Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in adults. Current treatment options typically consist of surgery followed by chemotherapy or more frequently radiotherapy, however, median patient survival remains at just over 1 year. Therefore, the need for novel curative therapies for GBM is vital. Characterization of GBM cells has contributed to identify several molecules as targets for immunotherapy-based treatments such as EGFR/EGFRvIII, IL13Rα2, B7-H3, and CSPG4. Cytotoxic T lymphocytes collected from a patient can be genetically modified to express a chimeric antigen receptor (CAR) specific for an identified tumor antigen (TA). These CAR T cells can then be re-administered to the patient to identify and eliminate cancer cells. The impressive clinical responses to TA-specific CAR T cell-based therapies in patients with hematological malignancies have generated a lot of interest in the application of this strategy with solid tumors including GBM. Several clinical trials are evaluating TA-specific CAR T cells to treat GBM. Unfortunately, the efficacy of CAR T cells against solid tumors has been limited due to several factors. These include the immunosuppressive tumor microenvironment, inadequate trafficking and infiltration of CAR T cells and their lack of persistence and activity. In particular, GBM has specific limitations to overcome including acquired resistance to therapy, limited diffusion across the blood brain barrier and risks of central nervous system toxicity. Here we review current CAR T cell-based approaches for the treatment of GBM and summarize the mechanisms being explored in pre-clinical, as well as clinical studies to improve their anti-tumor activity.

Natural killer (NK) cells rapidly reconstitute following allogeneic stem cell transplantation (allo‐SCT), at the time when alloreactive T cell immunity is being established. We investigated very early NK cell reconstitution in 82 patients following T cell‐depleted allo‐SCT. NK cell number rapidly increased, exceeding T cell reconstitution such that the NK:T cell ratio was over 40 by day 14. NK cells at day 14 (NK‐14) were donor‐derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL‐10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. NK‐14 cell number was inversely correlated with the incidence of grade II‐IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo‐SCT is an important determinant of the clinical outcome, suggesting that NK cells may suppress the development of the T cell‐mediated alloreactive immune response through production of IL‐10.

Transplantation is an effective treatment of many clinical disorders, but the mechanisms that regulate immunological tolerance are uncertain and remain central to improving patient outcome. Hemopoietic stem cell transplantation (SCT) often establishes “mixed chimerism” in which immune cells from both the donor and patient coexist in vivo in a setting of immunological tolerance. We studied immune function in 69 patients within 2 months following SCT; 37 were fully donor and 32 displayed mixed chimerism. The proportion of T regulatory (Treg) cells was increased during mixed chimerism and comprised equal numbers of donor and host-derived regulatory cells. This was associated with a tolerogenic PD-L1+ profile on dendritic cells. Importantly, effector T cells from patients with mixed chimerism exhibited reduced cytotoxicity against host target cells in vitro, but this was restored following depletion of CD4+ Treg cells. These data show that Treg cells play a major role in sustaining immunological tolerance during mixed chimerism. These insights should help to guide novel interventions to improve clinical transplantation.

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Key Points• A stem cell graft NK cell dose below 6.3 3 10 6 cells per kg associates with risk of disease relapse following T-cell-depleted allo-HSCT.• Clinical outcomes of patients undergoing allo-HSCT may be improved by setting an NK cell threshold within donor stem cell grafts.The graft-versus-leukemia (GVL) effect of allogeneic hemopoietic stem cell transplantation (allo-HSCT) is mediated by the donor immune system and acts to decrease the rate of disease relapse. Although studies of posttransplant immune reconstitution have identified correlates of clinical outcome, the number and profile of mature immune cells infused with the stem cell graft is also likely to be an important determinant and has been relatively poorly studied.We characterized immune cells within the stem cell graft of 107 patients who underwent T-cell-depleted allo-HSCT and related this to clinical outcome. The number of natural killer (NK) cells and T cells that were infused varied markedly between patients, but T-cell dosewas not an important factor in subsequent outcome. In contrast, the number of NK cells was a powerful determinant of the risk of disease relapse. Patients who received an NK cell dose below the median level of 6.3 3 10 6 cells per kg had a relapse rate of 40% at 2 years posttransplant compared with only 6% for those whose stem cell graft contained a dose above this value. Analysis of NK subsets showed that this effect was mediated primarily by the CD56 dim population of mature effector cells and that high-level expression of the activatory protein DNAM on donor NK cells was also strongly protective. These observations offer important insights into the mechanism of GVL and suggest that optimization studies of the number of NK cells within the stem cell graft should be considered as a means to reduce disease relapse.

Frequently, the results generated when testing novel antitumor immunotherapies in vitro do not correlate with data collected in in vivo models and/or in clinical settings. It is our hypothesis that this discrepancy is caused by the use of in vitro conditions, such as normoxia, a two-dimensional surface, optimal growth media, and lack of cell complexity and heterogeneity. These conditions do not accurately reflect the tumor microenvironment (TME) that the tested immunotherapeutic strategies experience in vivo. While there are many variables which can have an impact upon the antitumor efficacy of an immunotherapy, the immunosuppressive TME is one in which several of the conditions commonly found in vivo can be mimicked in vitro. These conditions, which include hypoxia, low pH, low glucose, presence of adenosine, cell complexity and heterogeneity, as well as the three-dimensional structure of TME, can all affect immune cell-tumor cell interactions. Here, we discuss the impact that these conditions, either individually or in combination, can have on these interactions. Furthermore, we propose that performing in vitro assays under TME-like conditions improves the clinical relevance of the yielded results. This, in turn, contributes to accelerate the speed, reduce the cost, and increase efficiency of screening novel immunotherapies and eventually the development of prospective clinical trials.

1. Bespoke (custom-built) Raspberry Pi cameras are increasingly popular research tools in the fields of behavioral ecology and conservation, because of their comparative flexibility in programmable settings, ability to be paired with other sensors, and because they are typically cheaper than commercially built models.2. Here, we describe a novel, Raspberry Pi-based camera system that is fully portable and yet weatherproof-especially to humidity and salt spray. The camera was paired with a passive infrared sensor, to create a movement-triggered camera capable of recording videos over a 24-hr period. We describe an example deployment involving "retro-fitting" these cameras into artificial nest boxes on Praia Islet, Azores archipelago, Portugal, to monitor the behaviors and interspecific interactions of two sympatric species of storm-petrel (Monteiro's storm-petrel Hydrobates monteiroi and Madeiran storm-petrel Hydrobates castro) during their respective breeding seasons.3. Of the 138 deployments, 70% of all deployments were deemed to be "Successful" (Successful was defined as continuous footage being recorded for more than one hour without an interruption), which equated to 87% of the individual 30-s videos.The bespoke cameras proved to be easily portable between 54 different nests and reasonably weatherproof (~14% of deployments classed as "Partial" or "Failure" deployments were specifically due to the weather/humidity), and we make further trouble-shooting suggestions to mitigate additional weather-related failures.4. Here, we have shown that this system is fully portable and capable of coping with salt spray and humidity, and consequently, the camera-build methods and scripts could be applied easily to many different species that also utilize cavities, burrows, and artificial nests, and can potentially be adapted for other wildlife monitoring situations to provide novel insights into species-specific daily cycles of behaviors and interspecies interactions.

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