Single cell imaging of T cell immunotherapy responses in vivo

T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single cell resolution in vivo. Here, optically-clear immunocompromised zebrafish were engrafted with fluorescent-labelled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single cell visualization of T cell-based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease. Overall design: Blood Lineage analysis of zebrafish kidney marrow samples from WT, rag2?/? and rag2?/?, il2rga-/- immunocompromised zebrafish