Human mucin 1 (MUC1) is a highly glycosylated transmembrane glycoprotein that is expressed on the luminal surface of ductal epithelial cells. Human adenocarcinomas overexpress MUC1 as a tumor-associated antigen (TAA) that presents to the immune system peptide epitopes and glycopeptide epitopes with tumor specific carbohydrates, such as mono- and disaccharides known as Tn, sialyl-Tn, and TF antigens. Studies in MUC1 transgenic (MUC1-Tg) mice have indicated that, compared to transgene negative wild-type (WT) mice, the MUC1-Tg immune system maintains a certain level of tolerance to the MUC1 peptide, reflected most notably in decreased CD4 T cell help. We made a novel observation that in contrast to suppressed responses to the MUC1-peptide vaccine, vaccination of MUC1-Tg mice with tumor-associated MUC1 glycopeptide resulted in effective anti-MUC1 immunity similar to that elicited in WT mice. We hypothesized that the tumor-associated glycopeptides were seen as foreign and therefore not subject to tolerance. To study CD4 T cell responses to MUC1 glycopeptides we generated glycopeptide GST(GalNAc;Tn)A specific, MHC-Class II restricted CD4 T cell hybridoma, RF6. We cloned the RF6 TCR (RFT; Vá4.1Já16-Vâ15Jâ1.3) and generated TCR transgenic mice RFT-Tg. Using the RFT-Tg mice and the previously generated MUC1 peptide-specific TCR transgenic VFT-Tg mice, we show that peptide-specific VFT CD4 T cells transferred to MUC1-Tg mice are suppressed through mechanisms of peripheral tolerance, which are not induced against MUC1-glycopeptide specific RFT CD4 T cells. We show that peptide-specific CD4 T cells are transiently activated upon transfer into MUC1-Tg mice, suggesting that MUC1-peptide epitope is presented in the periphery in healthy mice as well as in tumor bearing mice. In contrast, MUC1-glycopeptide epitopes are tumor specific and thus treated as foreign antigens in MUC1-Tg mice, resulting in effective activation of glycopeptide-specific CD4 T cells. Simultaneous activation of glycopeptide-specific T cells can break tolerance of peptide-specific T cells. Our findings with MUC1 apply to other TAA that contain some epitopes that are "self" and subject to self tolerance and other epitopes that are tumor specific ("foreign") and not affected by tolerance. Understanding this distinction is very important in the development of effective and safe cancer vaccines
