The association of a specific autoantibody response with distinct disease phenotypes is observed in both autoimmune diseases and cancer. Although the mechanisms underlying such an association remain unclear, it is likely that unique properties of disease-specific autoantigens that are expressed in the relevant target cells within the immunizing microenvironment play an important role. It has recently been observed that the majority of autoantigens targeted across the spectrum of systemic autoimmune diseases (but not non-autoantigens) are selectively cleaved by the cytotoxic lymphocyte granule protease granzyme B, generating unique fragments not observed during other forms of cell death. In spite of the strength and specificity of this association, the relevance of susceptibility to cleavage by granzyme B has remained unclear. Possibilities include a role for cleavage by granzyme B (perhaps altering the subsequent processing of the antigen), or a role for the granzyme B cleavage site itself in influencing antigen loading and presentation.
Our recent studies using hepatocellular carcinoma (HCC) and the HCC autoantigen, nucleophosmin/B23, as a model system have allowed us to define unique structural features of this molecule expressed exclusively in the relevant disease microenvironment. Thus, B23 is selectively susceptible to cleavage by granzyme B in extracts of neoplastic, compared to non-neoplastic liver. Additional studies in vitro and in tumors demonstrated that this increased susceptibility to cleavage by granzyme B arise when B23 translation is initiated at methionine7, generating a unique SDS-stable conformation in which the granzyme B cleavage site loop is exposed. The recent observation that the presence or absence of single protease cleavage sites can completely alter the final processing of that antigen and determine which peptides are loaded onto MHC class II molecules for presentation to T cells is, therefore, particularly relevant.
We propose that the unique conformation and protease cleavability of tumor B23 provides the first opportunity to directly address the processing of, and immune responses to, the relevant immunogenic form of an autoantigen and to define the function of the granzyme B cleavage loop in antigen processing and presentation. The proposed studies will therefore define whether the N-terminally truncated form of B23 (which assumes an SDS-stable oligomeric structure, and exposes an otherwise cryptic protease cleavage site) is processed differently from the wild-type form of the protein (which is expressed under almost all other circumstances). The different forms of the immunogen will be produced in vitro, and T cell clones recognizing B23 will be generated from patients with HCC and B23 autoantibodies. Further understanding of the mechanism(s) responsible for determining such changes in conformation may have practical implications in both the therapeutic modulation of this response in systemic autoimmune disease and enhancement of this response in tumor immunotherapy.