Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CAR ^High T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CAR ^High T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome’s sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (T_CM) cells while CD26high expression is present in effector Th1, Th2, Th17, and T_EM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.