Major histocompatibility complex (MHC) restriction of antigen presentation of a single mucin1 (MUC1) variable number of tandem repeats peptide (VNTR1) was examined by generating cytotoxic T lymphocytes (CTL) derived from peripheral blood mononuclear cells (PBMC) stimulated with a single repeat MUC1 peptide presented by allogeneic (MHC-independent) or autologous (MHC-dependent) Epstein-Barr Virus (EBV) immortalized B lymphocytes. The ability to generate greater CTL activity against MUC1-expressing tumor cells by stimulation with autologous versus allogeneic EBV-B supports the hypothesis that presentation of a single repeat of MUC1 peptide is MHC-restricted (MHC-dependent).

Prostate cancers express MUC1, but nearly all metastatic cells lack HLA class I molecules. Thus, a lymphocyte population that can sense its antigenic environment, while also able to react to stimuli of natural killer (NK) cells, may be a more versatile effector cell population for antitumor immune responses. Herein, we report that tumor-specific MUC1 peptide, interleukin 2, and interleukin 12 act synergistically to stimulate the ex vivo expansion of CD8 CD56 T cells and CD8 CD56 natural killer T (NKT) cells from the peripheral blood mononuclear cells of prostate cancer patients, as well as healthy male and female donors. Both the CD56 NKT cells and CD56 T cells lysed allogeneic mucin-bearing target cells, as well as NK target cells, but not lymphokine-activated killer target cells. However, the CD56 NKT cells displayed a 2-fold greater cytolytic activity than the CD56 T cells. The mucin-specific cytolytic activity and NK cytolytic activities for both lymphocyte populations were independent of HLA class I and CD1 molecules. The CD56 T cells up-regulated CD56 with continued antigenic stimulation in the presence of interleukin 12, suggesting that CD8 CD56 T cells are NKT cells. However, CD56 NKT cells expand poorly to continued stimulation. All mucin-stimulated NKT cells exhibited the activated/memory CD45RO phenotype. The NKT cell lines express the / T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the V 24V 11 TCR typically associated with NKT cells. Whereas CD161 is generally considered a marker of NKT cells, the mucin-stimulated NKT cells did not express this marker. Thus, we have described two phenotypically distinct NKT types that do not display a biased TCR repertoire, but do display specificity for a tumor-specific peptide antigen (CTL-like activity), as well as HLA class I-deficient target cells (NK-like activity).