Chronic lymphocytic leukemia (CLL) cells possess regulatory functions comparable to those of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated IL-10 production. However, the mechanisms governing IL-10 production by CLL cells are not fully understood. Here, we show that the CXC chemokine ligand 12 (CXCL12)-CXCR4-STAT3 axis regulates IL-10 production by CLL cells and their ability to suppress T-cell effector function through an IL-10 mediated mechanism. Knockdown of STAT3 significantly impaired the ability of CLL cells to produce IL-10. Furthermore, experiments to assess the role of lenalidomide, an immunomodulatory agent with direct antitumor effect as well as pleiotropic activity on the immune system, showed that this agent prevents a CXCL12-induced increase in p-S727-STAT3 and the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T-cell dysfunction. We conclude that the capacity of CLL cells to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and likely contributes to immunodeficiency in patients. Lenalidomide appears to be able to reverse CLL-induced immunosuppression through including abrogation of the CXCL12-CXCR4-S727-STAT3-mediated IL-10 response by CLL cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells.

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3β- (GSK-3β-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3β and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3β at Ser9 without affecting the expression level of total GSK-3β protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3β siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3β inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3β, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3β and further activating the Nrf2-ARE signaling pathway.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.