NK Cells and Cancer

NK cells play an important role in host immunity against cancer by exerting cytotoxicity and secreting a wide variety of cytokines to inhibit tumour progression. Their effector functions are regulated by the integration of opposing signals from activating and inhibitory receptors, which determine NK cell activity against tumour targets. NK cell cytotoxicity requires successful progression through discrete activation events that begin with NK cell adhesion to a tumour target cell and culminate in the polarized release of cytotoxic granules into the immunological synapse. Tumour cells can evade NK cell attack through numerous mechanisms such as shedding of activating ligands, upregulation of inhibitory ligands, or stimulation of inhibitory regulatory T lymphocytes. A better understanding of specific NK cell responses to tumour targets can generate better NK cell‐based immunotherapeutic strategies for cancer. This chapter discusses NK cell immunosurveillance of cancer, NK cell tumour recognition strategies, cancer immune evasion from NK cells, and different approaches to NK cell modulation for cancer therapy

Bringing function to the forefront of cell therapy: how do we demonstrate potency?

Unlike conventional pharmaceuticals, biologics and Advanced Therapy Medicinal Products (ATMPs) are required to meet a standard of “potency” as part of the final release criteria at completion of manufacture. During early phase clinical trials, most regulatory agencies have been willing to accept very immature potency assays with an expectation that these will be improved, qualified and validated during the clinical development of the drug to Marketing Authorisation Application (MAA) or Biologics License Application (BLA) submission.This model of continuous development of potency assay in parallel with drug development has already led to at least two notable problem cases; namely Iovance and Mesoblast. Both companies completed successful phase III clinical trials but, in both cases, the initial BLA was rejected on the basis that their potency assay for drug product release was inadequate. Fortunately these issues appear to have been overcome in March of this year, with Mesoblast receiving acceptance of their BLA for Remestemcel and Iovance obtaining a rolling BLA approval for Lifileucel

Bringing function to the forefront of cell therapy: how do we demonstrate potency?

Unlike conventional pharmaceuticals, biologics and Advanced Therapy Medicinal Products (ATMPs) are required to meet a standard of “potency” as part of the final release criteria at completion of manufacture. During early phase clinical trials, most regulatory agencies have been willing to accept very immature potency assays with an expectation that these will be improved, qualified and validated during the clinical development of the drug to Marketing Authorisation Application (MAA) or Biologics License Application (BLA) submission.This model of continuous development of potency assay in parallel with drug development has already led to at least two notable problem cases; namely Iovance and Mesoblast. Both companies completed successful phase III clinical trials but, in both cases, the initial BLA was rejected on the basis that their potency assay for drug product release was inadequate. Fortunately these issues appear to have been overcome in March of this year, with Mesoblast receiving acceptance of their BLA for Remestemcel and Iovance obtaining a rolling BLA approval for Lifileucel

Antiviral immunity and T-regulatory cell function are retained after selective alloreactive T-cell depletion in both the HLA-identical and HLA-mismatched settings

AbstractNonselective T-cell depletion reduces the incidence of severe graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, but the cost is delayed and disordered antigen-specific immune reconstitution and increased infection. We use a method of selective depletion of alloreactive T cells expressing the activation marker CD69 after coculture with stimulator cells in a modified or standard mixed lymphocyte reaction. The technique has been shown to reduce alloreactivity while retaining third-party responses in vitro and, in a mismatched murine model, led to donor T-cell engraftment with a virtual absence of graft-versus-host disease and increased survival. We show in a human HLA-mismatched and unrelated HLA-identical setting that this technique retains >80% of specific cellular antiviral activity by cytomegalovirus-tetramer analysis and cytomegalovirus/Epstein-Barr virus peptide-stimulated interferon-γ ELISpot assay. Furthermore, CD4+ CD25+ T-regulatory cells are not removed by this method of selective allodepletion and retain their function in suppressing allogeneic proliferative responses. Preservation of antiviral cytotoxic T lymphocytes in selectively allodepleted stem cell grafts would lead to improved antiviral immunity after transplantation. The retention of immunosuppressive CD4+ CD25+ T-regulatory cells could lead to more ordered immune reconstitution and further suppress alloreactive responses after transplantation

Silsesquioxane polymer as a potential scaffold for laryngeal reconstruction

Cancer, disease and trauma to the larynx and their treatment can lead to permanent loss of structures critical to voice, breathing and swallowing. Engineered partial or total laryngeal replacements would need to match the ambitious specifications of replicating functionality, outer biocompatibility, and permissiveness for an inner mucosal lining. Here we present porous polyhedral oligomeric silsesquioxane-poly(carbonate urea) urethane (POSS-PCUU) as a potential scaffold for engineering laryngeal tissue. Specifically, we employ a precipitation and porogen leaching technique for manufacturing the polymer. The polymer is chemically consistent across all sample types and produces a foam-like scaffold with two distinct topographies and an internal structure composed of nano- and micro-pores. Whilst the highly porous internal structure of the scaffold contributes to the complex tensile behaviour of the polymer, the surface of the scaffold remains largely non-porous. The low number of pores minimise access for cells, although primary fibroblasts and epithelial cells do attach and proliferate on the polymer surface. Our data show that with a change in manufacturing protocol to produce porous polymer surfaces, POSS-PCUU may be a potential candidate for overcoming some of the limitations associated with laryngeal reconstruction and regeneration

JAK/STAT Signalling in Huntington's Disease Immune Cells.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Both central and peripheral innate immune activation have been described as features of the disease. Isolated human HD monocytes have been shown to produce more cytokines upon LPS stimulation compared to control monocytes. Understanding alterations in the signalling cascades responsible and activated by this increase in pro-inflammatory cytokine production is crucial in understanding the molecular basis of this phenomenon. Here we investigated the signalling cascade most commonly activated by pro-inflammatory cytokines such as IL-6 - the JAK/STAT signalling cascade. Using flow cytometry, we show that one out of three key transcription factors activated by JAK/STAT signalling is altered in primary human HD innate immune cells, suggesting that this pathway may only play a minor, additive role in the immune cell dysfunction in HD

Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity?

Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice for most animal cell systems since the early history of cryopreservation. It has been used for decades in many thousands of cell transplants. These treatments would not have taken place without suitable sources of DMSO that enabled stable and safe storage of bone marrow and blood cells until needed for transfusion. Nevertheless, its effects on cell biology and apparent toxicity in patients have been an ongoing topic of debate, driving the search for less cytotoxic cryoprotectants. This review seeks to place the toxicity of DMSO in context of its effectiveness. It will also consider means of reducing its toxic effects, the alternatives to its use and their readiness for active use in clinical settings

HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation

The peripheral immune response is altered in Huntington's disease, but the underlying mechanisms are unclear. Using RNA interference to lower huntingtin levels in leucocytes from patients, Träger et al. reverse disease-associated phenotypes including cytokine elevation and transcriptional dysregulation, and argue for a direct effect of mutant huntingtin on NFκΒ signallin

Lessons learned from pre-clinical testing of xenogeneic decellularized esophagi in a rabbit model

Summary Decellularization of esophagi from several species for tissue engineering is well described, but successful implantation in animal models of esophageal replacement has been challenging. The purpose of this study was to assess feasibility and applicability of esophageal replacement using decellularized porcine esophageal scaffolds in a new pre-clinical model. Following surgical replacement in rabbits with a vascularizing muscle flap, we observed successful anastomoses of decellularized scaffolds, cues of early neovascularization, and prevention of luminal collapse by the use of biodegradable stents. However, despite the success of the surgical procedure, the long-term survival was limited by the fragility of the animal model. Our results indicate that transplantation of a decellularized porcine scaffold is possible and vascular flaps may be useful to provide a vascular supply, but long-term outcomes require further pre-clinical testing in a different large animal model

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs