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Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background., (Copyright 2004 Wiley-Liss, Inc.)

MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1-specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot analysis in these cells. The profile of activated T-cell reactivity with different MUC1-specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2-based O-glycans, as opposed to the core 1 structures that dominate in the cancer-associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin-like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites.

Generation of the HLA-A0201 (A2) influenza Matrix 58-66 epitope contained within the full-length Matrix protein is impaired in cells lacking the proteasome subunits low molecular protein 2 (LMP2) and LMP7. This Ag presentation block can be relieved by transfecting the wild-type LMP7 cDNA into LMP7-deficient cells. A mutated form of LMP7, lacking the two threonines at the catalytic active site, was equally capable of relieving the block in presentation of the influenza Matrix A2 epitope. These observations were extended by analyzing whether modification of the influenza Matrix protein could overcome the block in presentation of the A2 Matrix epitope. Expression of either a rapidly degraded form of the full-length Matrix protein or shorter Matrix fragments led to an efficient presentation of the A2 influenza Matrix epitope by LMP7-negative cells. These findings demonstrate two main points: 1) LMP7 incorporation into the proteasome is of greater importance for the generation of the influenza A2 Matrix epitope than the presence of the LMP7's catalytic site; and 2) the interplay between cytosolic proteases and stability of target proteins is of importance in optimization of Ag presentation. These observations may have relevance to the immunodominance of tumor and viral epitopes and raise the possibility that generation of shorter protein fragments could be a mechanism to ensure optimal Ag presentation by cells expressing low levels of LMP7.