Recent studies suggest that CD4+ T cells can exert potent anti-tumor effects and improve immunotherapy efficacy by aiding CD8+ T cells. However, characterizing the mechanism of CD4+ T cells' anti-tumor activity has been challenging due to inaccurate major histocompatibility complex class II (MHC-II) peptide prediction algorithms and the lack of high-quality reagents for immune monitoring. To address this, we developed MHC2-substitution of CLIP and analytical LCMS evaluation (MHC2-SCALE), a streamlined approach combining affinity optimized class II-associated invariant chain peptide (CLIP) exchange technology, high throughput 2D-LCMS analysis, and rapid generation of peptide-bound MHC-II monomers for subsequent multimer assembly. We validated MHC-II peptide candidates predicted by the immune epitope database (IEDB) algorithm, as well as uncovered many true and immunogenic MHC-II binders that were not predicted by IEDB. Thus, MHC2-SCALE expands the opportunities for discovering, tracking, and phenotyping antigen-specific CD4+ T cells in preclinical and clinical settings, thereby improving therapies for cancer, autoimmunity, or infectious diseases.
© 2025 The Author(s).