A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo.

Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34- AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.

T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes.

Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.

MZB1 promotes the secretion of J-chain-containing dimeric IgA and is critical for the suppression of gut inflammation.

IgA is the most abundantly produced antibody in the body and plays a crucial role in gut homeostasis and mucosal immunity. IgA forms a dimer that covalently associates with the joining (J) chain, which is essential for IgA transport into the mucosa. Here, we demonstrate that the marginal zone B and B-1 cell-specific protein (MZB1) interacts with IgA through the α-heavy-chain tailpiece dependent on the penultimate cysteine residue and prevents the intracellular degradation of α-light-chain complexes. Moreover, MZB1 promotes J-chain binding to IgA and the secretion of dimeric IgA. MZB1-deficient mice are impaired in secreting large amounts of IgA into the gut in response to acute inflammation and develop severe colitis. Oral administration of a monoclonal IgA significantly ameliorated the colitis, accompanied by normalization of the gut microbiota composition. The present study identifies a molecular chaperone that promotes J-chain binding to IgA and reveals an important mechanism that controls the quantity, quality, and function of IgA.

Efficient and Reliable MicroRNA Imaging in Living Cells via a FRET-Based Localized Hairpin-DNA Cascade Amplifier.

MicroRNAs (miRNAs) play critical roles in many biological processes and are vital biomarkers for disease diagnostics. Hence, it is of significance to develop miRNA biosensors with fast responses, high sensitivity, and excellent reliability in living cells. As one kind of DNA molecular machine, DNA amplifiers are very promising for intracellular miRNA imaging due to their nonenzymatic, isothermal working principle and excellent signal-amplification ability. However, the practical application of current DNA amplifiers is still an issue because of their slow kinetics, unsatisfactory efficiency, and false-positive signals. Herein, taking advantage of the spatial-confinement effect on a three-dimensional (3D) finite DNA nanostructure, a FRET-based localized hairpin-DNA cascade amplifier (termed as localized-HDCA) is developed for the rapid, efficient, and reliable imaging of intracellular tumor-related miRNA. The localized-HDCA system consists of two metastable hairpin DNAs (H1 and H2) localized on a DNA nanocube. Benefiting from the spatial-confinement effect in the confined space of DNA nanocubes, not only was the speed of the miRNA-triggered HDCA reaction significantly accelerated (7 times faster), but also the reaction efficiency was greatly improved (2.6 times higher). In addition, the FRET-based 3D finite DNA nanocubes provide this localized-HDCA with improved cell permeability and better nuclease resistance as well as the ability to avoid false-positive signals, which guarantee reliable miRNA imaging in living cells. With these advantages, this strategy is expected to be widely applied to the development of more efficient and robust DNA molecular machines for biomedical research and clinical diagnosis.

IGFBPL1 Regulates Axon Growth through IGF-1-mediated Signaling Cascades.

Activation of axonal growth program is a critical step in successful optic nerve regeneration following injury. Yet the molecular mechanisms that orchestrate this developmental transition are not fully understood. Here we identified a novel regulator, insulin-like growth factor binding protein-like 1 (IGFBPL1), for the growth of retinal ganglion cell (RGC) axons. Expression of IGFBPL1 correlates with RGC axon growth in development, and acute knockdown of IGFBPL1 with shRNA or IGFBPL1 knockout in vivo impaired RGC axon growth. In contrast, administration of IGFBPL1 promoted axon growth. Moreover, IGFBPL1 bound to insulin-like growth factor 1 (IGF-1) and subsequently induced calcium signaling and mammalian target of rapamycin (mTOR) phosphorylation to stimulate axon elongation. Blockage of IGF-1 signaling abolished IGFBPL1-mediated axon growth, and vice versa, IGF-1 required the presence of IGFBPL1 to promote RGC axon growth. These data reveal a novel element in the control of RGC axon growth and suggest an unknown signaling loop in the regulation of the pleiotropic functions of IGF-1. They suggest new therapeutic target for promoting optic nerve and axon regeneration and repair of the central nervous system.

EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity.

Regulated apoptosis of germinal centre (GC) B cells is critical for normal humoral immune responses. ELL-associated factor 2 (EAF2) regulates transcription elongation and has been shown to be an androgen-responsive potential tumour suppressor in prostate by inducing apoptosis. Here we show that EAF2 is selectively upregulated in GC B cells among various immune cell types and promotes apoptosis of GC B cells both in vitro and in vivo. EAF2 deficiency results in enlarged GCs and elevated antibody production during a T-dependent immune response. After immunization with type II collagen, mice lacking EAF2 produce high levels of collagen-specific autoantibodies and rapidly develop severe arthritis. Moreover, the mutant mice spontaneously produce anti-dsDNA, rheumatoid factor and anti-nuclear antibodies as they age. These results demonstrate that EAF2-mediated apoptosis in GC B cells limits excessive humoral immune responses and is important for maintaining self-tolerance.

Improved renal recovery with postresuscitation N-acetylcysteine treatment in asphyxiated newborn pigs.

Renal injury is one of the severe and common complications that occurs early in neonates with asphyxia, and reactive oxygen species have been implicated to play an important role on its pathogenesis. Improved renal recovery has been shown previously with N-acetyl-l-cysteine (NAC) in various acute kidney injuries. Using a subacute swine model of neonatal hypoxia-reoxygenation (H/R), we examined whether NAC can sustain its beneficial effect on renal recovery for 48 h. Newborn piglets were randomly assigned into a sham-operated group (without H/R, n = 6) and two H/R experimental groups (n = 8 each) with 2 h normocapnic alveolar hypoxia and 1 h 100% oxygen of reoxygenation followed by 21% oxygen for 47 h. Five minutes after reoxygenation, piglets received either normal saline (H/R control) or NAC (150-mg/kg bolus and 20 mg/kg per hour i.v. for 24 h) in a blinded, randomized fashion. All piglets were acidotic and in cardiogenic shock after hypoxia. Treating the piglets with NAC significantly increased both renal blood flow and oxygen delivery throughout the reoxygenation period. N-acetyl-l-cysteine treatment also improved the renal function with the attenuation of elevated urinary N-acetyl-ß-d-glucosaminidase activity and plasma creatinine concentration observed in H/R controls (both P < 0.05). The tissue levels of lipid hydroperoxides and caspase 3 in the kidney of NAC-treated animals were significantly lower than those of H/R controls. Conclusively, postresuscitation administration of NAC elicits a prolonged beneficial effect in improving renal functional recovery and reducing oxidative stress in newborn piglets with H/R insults for 48 h.

Apoptosis induced by methylene-blue-mediated photodynamic therapy in melanomas and the involvement of mitochondrial dysfunction revealed by proteomics.

Methylene blue (MB) is a widely studied agent currently under investigation for its properties relating to photodynamic therapy (PDT). Recent studies have demonstrated that MB exhibits profound phototoxicity affecting a variety of tumor cell lines. However, the mechanistic explanation for methylene-blue-mediated photodynamic therapy (MB-PDT) in the context of melanoma therapy is still obscure. In the present study, B16F1 melanoma cells were treated by MB-PDT under different conditions, and thereafter subjected to cell viability detection assays. MB-PDT could induce intense apoptotic cell death through a series of steps beginning with the photochemical generation of reactive oxygen species that activate the caspase-9/caspase-3 apoptosis pathway. Blocking activation of caspase-3 and induction of oxidative stress by caspase inhibitor and by glutathione, respectively, markedly reduced apoptotic cell death in vitro. Importantly, proteomics study defining altered protein expression in treated cells suggests the involvement of several mitochondrial proteins playing important roles in electron transfer chain, implying mitochondrial dysfunction during the treatment. Furthermore, a transplantable mouse melanoma model was utilized to estimate the effectiveness of MB-PDT in vivo. The treated mice displayed decreased tumor size and prolonged survival days, which was associated with enhanced apoptotic cell death. These results, offering solid evidence of the induction of mitochondria-related apoptosis in tumor cells, reveal new aspects of MB-PDT having potential to be a palliative treatment of melanoma.