How can Cytokine-induced killer cells overcome CAR-T cell limits.

The successful treatment of patients affected by B-cell malignancies with Chimeric Antigen Receptor (CAR)-T cells represented a breakthrough in the field of adoptive cell therapy (ACT). However, CAR-T therapy is not an option for every patient, and several needs remain unmet. In particular, the production of CAR-T cells is expensive, labor-intensive and logistically challenging; additionally, the toxicities deriving from CAR-T cells infusion, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), have been documented extensively. Alternative cellular therapy products such as Cytokine-induced killer (CIK) cells have the potential to overcome some of these obstacles. CIK cells are a heterogeneous population of polyclonal CD3+CD56+ T cells with phenotypic and functional properties of NK cells. CIK cell cytotoxicity is exerted in a major histocompatibility complex (MHC)-unrestricted manner through the engagement of natural killer group 2 member D (NKG2D) molecules, against a wide range of hematological and solid tumors without the need for prior antigen exposure or priming. The foremost potential of CIK cells lies in the very limited ability to induce graft-versus-host disease (GvHD) reactions in the allogeneic setting. CIK cells are produced with a simple and extremely efficient expansion protocol, which leads to a massive expansion of effector cells and requires a lower financial commitment compared to CAR-T cells. Indeed, CAR-T manufacturing involves the engineering with expensive GMP-grade viral vectors in centralized manufacturing facilities, whereas CIK cell production is successfully performed in local academic GMP facilities, and CIK cell treatment is now licensed in many countries. Moreover, the toxicities observed for CAR-T cells are not present in CIK cell-treated patients, thus further reducing the costs associated with hospitalization and post-infusion monitoring of patients, and ultimately encouraging the delivery of cell therapies in the outpatient setting. This review aims to give an overview of the limitations of CAR-T cell therapy and outline how the use of CIK cells could overcome such drawbacks thanks to their unique features. We highlight the undeniable advantages of using CIK cells as a therapeutic product, underlying the opportunity for further research on the topic.

Strikingly conserved gene expression changes of polyamine regulating enzymes among various forms of acute and chronic kidney injury.

The polyamines spermidine and spermine and their common precursor molecule putrescine are involved in tissue injury and repair. Here, we test the hypothesis that impaired polyamine homeostasis contributes to various kidney pathologies in mice during experimental models of ischemia-reperfusion, transplantation, rhabdomyolysis, cyclosporine treatment, arterial hypertension, diabetes, unilateral ureteral obstruction, high oxalate feeding, and adenine-induced injuries. We found a remarkably similar pattern in most kidney pathologies with reduced expression of enzymes involved in polyamine synthesis together with increased expression of polyamine degrading enzymes. Transcript levels of amine oxidase copper-containing 1 (Aoc1), an enzyme which catalyzes the breakdown of putrescine, were barely detectable by in situ mRNA hybridization in healthy kidneys. Aoc1 was highly expressed upon various experimental kidney injuries resulting in a significant reduction of kidney putrescine content. Kidney levels of spermine were also significantly reduced, whereas spermidine was increased in response to ischemia-reperfusion injury. Increased Aoc1 expression in injured kidneys was mainly accounted for by an Aoc1 isoform that harbors 22 additional amino acids at its N-terminus and shows increased secretion. Mice with germline deletion of Aoc1 and injured kidneys showed no decrease of kidney putrescine content; although they displayed no overt phenotype, they had fewer tubular casts upon ischemia-reperfusion injury. Hyperosmotic stress stimulated AOC1 expression at the transcriptional and post-transcription levels in metanephric explants and kidney cell lines. AOC1 expression was also significantly enhanced after kidney transplantation in humans. These data demonstrate that the kidneys respond to various forms of injury with down-regulation of polyamine synthesis and activation of the polyamine breakdown pathway. Thus, an imbalance in kidney polyamines may contribute to various etiologies of kidney injury.

Canonical BMP signaling in tubular cells mediates recovery after acute kidney injury.

Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease.

Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion.

Transient receptor potential vanilloid 4 (TRPV4) cation channels are functional in all renal vascular segments and mediate endothelium-dependent vasorelaxation. Moreover, they are expressed in distinct parts of the tubular system and activated by cell swelling. Ischaemia/reperfusion injury (IRI) is characterized by tubular injury and endothelial dysfunction. Therefore, we hypothesised a putative organ protective role of TRPV4 in acute renal IRI. IRI was induced in TRPV4 deficient (Trpv4 KO) and wild-type (WT) control mice by clipping the left renal pedicle after right-sided nephrectomy. Serum creatinine level was higher in Trpv4 KO mice 6 and 24 hours after ischaemia compared to WT mice. Detailed histological analysis revealed that IRI caused aggravated renal tubular damage in Trpv4 KO mice, especially in the renal cortex. Immunohistological and functional assessment confirmed TRPV4 expression in proximal tubular cells. Furthermore, the tubular damage could be attributed to enhanced necrosis rather than apoptosis. Surprisingly, the percentage of infiltrating granulocytes and macrophages were comparable in IRI-damaged kidneys of Trpv4 KO and WT mice. The present results suggest a renoprotective role of TRPV4 during acute renal IRI. Further studies using cell-specific TRPV4 deficient mice are needed to clarify cellular mechanisms of TRPV4 in IRI.

Tubular Epithelial NF-κB Activity Regulates Ischemic AKI.

NF-κB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type-specific functions of NF-κB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-κB signaling in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. NF-κB reporter activity and nuclear localization of phosphorylated NF-κB subunit p65 analyses in mice revealed that IRI induced widespread NF-κB activation in renal tubular epithelia and in interstitial cells that peaked 2-3 days after injury. To genetically antagonize tubular epithelial NF-κB activity, we generated mice expressing the human NF-κB super-repressor IκBαΔN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-κB-dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IκBαΔN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-κB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response.

PI3K/AKT signaling inhibits NOTCH1 lysosome-mediated degradation.

The pathways of NOTCH and PI3K/AKT are dysregulated in about 60% and 48% of T-cell acute lymphoblastic leukemia (T-ALL) patients, respectively. In this context, they interact and cooperate in controlling tumor cell biology. Here, we propose a novel mechanism by which the PI3K/AKT pathway regulates NOTCH1 in T-ALL, starting from the evidence that the inhibition of PI3K/AKT signaling induced by treatment with LY294002 or transient transfection with a dominant negative AKT mutant downregulates NOTCH1 protein levels and activity, without affecting NOTCH1 transcription. We showed that the withdrawal of PI3K/AKT signaling was associated to NOTCH1 phosphorylation in tyrosine residues and monoubiquitination of NOTCH1 detected by Ubiquitin capture assay. Co-immunoprecipitation assay and colocalization analysis further showed that the E3 ubiquitin ligase c-Cbl interacts and monoubiquitinates NOTCH1, activating its lysosomal degradation. These results suggest that the degradation of NOTCH1 could represent a mechanism of control by which NOTCH1 receptors are actively removed from the cell surface. This mechanism is finely regulated by the PI3K/AKT pathway in physiological conditions. In pathological conditions characterized by PI3K/AKT hyperactivation, such as T-ALL, the excessive AKT signaling could lead to NOTCH1 signaling dysregulation. Therefore, a therapeutic strategy directed to PI3K/AKT in T-ALL could contemporaneously inhibit the dysregulated NOTCH1 signaling. © 2015 Wiley Periodicals, Inc.

Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia.

UNLABELLED: Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. RESULTS: Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. INNOVATION: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. CONCLUSIONS: TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.