Identification of endogenous peptides recognized by in vivo or in vitro generated alloreactive cytotoxic T lymphocytes: distinct characteristics correlated with CD8 dependence

We studied the molecular basis for CD8 independence of in vivo generated (B M3.3) versus CD8 dependence of in vitro sensitized (KBS.C20/Des) alloreacti ve H-2K(b)-specific cytotoxic T lymphocytes (CTL). Using microcapillary hig h-performance liquid chromatography fractionation of H-2K(b) eluates, mass spectrometry and CTL reconstitution assays, we determined that BM3.3 and KB 5.C20 recognize, respectively, a single peptide (pBM1) expressed on 8,000 H -2K(b) molecules per allogeneic cell, and three distinct peptides (pKB1, 2, 3), each expressed on around 200 H-2K(b) molecules per allogeneic cell. CD 8 (in)dependence was intrinsic to the respective TCR/H-2K(b)-peptide intera ctions. KB5.C20 and BM3.3 TCR illustrate the correlation that appears to ex ist between CD8 dependence/low affinity and in vitro sensitization as oppos ed to low dependency on CD8 and high TCR affinity observed after in vivo se nsitization. The results suggest that CD8-dependent alloreactive CTL obtain ed in vitro with high frequency correspond to low-affinity TCR from the MHC -biased TCR repertoire unpurged by negative selection and have implications for cellular immunotherapeutic approaches.