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Short-lived protein translation products are proposed to be a major source of substrates for major histocompatibility complex (MHC) class I antigen processing and presentation; however, a direct link between protein stability and the presentation level of MHC class I-peptide complexes has not been made. We have recently discovered that the peptide Tyr((369-377)) , derived from the tyrosinase protein is highly presented by HLA-A2 on the surface of melanoma cells. To examine the molecular mechanisms responsible for this presentation, we compared characteristics of tyrosinase in melanoma cells lines that present high or low levels of HLA-A2-Tyr((369-377)) complexes. We found no correlation between mRNA levels and the levels of HLA-A2-Tyr((369-377)) presentation. Co-localization experiments revealed that, in cell lines presenting low levels of HLA-A2-Tyr((369-377)) complexes, tyrosinase co-localizes with LAMP-1, a melanosome marker, whereas in cell lines presenting high HLA-A2-Tyr((369-377)) levels, tyrosinase localizes to the endoplasmic reticulum. We also observed differences in tyrosinase molecular weight and glycosylation composition as well as major differences in protein stability (t(1/2) ). By stabilizing the tyrosinase protein, we observed a dramatic decrease in HLA-A2-tyrosinase presentation. Our findings suggest that aberrant processing and instability of tyrosinase are responsible for the high presentation of HLA-A2-Tyr((369-377)) complexes and thus shed new light on the relationship between intracellular processing, stability of proteins, and MHC-restricted peptide presentation., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Effector CTL contribute to tumoral immunity by killing tumor cells through secretion of cytotoxic granules and cytokines. Activation of CTL requires specific recognition of cognate peptide-MHC-I (pMHC) complexes on the tumor cell surface by the CTL TCR. It has been suggested that the half-life (t(1/2)) of the TCR/pMHC interaction modulates the activation of naïve CD8(+) T cells; however, it remains unknown whether CTL effector function can also be regulated by the TCR/pMHC t(1/2). Here, we have studied CTL activity in response to tumor cells loaded with pMHC that bind the TCR with different t(1/2). We observed that the TCR/pMHC t(1/2) can differentially regulate CTL effector function during the interaction with tumor cells and defines the nature of anti-tumoral CTL responses in vivo. Although prolonged TCR/pMHC t(1/2) promoted only partial expression of cytotoxic molecules, short t(1/2) induced partial polarization of lytic machinery toward target cells. In contrast, intermediate TCR/pMHC t(1/2) induced strong expression of cytotoxic molecules, efficient polarization of lytic machinery and subsequent release of toxic granules by CTL that killed tumor cells. Consistently, efficient in vivo CTL-mediated tumor clearance was only observed for tumors expressing intermediate t(1/2) pMHC ligands. These data suggest that there is an optimal TCR/pMHC t(1/2) for efficient CTL activity.

Based on the hypothesis that immune outcome can be influenced by the form of antigen administered and its ability to access various antigen-processing pathways, we targeted the 63 kDa fragment of protective antigen (PA) of Bacillus anthracis to various subcellular locations by DNA chimeras bearing a set of signal sequences. These targeting signals, namely, lysosome-associated membrane protein 1 (LAMP1), tissue plasminogen activator (TPA) and ubiquitin, encoded various forms of PA viz. lysosomal, secreted and cytosolic, respectively. Examination of IgG subclass distribution arising as a result of DNA vaccination indicated a higher IgG1:IgG2a ratio whenever the groups were immunized with chimeras bearing TPA, LAMP1 signals alone or when combined together. Importantly, high end-point titers of IgG antibodies were maintained until 24 wk. It was paralleled by high avidity toxin neutralizing antibodies (TNA) and effective cellular adaptive immunity in the systemic compartment. Anti-PA and TNA titers of approximately 10(5) and approximately 10(3), respectively, provided protection to approximately 90% of vaccinated animals in the group pTPA-PA63-LAMP1. A significant correlation was found between survival percentage and post-challenge anti-PA titers and TNA titers. Overall, immune kinetics pointed that differential processing through various compartments gave rise to qualitative differences in the immune response generated by various chimeras.