In recent years, chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in treating patients with relapsed or refractory leukemia and lymphoma. Even so, a significant proportion of patients relapse after treatment with CAR-T cells. The dysfunction of CAR-T cells, such as exhaustion, is one of the critical causes of relapse. Reducing CAR-T cell exhaustion is the key to improving efficacy and remains a global problem that urgently needs to be solved.

On January 14, 2022, Prof. HUANG He from the First Affiliated Hospital, Zhejiang University School of Medicine and Prof. QIAN Pengxu from the Zhejiang University School of Medicine co-published an article entitled Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti-Leukemia Efficacy of CAR-T Cells via SOCE-Calcineurin-NFAT and Glycolysis Pathways in the journal Advanced Science. This study reports that regulating the SOCE-Calcineurin-NFAT pathway in CAR-T cells significantly prevents CAR-T cell exhaustion, thereby yielding CAR-T products with enhanced anti-tumor potency.

Firstly, the study revealed hyperactivated calcium signaling via sustained tonic signaling in CAR-T cells. The researchers performed the transcriptome analysis among T cells and CAR-T cells in vitro respectively, and found that the pathway for regulation of the cytosolic calcium ion concentration was significantly upregulated in the CAR-T-d12 group. The researchers then measured the cytosolic calcium concentration in CAR-T cells at different culture times or in different stages, and found a significant increase of calcium concentration in CAR-T cells cultured for 12 days or at the effector stage. In summary, the researchers defined cytosolic calcium content as an indicator of CAR-T cell activation, which could also distinguish effector from memory CAR-T cells.

Figure1 Calcium signaling is enhanced via tonic signaling in CAR-T cells.

Secondly, the study revealed that the SOCE (store-operated calcium entry) inhibitor BTP-2 markedly diminished exhaustion and terminal differentiation of CAR-T cells. The researchers applied different types of calcium inhibitors, and found that the SOCE inhibitor BTP-2 could effectively regulate the intracellular calcium concentration. More importantly, BTP-2 could significantly prevent or reverse CAR-T cell activation, exhaustion, and terminal differentiation caused by spontaneous tonic signaling or tumor antigen stimulation. Furthermore, BTP-2 pretreated CAR-T cells showed superior tumor-killing capacity and prolonged persistence in the B-ALL mouse model.

Figure 2 BTP-2 pretreatment enhances the anti-tumor effect of CAR-T cells in vivo.

Finally, the study demonstrated that BTP-2 impeded CAR-T cell exhaustion through the calcium-calcineurin-NFAT and glycolysis pathways. RNA-seq analysis revealed several cellular metabolism-related pathways down-regulated in the BTP-2 treatment group. To verify the role of the SOCE-Calcineurin-NFAT pathway, the researchers detected the NFATc2 and found that BTP-2 treatment and FK506 (calcineurin/NFAT inhibitor) treatment both blocked NFAT translocation from the cytoplasm to the nucleus. Detection of Embden-Meyerhof-Pathway metabolites, glycolysis rate, and mRNA levels of glycolysis-related enzymes all confirmed the reduction of glycolysis levels in both the BTP-2-treated and FK-506-treated groups. In summary, these findings suggested that the SOCE-Calcineurin-NFAT pathway regulated the exhaustion and differentiation of CAR-T cells by suppressing glycolysis.  

Figure 3 BTP-2 reduces CAR-T cell exhaustion through the SOCE-Calcineurin-NFAT and glycolysis pathways.

Overall, this study unveils that attenuated calcium signaling renders CAR-T cells resistant to exhaustion, favors the persistence of highly functional CAR-T cells, and enhances anti-tumor efficacy. The regulatory roles of the SOCE-Calcineurin-NFAT pathway and the metabolic pathway (glycolysis) on CAR-T cell function are also emphasized. The study has considerable potential for clinical applications.

More information: Ph.D. candidates SHAO Mi, TENG Xinyi and GUO Xin are the co-first authors of this article. Prof. HUANG He, Prof. QIAN Pengxu and Dr. HU Yongxian are the co-corresponding authors of this article.

Source: the First Affiliated Hospital, Zhejiang University School of Medicine

Photo credit: the research team led by Prof. HUANG He