Nonclassical Antigen-Processing Pathways Are Required for MHC Class II–Restricted Direct Tumor Recognition by NY-ESO-1–Specific CD4+ T Cells

Tumor antigen-specific CD4+ T cells that directly recognize cancer cells are important for orchestrating anti-tumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4+ T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in ovarian cancer patients. While both subsets similarly recognized exogenous NY-ESO-1 protein pulsed on DP04+ target cells, only one type recognized target cells that intracellularly expressed NY-ESO-1 including cancer cells. Tumor-recognizing CD4+ T cells more efficiently recognized short 8-9-mer peptides than non-tumor-recognizing CD4+ T cells. In addition to endosomal/lysosomal proteases that are typically involved in the MHC-II antigen presentation, several pathways in MHC class I presentation pathways such as the proteasomal degradation and transporter-associated with antigen-processing (TAP)-mediated peptide transport were involved in the presentation of intracellular NY-ESO-1 on MHC-II. In addition, the presentation was significantly inhibited by primaquine, a small molecule that inhibits endosomal recycling. Consistently, pharmacological inhibition of new protein synthesis enhanced the presentation. Together, our data demonstrated that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple non-classical antigen-processing pathways. Harnessing direct tumor-recognizing ability of CD4+ T cells could be a promising strategy to enhance anti-tumor immune responses in the immunosuppressive tumor microenvironment.