Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism.

Chimeric antigen receptor (CAR)-T therapy has shown superior efficacy against hematopoietic malignancies. However, many patients failed to achieve sustainable tumor control partially due to CAR-T cell exhaustion and limited persistence. In this study, by performing single-cell multi-omics data analysis on patient-derived CAR-T cells, we identify CD38 as a potential hallmark of exhausted CAR-T cells, which is positively correlated with exhaustion-related transcription factors and further confirmed with in vitro exhaustion models. Moreover, inhibiting CD38 activity reverses tonic signaling- or tumor antigen-induced exhaustion independent of single-chain variable fragment design or costimulatory domain, resulting in improved CAR-T cell cytotoxicity and antitumor response. Mechanistically, CD38 inhibition synergizes the downregulation of CD38-cADPR -Ca2+ signaling and activation of the CD38-NAD+-SIRT1 axis to suppress glycolysis. Collectively, our findings shed light on the role of CD38 in CAR-T cell exhaustion and suggest potential clinical applications of CD38 inhibition in enhancing the efficacy and persistence of CAR-T cell therapy.

Mitochondrial isocitrate dehydrogenase impedes CAR T cell function by restraining antioxidant metabolism and histone acetylation.

The efficacy of chimeric antigen receptor (CAR) T cell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Long-lived memory CAR T cells are critical for improving tumor clearance and long-term protection. However, during rapid ex vivo expansion or in vivo tumor eradication, metabolic shifts and inhibitory signals lead to terminal differentiation and exhaustion of CAR T cells. Through a mitochondria-related compound screening, we find that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances memory CAR T cell formation and sustains anti-leukemic cytotoxicity in vivo. Mechanistically, IDH2 impedes metabolic fitness of CAR T cells by restraining glucose utilization via the pentose phosphate pathway, which alleviates oxidative stress, particularly in nutrient-restricted conditions. In addition, IDH2 limits cytosolic acetyl-CoA levels to prevent histone acetylation that promotes memory cell formation. In combination with pharmacological IDH2 inhibition, CAR T cell therapy is demonstrated to have superior efficacy in a pre-clinical model.

Rewiring mitochondrial metabolism to counteract exhaustion of CAR-T cells.

Short persistence and early exhaustion of T cells are major limits to the efficacy and broad application of immunotherapy. Exhausted T and chimeric antigen receptor (CAR)-T cells upregulate expression of genes associated with terminated T cell differentiation, aerobic glycolysis and apoptosis. Among cell exhaustion characteristics, impaired mitochondrial function and dynamics are considered hallmarks. Here, we review the mitochondrial characteristics of exhausted T cells and particularly discuss different aspects of mitochondrial metabolism and plasticity. Furthermore, we propose a novel strategy of rewiring mitochondrial metabolism to emancipate T cells from exhaustion and of targeting mitochondrial plasticity to boost CAR-T cell therapy efficacy.

Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti-Leukemia Efficacy of CAR-T Cells via SOCE-Calcineurin-NFAT and Glycolysis Pathways.

Chimeric antigen receptor (CAR) T cells are potent agents for recognizing and eliminating tumors, and have achieved remarkable success in the treatment of patients with refractory leukemia and lymphoma. However, dysfunction of T cells, including exhaustion, is an inevitable obstacle for persistent curative effects. Here, the authors initially found that calcium signaling is hyperactivated via sustained tonic signaling in CAR-T cells. Next, it is revealed that the store-operated calcium entry (SOCE) inhibitor BTP-2, but not the calcium chelator BAPTA-AM, markedly diminishes CAR-T cell exhaustion and terminal differentiation of CAR-T cells in both tonic signaling and tumor antigen exposure models. Furthermore, BTP-2 pretreated CAR-T cells show improved antitumor potency and prolonged survival in vivo. Mechanistically, transcriptome and metabolite analyses reveal that treatment with BTP-2 significantly downregulate SOCE-calcineurin-nuclear factor of activated T-cells (NFAT) and glycolysis pathways. Together, the results indicate that modulating the SOCE-calcineurin-NFAT pathway in CAR-T cells renders them resistant to exhaustion, thereby yielding CAR products with enhanced antitumor potency.

[Impacts of ODF2 gene knockdown on the sperm motility and fertility of male mice].

OBJECTIVE: To investigate the knockdown of the outer dense fiber protein 2 (ODF2) gene on the sperm motility and fertility of male mice. METHODS: We constructed three knockdown vectors with the target gene ODF2 and one control vector without the target gene. After infecting ICR mice, we determined the vector with the best knockdown effect by RT-PCR and Western blot and reinfected the mice with it. Then we obtained and analyzed the sperm motility parameters, pathological changes of the testis issue, and the litter size of the mice with gene knockdown. RESULTS: Compared with the normal controls, the mice infected with the vector with the best knockdown effect showed significantly decreased sperm motility parameters, pathomorphological abnormalities of the testis, and a reduced litter size (10.86 ± 1.28 vs 12.72 ± 2.05, P = 0.001). CONCLUSION: Decreased expression of the ODF2 gene deceases sperm motility parameters, impairs the morphology of the testis and affects the fertility of male mice.

Integrated Single-Cell Bioinformatics Analysis Reveals Intrinsic and Extrinsic Biological Characteristics of Hematopoietic Stem Cell Aging.

Hematopoietic stem cell (HSC) aging, which is accompanied by loss of self-renewal capacity, myeloid-biased differentiation and increased risks of hematopoietic malignancies, is an important focus in stem cell research. However, the mechanisms underlying HSC aging have not been fully elucidated. In the present study, we integrated 3 independent single-cell transcriptome datasets of HSCs together and identified Stat3 and Ifngr1 as two markers of apoptosis-biased and inflammatory aged HSCs. Besides, common differentially expressed genes (DEGs) between young and aged HSCs were identified and further validated by quantitative RT-PCR. Functional enrichment analysis revealed that these DEGs were predominantly involved in the cell cycle and the tumor necrosis factor (TNF) signaling pathway. We further found that the Skp2-induced signaling pathway (Skp2→Cip1→CycA/CDK2→DP-1) contributed to a rapid transition through G1 phase in aged HSCs. In addition, analysis of the extrinsic alterations on HSC aging revealed the increased expression levels of inflammatory genes in bone marrow microenvironment. Colony formation unit assays showed that inflammatory cytokines promoted cellular senescence and that blockade of inflammatory pathway markedly rejuvenated aged HSC functions and increased B cell output. Collectively, our study elucidated the biological characteristics of HSC aging, and the genes and pathways we identified could be potential biomarkers and targets for the identification and rejuvenation of aged HSCs.

Signaling pathways in the regulation of cytokine release syndrome in human diseases and intervention therapy.

Cytokine release syndrome (CRS) embodies a mixture of clinical manifestations, including elevated circulating cytokine levels, acute systemic inflammatory symptoms and secondary organ dysfunction, which was first described in the context of acute graft-versus-host disease after allogeneic hematopoietic stem-cell transplantation and was later observed in pandemics of influenza, SARS-CoV and COVID-19, immunotherapy of tumor, after chimeric antigen receptor T (CAR-T) therapy, and in monogenic disorders and autoimmune diseases. Particularly, severe CRS is a very significant and life-threatening complication, which is clinically characterized by persistent high fever, hyperinflammation, and severe organ dysfunction. However, CRS is a double-edged sword, which may be both helpful in controlling tumors/viruses/infections and harmful to the host. Although a high incidence and high levels of cytokines are features of CRS, the detailed kinetics and specific mechanisms of CRS in human diseases and intervention therapy remain unclear. In the present review, we have summarized the most recent advances related to the clinical features and management of CRS as well as cutting-edge technologies to elucidate the mechanisms of CRS. Considering that CRS is the major adverse event in human diseases and intervention therapy, our review delineates the characteristics, kinetics, signaling pathways, and potential mechanisms of CRS, which shows its clinical relevance for achieving both favorable efficacy and low toxicity.

Dasatinib enhances anti-leukemia efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion.

Relapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cells persistence. Sustained tonic signaling or strong activation drives CART cell differentiation and exhaustion, which limit the therapeutic efficacy and persistence of CART cells. Here, we identified dasatinib as the optimal candidate to prevent or reverse both CD28/CART and 4-1BB/CART cell differentiation and exhaustion during ex vivo expansion, which profoundly enhanced the therapeutic efficacy and in vivo persistence. Moreover, strong activation-induced CART cells differentiation, exhaustion and apoptosis driven by CD3/CD28 stimulation or antigen exposure were dramatically prevented or reversed by dasatinib treatment. Mechanistically, dasatinib markedly reduced the phosphorylation of Src and Lck, and downregulated the expression of genes involved in CAR signaling pathways, which resulted in the optimization of cell differentiation, exhaustion and apoptosis-related gene expression. Our study proposes a promising pharmacological approach for optimizing CART cells manufacture, and provides an experimental basis for reinvigorating CART cells in clinical application.

CRS-related coagulopathy in BCMA targeted CAR-T therapy: a retrospective analysis in a phase I/II clinical trial.

Chimeric antigen receptor (CAR) T-cell therapy that targets B-cell maturation antigen (BCMA) has shown promising effects in the treatment of patients with refractory/relapsed multiple myeloma (R/R MM) patients. In this retrospective analysis of phase I/II clinical trial (ChiCTR1800017404), 37 patients with R/R MM received their first BCMA-targeted CAR T-cells following lymphodepletion chemotherapy. The response rate was high (97%), while accompanied by a high incidence of adverse events including coagulation dysfunction. Of 37 patients, all (100%) had cytokine release syndrome (CRS) and 34 (91%) developed at least one abnormal coagulation parameter. The values of coagulation parameters were positively correlated with the severity of CRS as well as with the levels of some cytokines, such as interleukin (IL)-6, IL-10, and interferon (IFN)-γ, etc. Furthermore, levels of the plasma tissue factor (TF), Factor X (FX), Factor XII (FXII), and P-selectin also showed a positive correlation with severity of CRS as well as some specific cytokines, which indicates that these factors are likely to play important roles in CRS-related coagulopathy. Our study suggests that there exists relationship in some extent between coagulation disorder and CRS. Moreover, coagulation dysfunction can be managed with daily monitoring and early intervention despite high incidence.

Single-Cell Transcriptomic Analysis Reveals BCMA CAR-T Cell Dynamics in a Patient with Refractory Primary Plasma Cell Leukemia.

Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in clinical patients remains unexplored. Here, we longitudinally profiled the transcriptomes of 55,488 T cells including CAR-T products, CAR-T cells, and endogenous T cells at the peak and remission phases in a plasma cell leukemia (PCL) patient treated with BCMA CAR-T cells by single-cell transcriptomic analysis. Our results showed distinct CAR-T and endogenous T cell subsets indicating stage-specific expression in proliferation, cytotoxicity, and intercellular signaling pathways. Furthermore, we found that CAR-T cells at peak phase gradually convert to a highly cytotoxic state from a highly proliferative state along a development trajectory. Moreover, re-analysis of a single cell study from CD8+ CD19 CAR-T confirmed our findings. These commonalities suggest conserved mechanisms for CAR-T treatment across hematological malignancies. Taken together, our current study provides insight into CAR-T cell dynamics during CAR-T therapy and proves that both BCMA CAR-T and CD19 CAR-T have similar transcriptional characteristics, especially at the CAR-T peak phase.

Lymphodepletion chemotherapy revitalizes chimeric antigen receptor T cells contributing to regression of relapsed B-cell lymphoma: A case report.

INTRODUCTION: Chimeric antigen receptor T cells (CAR-T) targeting CD19 have shown great potential for treatment of B-cell malignancies. For those patients who can not achieve complete remission (CR) or suffer from relapse after CAR-T therapy, further therapeutic strategies still remain elusive. Whether existing CAR-T cells can revitalize in vivo and eradicate tumor cells is still unknown. PATIENT CONCERNS: We report a case of diffused large B-cell lymphoma patient who had achieved CR after CD19 targeted CAR-T therapy but relapsed after 5 months. DIAGNOSIS: Five months after CAR-T cell infusion, the patient was confirmed a relapse by follow-up PET/CT scan and a mass biopsy. Flow cytometry showed a dramatically decreased percentage of CAR-T cells in peripheral blood (PB). INTERVENTIONS: A second anti-CD19 CAR-T therapy was planned with deliberation. Firstly, the patient received lymphodepletion chemotherapy with fludarabine (25 mg/m, d1-d3) and cyclophosphamide (500 mg/m d2-d3). OUTCOMES: After fludarabine and cyclophosphamide (FC) lymphodepletion chemotherapy, pre-existing CAR-T cells were revitalized and the patient developed grade 2 cytokine release syndrome (CRS) contributing to the regression of relapsed B-cell lymphoma. CONCLUSIONS: This case suggested that FC chemotherapy could revitalize CAR-T cells contributing to the regression of relapsed B-cell lymphoma. Nevertheless, further researches are required in the future as this report described only a single case.