T-lymphocyte mediated killing of TAP-deficient tumor cells and immunodominance of minor histocompatibility antigens

Abstract: Target structures and regulation of cytotoxic T Iymphocyte (CTL) mediated responses is the subject of this thesis. One main finding is that cells devoid of a functional transporter associated with antigen processing (TAP) appear to express novel epitopes that can be recognized by syngenic CTL. Since downregulation of TAP-function occur in many human tumors, and some virus infections, these epitopes represent a novel CTL target with potential applicability in immunotherapy. Another main finding is that subdominance of certain epitopes derived from minor histocompatibility (mH) antigens does not appear to be caused by insufficient antigen presentation. We propose that immunodominance in this CTL response is mediated by responding T-lymphocytes. In vitro, the CTL response against subdominant epitopes was inhibited by T-cells responding against dominant epitopes presented by the same APC. A murine mutant Iymphoma, RMA-S, had prior to this study been selected for low levels of cell surface MHC class I expression. In paper I, RMA-S cells were found to be resistant to killing by mH-, viral- and tumor-specific CTL, while it was still sensitive to killing by allo-H-2 specific CTL. This correlated with rejection patterns in vivo. The study suggested that RMA-S had a defect in the presentation of cellularly derived, antigens. RMA-S was later shown, by other investigators, to be deficient in TAP-function. In paper II, we found surprisingly that RMA-S cells could elicit a potent CTL response in syngenic C57BL/6 (B6) mice, if transfected with the gene for the costimulatory molecule B7-1 (CD80). Efficient killing required the presence of MHC class I molecules, and the absence of TAP-function, in the target cells. CTL elicited by RMA-S.B7- 1 killed TAP-deficient murine transformed and non-transformed cells, while the corresponding TAP-expressing cells were considerably less sensitive, or resistant, to lysis. These CTL also killed the human TAP-deficient cell line T2 transfected with the gene encoding the murine MHC class I molecule H-2-Kb. Immunization with RMA-S.B7-1, protected syngenic B6 mice from in vivo outgrowth of non-transfected RMA-S tumor cells. The epitopes recognized were termed "T cell epitopes associated with TAP-deficiency" (TAD). In follow-up studies, presented in the thesis, we found that B6 CTL elicited by syngenic TAP deficient cells paradoxically killed several TAP-expressing murine tumor cell lines, while non transformed, TAP-expressing B6 Con A blasts, remained resistant to Iysis. At least for the TAP expressing tumor cell line RMA, the killing was directed against TAD. These findings suggest that TAD can be expressed by several different tumor cell lines, and may constitute a novel shared tumor associated antigen (TAA). In paper III, we used an established transplantation model to identify dominant and subdominant epitopes from mH antigens as cellularly eluted peptides. The CTL response of B6 mice against cells of BALB.B origin, a mH barrier of at least 40 antigens, was directed against three peaks from an HPLC separated peptide eluate from BALB.B cells. One of these immunodominant epitopes was found to be associated with the mH locus H-28C. Furthermore, three subdominant epitopes associated with the loci H-8C, H-l9C and H-25C were identified. These epitopes elicited CTL when present alone in cells from mH congenic mice, but not when present in BALB.B cells, also expressing the dominant epitopes. Restriction elements for all epitopes were determined by the recognition of eluate fractions loaded on the human cell line T2, transfected with the genes encoding the murine H-2 Kb or -Db molecules. In paper IV, subdominant BALB.B epitopes were found to be presented by BALB.B dendritic cells (DC). Furthermore, they were shown to be present in cell surface eluates of BALB.B cells in amounts sufficient for CTL activation. This suggested that the mechanism for immunodominance in the CTL response against this mH barrier is not likely to be explained by insufficient presentation of subdominant epitopes. In vitro, we demonstrated that the CTL response against the subdominant BALB.B epitopes was inhibited by an immune response against dominant BALB.B epitopes. This suggests that immunodominance in this CTL response is mediated by responding T-lymphocytes. In paper V, we investigated immunodominance in the CTL response against human histocompatibility antigens. We studied the recognition pattern of CTL clones from patients suffering from GvHD, or bone marrow rejection, following bone marrow transplantation (BMT). The recognition pattern suggests an immunodominance hierarchy. In the presence of an MHC class I mismatch, all CTL clones were directed against the mismatched molecule. In the presence of a mismatch for the mH antigen HA-1, CTL clones were directed against HA-1, in one case totally overshadowing the recognition of an H-Y mismatch. The potential development and application of the findings are outlined.