Differential Optical Imaging of Antigen Presentation Machinery Using Molecular Optical Reporters

Detection of antigen presentation is central to understanding immunological processes and developing therapeutics for cancer, infectious diseases, and allergies. However, methods with the ability to dynamically and noninvasively distinguish between major histocompatibility complex class I (MHC-I) and MHC-II antigen presentations remain lacking. Herein, we develop activatable molecular optical reporters (MORs) for real-time differential imaging of antigen presentations in lymph nodes (LNs). These MORs are engineered to passively target LNs and activated through proteolytic cleavage by key enzymes in the MHC-I and MHC-II pathways, the immunoproteasome (iP) and cathepsin S (CTSS), respectively, triggering their chemiluminescent or fluorescent signals. Coupled with minimized signal crosstalk and high sensitivity, MORs delineate the subtle differences in the antigen presentation machinery across various disease models, including cancer and bacterial or viral infection, a feat unattainable for existing imaging methods. After systemic administration, MORs also allow real-time visualization of antigen presentation in the tumor microenvironment. Besides, MORs are validated to have potential for preclinical application in immunotherapeutics screening and clinical application in tissue biopsy. Thus, our study not only presents the first example of real-time, in vivo differential imaging of antigen presentation pathways but also opens new avenues for optical probes in immune contexture analysis.