Highlights d scRNA-seq analyses highlight conserved myeloid subsets in human and murine CRC d Two distinct TAM subsets show inflammatory and angiogenic signatures, respectively d Two distinct TAM subsets show differential sensitivity to CSF1R blockade d Anti-CD40 activates specific cDC1s and expands Th1-like and CD8 + memory T cells

Immunodominant epitopes are few selected epitopes from complex antigens that initiate T cell responses. Here, to provide further insights into this process, we use a reductionist cell-free antigen processing system composed of defined components. We use the system to characterize steps in antigen processing of pathogen-derived proteins or autoantigens and we find distinct paths for peptide processing and selection. Autoantigen-derived immunodominant epitopes are resistant to digestion by cathepsins, whereas pathogen-derived epitopes are sensitive. Sensitivity to cathepsins enforces capture of pathogen-derived epitopes by Major Histocompatibility Complex class II (MHC class II) prior to processing, and resistance to HLA-DM-mediated-dissociation preserves the longevity of those epitopes. We show that immunodominance is established by higher relative abundance of the selected epitopes, which survive cathepsin digestion either by binding to MHC class II and resisting DM-mediated-dissociation, or being chemically resistant to cathepsins degradation. Non-dominant epitopes are sensitive to both DM and cathepsins and are destroyed.

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4+ T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: MHC class II, cathepsins, and HLA-DM. Our minimalist system successfully identified the physiologically selected immunodominant epitopes of model antigens, HA1 from influenza virus (A/Texas/1/77) and type II collagen. When applied for de novo epitope identification to a malaria antigen, or HA1 from H5N1 virus (Avian Flu), the system selected a single epitope from each protein that were confirmed to be immunodominant by their capacity to activate CD4+ T cells in HLA-DR1 positive human volunteers or transgenic mice immunized with the corresponding proteins. Thus, we provide a powerful new tool for the identification of physiologically relevant helper T cell epitopes from antigens.