In western North America, quaking aspen stands (Populus tremuloides Michx.) have predominantly been described as low flammability, “fireproof” forests, but the specific relationship between aspen stand composition, fuel characteristics, and potential fire behavior is not fully understood. We investigated surface and canopy fuel characteristics in 80 aspen stands in Utah, U.S., that spanned gradients of tree species composition from aspen to conifer dominance and stand development from early to late stages. We quantified fuel type and load, measured fuel moisture content in representative stands across two summer seasons, and modeled flame lengths in each stand. Fuel type and load varied greatly across stands, though late development, conifer-dominated stands had significantly higher (∼2–5 times) fine dead woody and litter load and significantly lower (∼2–5 times) live understory herbaceous load compared to pure aspen stands. Fuel moisture content did not vary by stand type. Modeled flame lengths were lowest in pure aspen stands, and flame lengths increased linearly with decreasing aspen composition, suggesting that potential surface fire behavior increases as a seral aspen stand progresses through succession to conifer dominance.

Quaking aspen (Populus tremuloides Michx.) ecosystems are highly valued in the southwestern United States because of the ecological, economic, and aesthetic benefits they provide. Aspen has experienced extensive mortality in recent decades, and there is evidence that many areas in Arizona, United States lack adequate recruitment to replace dying overstory trees. Maintaining sustainable levels of regeneration and recruitment (i.e. juveniles) is critical for promoting aspen ecosystem resilience and adaptive capacity, but questions remain about which factors currently limit juvenile aspen and which strategies are appropriate for managing aspen in an increasingly uncertain future. To fill these critical knowledge gaps, we sampled aspen populations across Arizona and collected data representing a suite of biotic and abiotic factors that potentially influence juvenile aspen. Specifically, we addressed two questions: (i) Is aspen sustainably regenerating and recruiting in Arizona? and (2) Which biotic and abiotic factors significantly influence aspen regeneration and recruitment? We found that many aspen populations in Arizona lack sustainable levels of juvenile aspen, and the status of recruitment was especially dire, with 40% of study plots lacking a single recruiting stem. Aspen regeneration was less abundant on warmer sites than cooler ones, highlighting the threat that a rapidly warming climate poses to aspen sustainability. Aspen recruitment was significantly more abundant in areas with recent fire than in areas without fire, and recruitment had a strong positive relationship with fire severity. The most important limiting factors for aspen recruitment were ungulate browse, especially by introduced Rocky Mountain elk (Cervus canadensis nelsoni), and the invasive insect, oystershell scale (Lepidosaphes ulmi). We conclude with a discussion of how management can promote sustainability of aspen populations by addressing the array of threats that aspen faces, such as a warming climate, chronic ungulate browse, and outbreaks of oystershell scale.

Quaking aspen (Populus tremuloides Michx.) landscapes are valued for their biodiversity, water retention, fire mitigation, aesthetics, and recreation opportunities. Across North America, some aspen populations are experiencing population declines as they face uninhibited ungulate browsing, drought, fire suppression, insects, disease, and inappropriate management. Increased human development and recreational use within aspen landscapes can serve as additive stressors, though there is a dearth of literature examining these elements. At a popular recreational area in Utah, USA, identifying the cause of apparent decline within a larger aspen community is complicated by development upstream and recreation-related activities. We sought to (1) assess the overall condition of the aspen at the site, (2) understand key variables that influence aspen conditions, and (3) elucidate how aspen fitness varies across the site. We collected data from forty-five plots using established aspen sampling methods, including ungulate presence, tree characteristics, soil chemistry, and environmental descriptors. Results suggest that a combination of higher levels of browsing and elevated soil sodium may be causing premature mortality and limiting aspen recruitment in a portion of the study area. These findings will inform future management at this site, as well as similar recreational forest settings experiencing compound stressors.

AbstractSpecies of conservation concern are often habitat specialists, posing significant risk to those species when specific plant communities are threatened. As a result, practitioners habitually focus conservation efforts on these communities while ignoring ecological mechanisms that explain the wildlife–plant relationships. In doing so, practitioners may overlook alternative vegetation communities that could maintain wildlife populations under alternative conditions (e.g., climate change). Here, we term these areas surrogate habitat, defined as vegetation communities or resource sites that provide similar critical resources as conventional sites, and assess their potential for conservation using a case study of greater sage‐grouse (Centrocercus urophasianus) on Parker Mountain, Utah (1998–2009). Sage‐grouse are a sagebrush‐obligate species and a species of conservation concern. Range‐wide conservation efforts have long emphasized management of seasonal habitats within semiarid sagebrush ecosystems, specifically management of mesic or wet meadow sites that provide brood‐rearing habitat required for population persistence. Despite this requirement, no conventional mesic habitat exists on Parker Mountain, yet it supports one of Utah's largest sage‐grouse populations. Rather, the Parker sagebrush system abuts quaking aspen (Populus tremuloides) stands that may provide brood‐rearing habitat analogous to wet meadow sites. It is unclear, however, to what extent sage‐grouse use these aspen stands because sage‐grouse commonly avoid tall structures (e.g., trees) and their associated avian predators. Thus, we tested whether (1) sage‐grouse selected for surrogate habitat (i.e., aspen edge) and (2) selection behaviors related to surrogate habitat had demographic effects on the population. As we predicted, sage‐grouse selected for these areas, and the sage‐grouse that spent increased time closer to aspen edges did not experience increased mortality. Together, this demonstrates that the aspen–sagebrush edge provided a surrogate for the wet meadows used by other populations. More broadly, this suggests that conservation practitioners should move beyond simplistic wildlife–habitat associations toward a more holistic view of animal ecology focused on the wildlife–resource association, an approach that becomes particularly useful in areas where conventional obligate habitat may be degraded or lost. This work also implores us to examine alternative habitat potential rather than applying one‐size‐fits‐all models to threatened species conservation.

Knowledge of Eurasian aspen’s (Populus tremula L.) ecological and growth characteristics is of high importance to plant and wildlife community ecology, and noncommercial forest ecosystem services. This research assessed these characteristics, identified aspen’s habitat optimum, and examined causality of its current scarce distribution in central Europe. We analyzed a robust database of field measurements (4,656,130 stands) for forest management planning over 78,000 km2 of the Czech territory. Our analysis we used GIS techniques, with basic and multivariate statistics such as general linear models, ordination, and classification. Results describe a species of broad ecological amplitude that has heretofore attracted little research attention. Spatial analysis showed significant differences between aspen and other forest non-forest cover types. Additionally, we found significant association between the proportion of aspen in a stand, the size of forest property, and the forest category. The results demonstrate historic reasons for aspen’s widespread presence, though contemporary occurrence is limited. This study advances the concept of a quantitatively based aspen ecological optimum (niche), which we believe may be beneficial for numerous aspen associates in the context of anticipated warming. Irrespective of local ecology (i.e., the realized aspen niche), the study confirms that profit-driven policy in forestry is chiefly responsible for historic aspen denudation in central Europe. Even so, we demonstrate that ample habitat is present. Further solutions for improving aspen resilience are provided to support these keystone systems so vital to myriad dependent flora and fauna.

Quaking aspen (Populus tremuloides Michx.) has high conservation value on the southwestern edge of its range, which extends from the southwestern United States (i.e., Arizona, New Mexico, and Texas) to central Mexico. This value is driven by aspen’s ecological importance, positive impact on local economies, and aesthetic and cultural values. Generally, the scant aspen populations that remain in the Southwest lack resilience and adaptive capacity, and managers are unsure how best to maintain the species in an uncertain future. This systematic review seeks to address that need by reviewing existing literature from the Southwest on which biotic and abiotic factors influence aspen forest dynamics and by synthesizing that literature with a discussion of how management can promote aspen ecosystem resilience and adaptive capacity. We found that fire and silvicultural treatments promote aspen regeneration, but chronic ungulate browse inhibits recruitment. Moreover, drought is a driver of overstory mortality and has a negative influence on recruitment. In the second half of this review, we propose three management objectives for increasing aspen resilience and adaptive capacity: (1) promote diversity in age structure, (2) mitigate ungulate impacts, and (3) enhance complexity. We consider how various management strategies could meet these objectives and highlight potential threats to aspen forest health and resilience.

This supplementary file contains Supplemental Figures 1-6. Supplemental Figure 1 shows that 7-day ex vivo culture of NK cells with IL-15 and 5 μM CHIR99021 leads to an increase in the frequencies of NK cells with heterogeneous adaptive NK cell phenotypes (defined by expression of CD57, PLZF, SYK and FcεR1γ) relative to DMSO controls. Supplemental Figure 2 contains a detailed phenotypic characterization of receptor expression and cytotoxic granule component levels in sorted CD3-CD56dimCD57- and CD3-CD56dimCD57+ NK cells cultured for 7 days with IL-15 and either DMSO or 5 μM CHIR99021. Supplementary Figure 3 shows an analysis of NK cell phenotype, viability and proliferation after 7-day culture with IL-15 and either DMSO or CHIR99021 at several concentrations (1 μM, 3 μM and 5 μM). Supplemental Figure 4 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and NKG2C. Supplemental Figure 5 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and KIR. Supplemental Figure 6 shows the frequency of ex vivo expanded NK cells expressing CD57 and/or NKG2C 14 days after adoptive transfer into NSG mice.

The Great Basin is an arid province located in the interior western United States. The region encompasses millions of hectares and quaking aspen (Populus tremuloides Michx.) forests comprise a minor portion of the total area. However, montane aspen forests play a disproportionately large role in providing ecosystem services in the region, including water retention, biodiversity, wildlife habitat, livestock forage, and recreational uses. With warming temperatures, increasing evaporative demand, and heightened precipitation variability, the future of aspen has become a critical concern. Using dendroecological approaches, we assessed growth patterns of 20 aspen stands across three geographically isolated "sky island" mountain ranges spanning portions of the north central Great Basin. We anticipated that the growth of Great Basin aspen would be strongly influenced by regional climatic patterns and largely in synchrony. Results revealed a more complex growth dynamic that varied among mountain ranges and across environmental gradients. In particular, aspen climate-growth relationships in the slightly dryer Ruby Mountains were strongly and positively correlated (r > 0.5) with previous fall to winter moisture availability. The Jarbidge Mountains had a positive but modest relationship with previous fall to winter moisture availability (r > 0.3). Climate-growth response in the Santa Rosa Mountains, the wettest range, showed no significant response to moisture availability during any time period examined but had greater tree-ring growth with warmer May temperatures. Although tree-ring centennial (1910 - 2010) growth trends were positive for all three mountain ranges, only the Santa Rosa Mountains maintained a positive recent growth trend (1970 - 2010). Moreover, distinct temporal shifts in tree growth-climate relationships in each mountain range suggest potentially unique aspen population adaptations to climate variability. For instance, in two of the mountain ranges, there was a shift from positive/neutral to negative growth relationships with temperature starting around the 1963 - 1987 time period, while tree growth also began simultaneously responding more positively to moisture availability. These growth shifts and observed enhanced sensitivities to monthly and seasonal climate variables over time may reflect dynamic tree growth responses caused by ongoing global climate change, but that may be tempered by local or regional factors, such as the relative availability and timing of soil moisture provided by spring snowmelt. A better understanding of biogeographic variation and causality in aspen growth could provide multiple management pathways governed by resilience characteristics in the face of future anthropogenic and climatic threats.

Quaking aspen (Populus tremuloides Michx.) stands have historically been referred to as “firebreak” forest types that can reduce fire activity, but high-intensity and high-severity fires have been observed to burn through aspen stands. Clearly, fire activity in aspen is highly variable, which may be due to the wide variation in aspen stand composition and structure and because the species occurs across wide geographic, environmental, and climatic gradients. In the western U.S., there is growing interest in promoting aspen stands within wildland-urban interface communities to reduce fire risk, but studies that refer to the low flammability of aspen stands rely on limited citations. If promoting aspen to reduce fire risk is a desirable forest management practice, consolidating the available literature is necessary to understand when, where, and how management might achieve this goal. Here, we synthesized literature and conducted a survey of forest and fire managers to assess current understanding of how fire interacts with aspen stands, as well as to examine possible factors that influence fire occurrence, behavior, and severity in aspen communities. We found evidence that the presence of aspen reduces fire occurrence, fire behavior, and fire severity, but this effect is dependent on many factors, including the percentage of aspen vs conifers in the overstory, load and type of understory fuels, weather, and season. We did not find any quantitative management guidelines on how to create, maintain, or use aspen stands to reduce fire risk. The large gap between “common knowledge” and empirical evidence regarding aspen’s ability to inhibit fire requires further research.

Communities of Populus tremuloides are widespread in the north and central-west part of North America. A well-known aspen site is the Pando aspen clone, which is situated adjacent to Fish Lake, Utah. This stand of genetically identical trees is putatively the most massive organism on earth. Our pedoanthracological study is focused on the reconstruction of forest history in the Pando aspen stand. Results document presence and abundance of charcoal species during a period of nearly 9,000 years. While we make no attempt to explicitly identify the Pando genotype throughout this period, we can determine trends in dominant tree species which allows for the possibility of a single long-lived aspen specimen. The dominance of Populus charcoals was recorded in all soil profiles within the current Pando stand area. The radiocarbon data documented an increase of fire events over the last 2,000 years, which were probably related to human activities. Results of our study document the first multi-millennial pattern of aspen cover at a single locale. We relate this long-term pattern to restoration practices in contemporary stable aspen communities, since these practices are compatible with endemic disturbance processes. We believe that patterns and processes gleaned from this work will be instructive for forest management broadly, as well as preservation of aspen communities across in North American and globally.

Upland aspen ( Populus spp . ) forests contribute significantly to biodiversity in their circumboreal role as keystone species. As aspen ecosystems flourish or diminish, myriad dependent species follow suit. The 43‐hectare Pando aspen ( Populus tremuloides Michx.) clone in Utah, USA, is thought to be the largest living organism on earth, but is faltering due to chronic herbivory. Long‐term resilience in aspen communities, including Pando, rests on successful recruitment of vegetative suckers that are nutritiously desirable to browsing ungulates. Here, I evaluate aspen reproduction alongside numerous vital indicators of Pando's status in the first trend assessment of this embattled iconic forest. I remeasured 64 plots from 2017 using 19 indicators to determine current conditions. Findings show that the genetically uniform Pando is “breaking up” because of herbivory and fencing. Initial successes within fenced zones are tempered by nearly half of Pando that remains unprotected from chronic wild and domestic herbivory. I propose a strategy of process‐based stewardship informed by adaptive monitoring to restore this famed “one‐tree forest.” Lessons from Pando may be applied to struggling, often species rich, aspen systems facing similar challenges globally.

Black locust (Robinia pseudoacacia L.) has been widely used to restore degraded land in northern China for many decades, and the forest has become an important ecosystem in China. However, there is still knowledge gap about how the range shift of black locust in response to future climate change, which is the first step for adaptive management of black locust. Here, a global niche model of black locust was established by means of maximum entropy model (MaxEnt), 1174 global occurrences data, as well as 13 climatic variables. Then, the global niche model was projected to China under current climate (2000) and four future climate scenarios (2080). The results showed that the range of black locust is mainly controlled by temperature related variables rather than precipitation related variables. The latitude of potential range of black locust is mainly between 23° and 40° in China with the area of occupation being about 26.7% (25.7 × 105 km2) of China's total land area. Future climate is conducive to the northward expansion of black locust in China with a speed of 21 km/decade, as well as an upward shift with a speed of 9.6 m/decade across climate scenarios. Relatively high stable ranges (87–94%) and quick range shift speed implies that little vulnerability of black locust in response to climate change, as well as little risk of extinction in China.

induced pluripotent stem cell (iPSC)-derived NK (iNK) cell novel ubiq-uitously targets cancer cells canonical stress ligand recognition. previously FT536 the conserved a 3 domain of the pan-tumor associated antigens MICA and MICB (MICA/B), and is a renewable statistically significant anti-tumor activity which is Conclusions Collectively, these studies demonstrate that FT536 is a highly potent, multi-tumor targeting CAR-iNK cell product that is uniform in composition and can be effectively and safely used off-the-shelf for on-demand treatment of multiple solid and hematological malignancies. An IND submission is planned for 2021, with an initial Phase 1 clinical trial to follow.

BackgroundOvarian cancer is a leading cause of cancer-related deaths among women due to the development of therapeutic resistance. Natural killer (NK) cells are cytotoxic lymphocytes that can kill neoplastic cells without prior sensitization. A key anti-tumor function of human NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC), mediated exclusively by the IgG Fcy (FcyR) receptor CD16A. The mechanism of action of several clinically successful antitumor therapeutic monoclonal antibodies (mAbs) involves ADCC; however, their therapeutic efficacy is reduced due to regulatory checkpoints of CD16A, which include its low IgG binding affinity and rapid downregulation upon NK cell activation by the membrane metalloprotease ADAM17.1–3 CD64, the highest affinity FcyR, is expressed on myeloid-derived cells and not lymphocytes and is also not cleaved by ADAM17. To enhance ADCC, we generated CD64/16A, a novel recombinant FcyR consisting of extracellular CD64 and intracellular and transmembrane CD16A to mediate high affinity IgG binding and potent cell signaling.4 5MethodsEngineered NK cells expressing CD64/16A were derived from human induced pluripotent stem-cells (iPSCs), referred to here as iNKs, which are clonal and clinically scalable NK cells. ADCC and natural cytotoxicity of three ovarian cancer cell lines were measured in vitro using Delfia EuTDA and IncuCyte cytotoxicity assays, and production of anti-tumor cytokines was determined via flow cytometry. Finally, tumor cell killing was assessed in vivo using a human xenograft mouse model of peritoneal metastatic ovarian cancer.ResultsOur data show that iNK-CD64/16A cells uniquely facilitate mAb absorption, robustly produce IFN-y and TNF-alpha, and kill several ovarian cancer cell lines in the presence of the therapeutic mAbs trastuzumab or cetuximab, which target HER2 or EGFR, respectively. We found that iNK-CD64/16A cells can capture soluble mAbs and retain antibody arming and ADCC efficacy after cryopreservation. Additionally, iNK-CD64/16A cells robustly mediate ADCC and reduce overall tumor burden of HER2+ tumor cells in vivo in the described metastatic ovarian cancer xenograft model.ConclusionsOur findings provide new insights into using high affinity Fc receptor-based adoptive NK cell therapies and lay the preclinical foundation necessary for developing an ‘off-the-shelf’ cellular therapy that can be combined with therapeutic tumor-targeting mAbs for the treatment of ovarian cancer. Importantly, iNK-CD64/16A cells can serve as a docking platform for therapeutic antibodies that can be switched and mixed for universal tumor antigen targeting to treat multiple malignancies.AcknowledgementsGrant supportNIH R01CA203348, HHMI Medical Research Fellows Program/Burroughs Wellcome FundReferencesJing Y, Ni Z, Wu J, et al. Identification of an ADAM17 cleavage region in human CD16 (FcgammaRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One 2015;10(3):e0121788.Wu J, Mishra HK, Walcheck B. Role of ADAM17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy. J Leukoc Biol 2019;105(6):1297–303.Dixon KJ, Wu J, Walcheck B. Engineering Anti-Tumor Monoclonal Antibodies and Fc Receptors to Enhance ADCC by Human NK Cells. Cancers (Basel) 2021;13(2).Snyder KM, Hullsiek R, Mishra HK, et al. Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells. Front Immunol 2018;9:2873.Walcheck B, Wu J. iNK-CD64/16A cells: a promising approach for ADCC? Expert Opin Biol Ther 2019;19(12):1229–32.Ethics ApprovalThis study was approved by the University of Minnesota’s Institutional Animal Care and Use Committee, protocol number 1902–36768A.

Background Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment, but it is associated with significant dose-limiting toxicities, restricted tumor targeting (limited by specific antigen expression), and, notably, a lack of multi-antigen targeting capability to mitigate tumor associated immune evasion and heterogeneity. Furthermore, dysfunctional starting material, product inconsistency, and small manufacturing lot size limits the application and on-demand availability of CAR-T cell therapy. Methods To overcome these considerable limitations, we have developed FT536, a first-of-kind, induced pluripotent stem cell (iPSC)-derived NK (iNK) cell with a novel CAR that ubiquitously targets cancer cells through canonical stress ligand recognition. We have previously reported FT536 recognizes the conserved α3 domain of the pan-tumor associated antigens MICA and MICB (MICA/B), and is derived from a renewable master iPSC line that contains multiplexed genetic edits to enhance effector cell functionality, persistence, and multi-antigen targeting capabilities via high affinity non cleavable CD16 (hnCD16) mediated antibody dependent cellular cytotoxicity (ADCC). Here we preview the nonclinical study for the investigational new drug (IND) application for FT536. Results Utilizing a manufacturing process analogous to pharmaceutical drug product development, we demonstrate FT536 can be consistently and uniformly produced with a greater than 4x10E7 fold cellular expansion per manufacturing campaign. Furthermore, FT536 can be cryopreserved at clinical scale to support off-the-shelf clinical application, with rapid product thaw and immediate patient infusion in an out-patient setting. Functional evaluation demonstrated that FT536 uniquely possesses potent and persistent antigen specific cytolytic activity against an array of solid and hematological tumor lines. Through its hnCD16 modality, FT536 can be utilized in combination with monoclonal antibodies to provide multi-antigen targeting capabilities and in conjunction with chemotherapeutics and/or radiation that augment surface MICA/B expression. In addition, directly thawed and infused FT536 demonstrated significant tumor growth inhibition in multiple solid and liquid in vivo xenograft models, in which tumor control was further enhanced in combination with a therapeutic antibody (figure 1). Finally, ongoing studies utilizing a lung adenocarcinoma model have highlighted the sustained persistence of FT536 in lung tissue up to 33 days following a single dose infusion without the need for exogenous cytokine support. Conclusions Collectively, these studies demonstrate that FT536 is a highly potent, multi-tumor targeting CAR-iNK cell product that is uniform in composition and can be effectively and safely used off-the-shelf for on-demand treatment of multiple solid and hematological malignancies. An IND submission is planned for 2021, with an initial Phase 1 clinical trial to follow.

Background Glioblastoma multiforme (GBM) is a primary brain tumor with a high mortality rate and median survival of ~14 months. Although progress has been made in the development of available therapies, the treatment of GBM remains palliative. 1 Emerging results from preclinical studies support the concept that GBM cells may be highly susceptible to natural killer (NK) cell cytotoxicity. 2 3 However, sourcing donor-derived NK cells for adoptive cell therapy is limited by cell number and quality. To overcome these barriers, we developed a robust manufacturing system for the generation of high-quality off-the-shelf NK cells derived from induced pluripotent stem cells (iPSCs). 4 Methods We generated triple gene-edited iPSCs designed for mass production of NK cells expressing a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound IL-15/IL-15R fusion protein along with knockout of the nicotinamide adenine dinucleotide (NAD+) hydrolase CD38. NK cells derived from these uniformly engineered iPSCs, termed iADAPT NK cells, displayed enhanced metabolic fitness, resistance to oxidative stress, broad natural cytotoxicity, and robust antibody-dependent cellular cytotoxicity (ADCC). To assess the cytotoxic capacity of iADAPT NK cells, we performed 3-dimensional (3-D) live imaging assays where iADAPT NK cell infiltration and cytotoxicity in response to 9 different primary, patient derived GBM spheroids was measured in real time over the course of 5 days. The in vivo persistence and antitumor function of iADAPT NK cells were also assessed using xenogeneic adoptive transfer models. Results In 3-D live imaging assays, iADAPT NK cell efficiently infiltrated and eliminated patient-derived GBM spheroids (figure 1A, B). These in vitro results were recapitulated in vivo in xenogeneic experiments where human GBM cells were implanted intracranially into immunodeficient mice (n=19) followed by adoptive transfer of either 1.5x106 or 3x106 iADAPT NK cells. We show that adoptive transfer of iADAPT NK cells promoted survival in a dose-dependent manner (figure 1C). Importantly, we also found that iADAPT NK cells persisted at high levels in the brain for at least 21 days in the absence of exogenous cytokine support (figure 1D). Abstract 169 Figure 1 Conclusions Triple gene-edited iPSCs can be used to robustly manufacture iADAPT NK cells. These off-the-shelf engineered NK cells exhibit potent cytotoxicity against primary, patient derived GBM cells. Work is in progress to further engineer iADAPT NK cells with chimeric antigen receptors incorporating defined targeting motifs to further enhance cytotoxicity against GBM cells. Our preclinical data provides proof-of-concept for a planned phase I clinical trial. References Jin J, Grigore F, Chen CC, Li M. Self-renewal signaling pathways and differentiation therapies of glioblastoma stem cells (Review). Int J Oncol 2021; 59 :45. Castriconi R, Daga A, Dondero A, Zona G, Luigi Poliani P, Melotti A, Griffero F, Marubbi D, Spaziante R, Bellora F, et al. NK cells recognize and kill human glioblastoma cells with stem cell-like properties. J Immunol 2009; 182 :3530–3539. Shiam H, Shanley M, Basar R, Daher M, Gumin J, Zamler DB, Uprety N, Wang F, Huang Y, Gabrusiewicz K, et al. Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma cells. J Clin Invest 2021; 131 :e142116. Cichocki F, Bjordahl R, Gaidarova S, Mahmood S, Abujarour R, Wang H, Tuininga K, Felices M, Davis ZB, Bendzick L, et al. iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy. Sci Transl Med 2020; 12 :eaaz5618. Ethics Approval This project has been approved by the University of Minnesota IACUC. Approval ID: 1812-36595A

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.

Natural killer (NK) cells are innate lymphocytes that target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and that recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells exclusively mediate ADCC through the IgG Fc receptor, CD16A, and studies have demonstrated that increasing the binding affinity between CD16A and therapeutic monoclonal antibodies (mAbs), mediated by the high-affinity 158V polymorphism, can augment clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we sought to arm iPSC-derived NK (iNK) cells expressing a high-affinity recombinant FcγR with various mAbs as unique tumor-targeting strategy for various malignancies.

Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape.

The influence of site preparation on understorey plant diversity was assessed in the commercial floodplain forest in the Thaya River alluvium near Br?eclav, Czech Republic. Phytosociological surveys were carried out during the summer of 2013 in 10-50 years old forest stands with and without mechanical site preparation (MSP). One hundred sixty releves (eighty from Quercus robur stands and eighty from Fraxinus angustifolia subsp. danubialis stands) were selected for analyses. Phytocoenological releves were analyzed using multidimensional statistics and plant community characteristics (fidelity, species richness, the percentage of non-native or endangered plant species). The results showed that mechanical site preparation significantly affects herbaceous species composition and richness, in the particular richness of non-native species and neophytes. We found a total richness of 231 herb and woody species, of which 49 (21.2 %) were non-native. Our results demonstrate that reforestation with the MSP decreased species richness of the understorey and increased the number of non-native species. The sites without MSP treatment contained significantly fewer non-native species. The invasive plants Aster lanceolatus and Acer negundo were more abundant in sites impacted by mechanical soil disturbance. Our results also show that a higher percentage of non-native plants survive longer in forest stands subjected to MSP. The abundance and cover of neophytes within MSP treatments are significantly higher in stands dominated by Fraxinus than Quercus stands.