Characterizing the Immunogenicity of DM-Sensitive and DM-Resistant Antigens

Introduction: The separation of graft-versus-leukemia (GvL) effect from graft-versus-host-disease (GvHD) is a major objective after allogeneic stem cell transplantation. We recently described two types of endogenous HLA class II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, presentation of DM-sensitive antigens rely on co-expression of HLA-DO - the natural inhibitor of HLA-DM. Since the expression of HLA-DO is not upregulated by inflammatory cytokines and restricted to B-cells, dendritic cells and thymic epithelial cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues. Therefore, usage of CD4 T-cells directed against DM-sensitive antigens might allow separation of GvL from GvHD. However, it remains elusive whether immunogenicity and anti-tumorigenic potential of DM-sensitive and DM-resistant antigens have comparable properties in vivo. Methods: Therefore, we sought to create an in vivo system using a DM-sensitive and a DM-resistant variant of the same model antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM or HLA-DO, respectively) to allocate the two model antigens ovalbumin (OVA) and murine Y-chromosome antigen DBY to their category. Furthermore, we introduced one to three amino acid substitutions within the MHC II restricted T-cell epitopes of the two antigens and tested DM-sensitivity or DM-resistance by T-cell activation using proliferation and IFN-g secretion as read-out in vitro. Finally, we vaccinated B6 mice with the generated epitope variants and measured expansion, phenotype and reactivity of OVA- or DBY-specific CD4 T-cells in vivo. Results: By testing T-cell recognition of OVA or DBY on murine B-cell lines overexpressing H2-M and H2-O, respectively, we could show that OVA leads to a more potent T-cell activation in the presence of H2-O demonstrating its DM-sensitive character. In contrast the wildtype epitope of DBY does not rely on H2-O expression for strong T-cell activation and was therefore assessed as DM-resistant antigen. By introducing one to three amino acid substitutions within the T-cell epitope we could generate one further DM-sensitive variant of OVA but also two DM-resistant counterparts. Likewise, we designed both DM-resistant and DM-sensitive epitope variants of murine DBY. To assess T-cell receptor avidity to our epitope variants presented on natural antigen presenting cell