory or induced death of TCRhi Perf(+) cells. Differentiation of TCRhi CDS+ cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.