Altered Peptide Ligands Impact the Diversity of Polyfunctional Phenotypes in T Cell Receptor Gene-Modified T Cells.

Abstract

The use of Tcell receptor (TCR) gene-modified Tcells in adoptive cell transfer has had promising clinical success, but often, simple preclinical evaluation does not necessarily accurately predict treatment efficacy or safety. Preclinical studies generally evaluate one or a limited number of type 1 cytokines to assess antigen recognition. However, recent studies have implicated other "typed" Tcells in effective anti-tumor/viral immunity, and limited functional evaluations may underestimate cross-reactivity. In this study, we use an altered peptide ligand (APL) model and multi-dimensional flow cytometry to evaluate polyfunctionality of TCR gene-modified Tcells. Evaluating six cytokines and the lytic marker CD107a on a per cell basis revealed remarkably diverse polyfunctional phenotypes within a single Tcell culture and among peripheral blood lymphocyte (PBL) donors. This polyfunctional assessment identified unexpected phenotypes, including cells producing both type 1 and type 2 cytokines, and highlighted interferon γneg (IFNγneg) antigen-reactive populations overlooked in our previous studies. Additionally, APLs skewed functional phenotypes to be less polyfunctional, which was not necessarily related to changes in TCR-peptide-major histocompatibility complex (pMHC) affinity. A better understanding of gene-modified Tcell functional diversity may help identify optimal therapeutic phenotypes, predict clinical responses, anticipate off-target recognition, and improve the design and delivery of TCR gene-modified Tcells.