Hypochlorous acid (HOCl)-treated whole tumor cell lysates (Ox-L) have been shown to be more immunogenic when used as an antigen source for therapeutic dendritic cell (DC)-based vaccines, improving downstream immune responses both in vitro and in vivo. However, the mechanisms behind the improved immunogenicity are still elusive. To address this question, we conducted a proteomic and immunopeptidomics analyses to map modifications and alterations introduced by HOCl treatment using a human melanoma cell line as a model system. First, we show that one-hour HOCl incubation readily induces extensive protein oxidation, mitochondrial biogenesis, and increased expression of chaperones and antioxidant proteins, all features indicative of an activation of oxidative stress-response pathways. Characterization of the DC proteome after loading with HOCl treated tumor lysate (Ox-L) showed no significant difference compared to loading with untreated whole tumor lysate (FT-L). On the other hand, detailed immunopeptidomic analyses on monocyte-derived DCs (mo-DCs) revealed a great increase in human leukocyte antigen class II (HLA-II) presentation in mo-DCs loaded with Ox-L compared to the FT-L control. Further, 2026 HLA-II ligands uniquely presented on Ox-L-loaded mo-DCs were identified. In comparison, identities and intensities of HLA class I (HLA-I) ligands were overall comparable. We found that HLA-II ligands uniquely presented by DCs loaded with Ox-L were more solvent exposed in the structures of their source proteins, contrary to what has been hypothesized so far. Analyses from a phase I clinical trial showed that vaccinating patients using autologous Ox-L as an antigen source efficiently induces polyfunctional vaccine-specific CD4+ T cell responses. Hence, these results suggest that the increased immunogenicity of Ox-L is, at least in part, due to qualitative and quantitative changes in the HLA-II ligandome, potentially leading to an increased HLA-II dependent stimulation of the T cell compartment (i.e., CD4+ T cell responses). These results further contribute to the development of more effective and immunogenic DC-based vaccines and to the molecular understanding of the mechanism behind HOCl adjuvant properties.
This is an open access article distributed under the Creative Commons Attribution License
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited