Use of novel fluorescent dyes enables in depth analysis of checkpoint inhibitors CTLA-4 (CD152) and PD-1 through spectral flow cytometry

Abstract

Abstract PD-1 and CTLA-4 (CD152) are prominent checkpoint inhibitors in T cell responses and their inhibition has emerged as a successful route to boost anti-tumor immune responses in cancer patients. Clinical trials involving CAR-T cell therapy with anti-CTLA-4 and PD-1 antibodies seem to be improving the tumor directed T cell response. Regulation of surface vs cytoplasmic CTLA-4 pools is crucial to balancing stimulatory and inhibitory signals in T-cell responses. Surface CTLA-4 contributes to tumor escape by inhibiting T cell responses, and upregulation of CTLA-4 expression from the cytoplasm to the surface is highly regulated. Therefore, it might be of interest to monitor these CTLA-4 pools as this might indicate the efficacy of CAR-T cell therapeutic approaches. Here we show how novel fluorescent dyes enable the composition of a spectral flow cytometry panel to interrogate the presence of surface versus cytoplasmic CTLA-4 pools and the dynamics of PD-1 expression in a single panel. Combining these parameters into a single panel allows for less workflow steps and permit conservative sample use.