TAP-INDEPENDENT MHC CLASS-I PEPTIDE ANTIGEN PRESENTATION TO ALLOREACTIVE CTL IS ENHANCED BY TARGET-CELL INCUBATION AT SUBPHYSIOLOGICAL TEMPERATURES

We investigated the peptide dependency of a group of CD8(+) anti-HLA-B 7 alloreactive CTL, The CTL killed target cells after acid denaturatio n of more than 98% of target cell surface peptide/MHC class I complexe s. The CTL also killed TAP(-) HLA-B7-transfected T2 (T2B7) cells. The killing was enhanced by target cell incubation at 26 degrees C. Despit e these findings, which suggested peptide-independent allorecognition, CTL-mediated cytolysis was reduced or abolished by several point muta tions affecting the HLA-B7 peptide-binding groove. Acid denaturation o f HLA complexes on T2B7 cells prohibited CTL recognition. CTL recognit ion was restored by T2B7 cell incubation with beta(2)-microglobulin an d a single HPLC fraction containing peptides extracted from TAP(+)HLA- B7(+) cells, but not by any of a panel of 17 synthetic HLA-B7-binding peptides. These findings indicated that CTL allorecognition was peptid e specific. Sensitizing peptide was extracted from T2B7 cells only aft er incubation at 26 degrees C. The amount of peptide detected in TAP() cells was at least 10-fold and 100-fold greater than that detected i n TAP(-) cells incubated at 26 degrees C and at 37 degrees C, respecti vely. TAP-independent peptide epitope presentation was sensitive to tr eatment with brefeldin A, but not sensitive to treatment with chloroqu ine, consistent with an endogenous peptide source. We propose that sub physiologic temperature incubation can enhance peptide/MHC class I pre sentation in the total absence of TAP function.