PRODH2-Mediated Proline Metabolism Boosts CAR T-cell Effector Function

Genetically engineered T-cell therapy is a rapidly growing area of clinical development, with chimeric antigen receptor (CAR) T cell–based immunotherapies demonstrating remarkable efficacy in hematologic cancers, but durable treatment responses are limited by factors that impact T-cell function. To identify genetic targets that can be coexpressed in CAR T cells to boost effector function, Ye, Park, Peng, Yang, and colleagues performed a genome-scale gain-of-function CRISPR screen in which CD8⁺ T cells isolated from Cas9⁺-expressing transgenic T-cell receptor (OT-1) mice were transduced with a lentiviral library of catalyt­ically dead guide RNAs, designed to activate transcription upon binding of target promoters. Transduced CD8⁺ T cells were cocultured with cancer cells previously pulsed with the cognate antigen of OT-1 T cells, and the degree of T-cell killing was measured by expression of CD107a, a marker presented on the T-cell surface following degranulation. Sequencing of CD107a^hi T cells revealed enrichment of cells overexpressing the gene encoding proline dehydrogenase 2, Prodh2. Accordingly, genomic knock-in or lentiviral overexpression of Prodh2 enhanced CAR T-cell proliferation and cytolytic activity in vitro and in vivo in multiple tumor models, particularly multiple myeloma. RNA sequencing analysis revealed that Prodh2 overexpression in CAR T cells altered expression of genes involved in the cell cycle, T-cell activation, and metabolic processes, in agreement with flow cytometry data supporting enhanced effector T-cell function as measured by increased production of IFNγ, TNFα, and granzyme B after coculture with cognate cancer cells. Functional studies involving metabolic perturbations demonstrated the contribution of proline metabolism to enhanced cytolytic activity observed upon PRODH2 overexpression, which also increased mitochondria biogenesis and oxidative phosphorylation. Concordant with mitochondrial phenotypes linked to enhanced memory-like features, PRODH2 knock-in CAR T cells maintained effector phenotypes over the course of prolonged coculture and repeated antigen stimulation. In summary, this study highlights a promising target that can augment CAR T-cell function to control tumor growth.

Ye L, Park JJ, Peng L, Yang Q, Chow RD, Dong MB, et al. A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy. Cell Metab 2022;34:595–614.e14.

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