Quantifying the Variability of Ground Light Sources and Their Relationships with Spaceborne Observations of Night Lights Using Multidirectional and Multispectral Measurements.

With the transition to LED lighting technology, multispectral night-time sensors are needed to quantify the changing nightscapes, given the limitations of the panchromatic sensors. Our objective was to quantify the contribution of lighting sources as measured on the ground and examine their correspondence with night-time brightness and color as measured from space. We conducted ground-based measurements of night-time brightness using the multidirectional (top, rear, right, front, left) and multispectral LANcube v2, which was mounted on the roof of a car, over 458 km of roads in central Israel and in Brisbane, Australia. For spaceborne measurements, we used the SDGSAT-1 multispectral Glimmer sensor. We found that spaceborne measurements of apparent radiance were best explained when including all ground-based directional measurements, with greater explanatory power for highways (R2 = 0.725) than for urban roads (R2 = 0.556). Incoming light in the five directions varied between road classes and land use. In most cases, the variability in night-time brightness and color was greater for urban road sections than for highways. We conclude that due to the spectral mixture of lighting sources, at a medium spatial resolution, the impact of the transition to LED lighting may be more easily recognized from space over highways than in dense urban settings.

Accelerated shifts in terrestrial life zones under rapid climate change.

Rapid climate change is impacting biodiversity, ecosystem function, and human well-being. Though the magnitude and trajectory of climate change are becoming clearer, our understanding of how these changes reshape terrestrial life zones-distinct biogeographic units characterized by biotemperature, precipitation, and aridity representing broad-scale ecosystem types-is limited. To address this gap, we used high-resolution historical climatologies and climate projections to determine the global distribution of historical (1901-1920), contemporary (1979-2013), and future (2061-2080) life zones. Comparing the historical and contemporary distributions shows that changes from one life zone to another during the 20th century impacted 27 million km2 (18.3% of land), with consequences for social and ecological systems. Such changes took place in all biomes, most notably in Boreal Forests, Temperate Coniferous Forests, and Tropical Coniferous Forests. Comparing the contemporary and future life zone distributions shows the pace of life zone changes accelerating rapidly in the 21st century. By 2070, such changes would impact an additional 62 million km2 (42.6% of land) under "business-as-usual" (RCP8.5) emissions scenarios. Accelerated rates of change are observed in hundreds of ecoregions across all biomes except Tropical Coniferous Forests. While only 30 ecoregions (3.5%) had over half of their areas change to a different life zone during the 20th century, by 2070 this number is projected to climb to 111 ecoregions (13.1%) under RCP4.5 and 281 ecoregions (33.2%) under RCP8.5. We identified weak correlations between life zone change and threatened vertebrate richness, levels of vertebrate endemism, cropland extent, and human population densities within ecoregions, illustrating the ubiquitous risks of life zone changes to diverse social-ecological systems. The accelerated pace of life zone changes will increasingly challenge adaptive conservation and sustainable development strategies that incorrectly assume current ecological patterns and livelihood provisioning systems will persist.

Potent Activation of Human T Cells by mRNA Encoding Constitutively Active CD40.

New strategies for augmenting the actual performance of therapeutic T cells in vivo are needed for improving clinical outcome of adoptive cell therapy. Cumulative findings suggest that CD40 plays an intrinsic role in T cell costimulation. Recently, we demonstrated the ability of truncated, auto-oligomerizing CD40 derivatives to induce strong activation of APCs in a ligand-independent manner. We reasoned that constitutively active CD40 (caCD40) can similarly exert enhancing effects on human antitumor T cells. To test this assumption, we transfected human T cells with in vitro-transcribed caCD40 mRNA. In polyclonal T cells, caCD40 triggered IFN-γ secretion and upregulated CD25 and 4-1BB. In antimelanoma tumor-infiltrating lymphocytes (TILs), caCD40 induced massive production of IFN-γ, exerting a pronounced synergistic effect when coexpressed with constitutively active TLR4 devoid of its extracellular ligand binding. In unselected "young" TILs, caCD40 reproducibly increased surface expression of CD25, OX40, 4-1BB, CD127, and CD28. Three days post-mRNA electroporation of CD8 TILs, caCD40 elevated IFN-γ and TNF-α production and cytolytic activity in the presence of autologous but not HLA-I-mismatched melanoma. Enhanced killing of autologous melanoma by young TILs was observed 4 d posttransfection. These findings suggest that caCD40 can function as a potent T cell adjuvant and provide essential guidelines for similar manipulation of other key members of the TNFR family.

Global assessment of marine biodiversity potentially threatened by offshore hydrocarbon activities.

Increasing global energy demands have led to the ongoing intensification of hydrocarbon extraction from marine areas. Hydrocarbon extractive activities pose threats to native marine biodiversity, such as noise, light, and chemical pollution, physical changes to the sea floor, invasive species, and greenhouse gas emissions. Here, we assessed at a global scale the spatial overlap between offshore hydrocarbon activities and marine biodiversity (>25,000 species, nine major ecosystems, and marine protected areas), and quantify the changes over time. We discovered that two-thirds of global offshore hydrocarbon activities occur in areas within the top 10% for species richness, range rarity, and proportional range rarity values globally. Thus, while hydrocarbon activities are undertaken in less than one percent of the ocean's area, they overlap with approximately 85% of all assessed species. Of conservation concern, 4% of species with the largest proportion of their range overlapping hydrocarbon activities are range restricted, potentially increasing their vulnerability to localized threats such as oil spills. While hydrocarbon activities have extended to greater depths since the mid-1990s, we found that the largest overlap is with coastal ecosystems, particularly estuaries, saltmarshes and mangroves. Furthermore, in most countries where offshore hydrocarbon exploration licensing blocks have been delineated, they do not overlap with marine protected areas (MPAs). Although this is positive in principle, many countries have far more licensing block areas than protected areas, and in some instances, MPA coverage is minimal. These findings suggest the need for marine spatial prioritization to help limit future spatial overlap between marine conservation priorities and hydrocarbon activities. Such prioritization can be informed by the spatial and quantitative baseline information provided here. In increasingly shared seascapes, prioritizing management actions that set both conservation and development targets could help minimize further declines of biodiversity and environmental changes at a global scale.

Cancer and mortality in relation to traffic-related air pollution among coronary patients: Using an ensemble of exposure estimates to identify high-risk individuals.

BACKGROUND: Moderate correlations were previously observed between individual estimates of traffic-related air pollution (TRAP) produced by different exposure modeling approaches. This induces exposure misclassification for a substantial fraction of subjects. AIM: We used an ensemble of well-established modeling approaches to increase certainty of exposure classification and reevaluated the association with cancers previously linked to TRAP (lung, breast and prostate), other cancers, and all-cause mortality in a cohort of coronary patients. METHODS: Patients undergoing percutaneous coronary interventions in a major Israeli medical center from 2004 to 2014 (n = 10,627) were followed for cancer (through 2015) and mortality (through 2017) via national registries. Residential exposure to nitrogen oxides (NOx) -a proxy for TRAP- was estimated by optimized dispersion model (ODM) and land use regression (LUR) (rPearson = 0.50). Mutually exclusive groups of subjects classified as exposed by none of the methods (high-certainty low-exposed), ODM alone, LUR alone, or both methods (high-certainty high-exposed) were created. Associations were examined using Cox regression models. RESULTS: During follow-up, 741 incident cancer cases were diagnosed and 3051 deaths occurred. Using a ≥25 ppb cutoff, compared with high-certainty low exposed, the multivariable-adjusted hazard ratios (95% confidence intervals) for lung, breast and prostate cancer were 1.56 (1.13-2.15) in high-certainty exposed, 1.27 (0.86-1.86) in LUR-exposed alone, and 1.13 (0.77-1.65) in ODM-exposed alone. The association of the former category was strengthened using more extreme NOx cutoffs. A similar pattern, albeit less strong, was observed for mortality, whereas no association was shown for cancers not previously linked to TRAP. CONCLUSIONS: Use of an ensemble of TRAP exposure estimates may improve classification, resulting in a stronger association with outcomes.

The Human and Physical Determinants of Wildfires and Burnt Areas in Israel.

Wildfires are expected to increase in Mediterranean landscapes as a result of climate change and changes in land-use practices. In order to advance our understanding of human and physical factors shaping spatial patterns of wildfires in the region, we compared two independently generated datasets of wildfires for Israel that cover approximately the same study period. We generated a site-based dataset containing the location of 10,879 wildfires (1991-2011), and compared it to a dataset of burnt areas derived from MODIS imagery (2000-2011). We hypothesized that the physical and human factors explaining the spatial distribution of burnt areas derived from remote sensing (mostly large fires, >100 ha) will differ from those explaining site-based wildfires recorded by national agencies (mostly small fires, <10 ha). Small wildfires recorded by forestry agencies were concentrated within planted forests and near built-up areas, whereas the largest wildfires were located in more remote regions, often associated with military training areas and herbaceous vegetation. We conclude that to better understand wildfire dynamics, consolidation of wildfire databases should be achieved, combining field reports and remote sensing. As nearly all wildfires in Mediterranean landscapes are caused by human activities, improving the management of forest areas and raising public awareness to fire risk are key considerations in reducing fire danger.

Where have all the people gone? Enhancing global conservation using night lights and social media.

Conservation prioritization at large scales is complex, combining biological, environmental, and social factors. While conservation scientists now more often aim to incorporate human-related factors, a critical yet unquantified challenge remains: to identify which areas people use for recreation outside urban centers. To address this gap in applied ecology and conservation, we developed a novel approach for quantifying human presence beyond populated areas by combining social media "big data" and remote sensing tools. We used data from the Flickr photo-sharing website as a surrogate for identifying spatial variation in visitation globally, and complemented this estimate with spatially explicit information on stable night lights between 2004 and 2012, used as a proxy for identifying urban and industrial centers. Natural and seminatural areas attracting visitors were defined as areas both highly photographed and non-lit. The number of Flickr photographers within protected areas was found to be a reliable surrogate for estimating visitor numbers as confirmed by local authority censuses (r = 0.8). Half of all visitors' photos taken in protected areas originated from under 1% of all protected areas on Earth (250 of -27 000). The most photographed protected areas globally included Yosemite and Yellowstone National Parks (USA), and the Lake and Peak Districts (UK). Factors explaining the spatial variation in protected areas Flickr photo coverage included their type (e.g., UNESCO World Heritage sites have higher visitation) and accessibility to roads and trails. Using this approach, we identified photography hotspots, which draw many visitors and are also unlit (i.e., are located outside urban centers), but currently remain largely unprotected, such as Brazil's Pantanal and Bolivia's Salar de Uyuni. The integrated big data approach developed here demonstrates the benefits of combining remote sensing sources and novel geo-tagged and crowd-sourced information from social media in future efforts to identify spatial conservation gaps and pressures in real time, and their spatial and temporal variation globally.

Sensitivity analysis of conservation targets in systematic conservation planning.

Systematic conservation planning has rapidly advanced in the past decade and has been increasingly incorporated in multiple studies and conservation projects. One of its requirements is a quantitative definition of conservation targets. While the Convention on Biological Diversity aims to expand the world's protected area network to 17% of the land surface, in many cases such uniform policy-driven targets may not be appropriate for achieving persistence of various species. Targets are often set arbitrarily, often because information required for the persistence of each species is unavailable or unknown in the focal region. Conservation planners therefore need to establish complementary novel approaches to address the gaps in setting targets. Here, we develop and present a novel method that aims to help guide the selection of conservation targets, providing support for decision makers, planners, and managers. This is achieved by examining the overall flexibility of the conservation network resulting from conservation prioritization, and aiming for greater flexibility. To test this approach we applied the decision support tool Marxan to determine marine conservation priority areas in the eastern Mediterranean Sea as a case study. We assessed the flexibility of the conservation network by comparing 80 different scenarios in which conservation targets were gradually increased and assessed by a range of calculated metrics (e.g., the percentage of the total area selected, the overall connectivity). We discovered that when conservation targets were set too low (i.e., below 10% of the distribution range of each species), very few areas were identified as irreplaceable and the conservation network was not well defined. Interestingly, when conservation targets were set too high (over 50% of the species' range), too many conservation priority areas were selected as irreplaceable, an outcome which is realistically infeasible to implement. As a general guideline, we found that flexibility in a conservation network is adequate when ~10-20% of the study area is considered irreplaceable (selection frequency values over 90%). This approach offers a useful sensitivity analysis when applying target-based systematic conservation planning tools, ensuring that the resulting protected area conservation network offers more choices for managers and decision makers.

Emerging conservation challenges and prospects in an era of offshore hydrocarbon exploration and exploitation.

Globally, extensive marine areas important for biodiversity conservation and ecosystem functioning are undergoing exploration and extraction of oil and natural gas resources. Such operations are expanding to previously inaccessible deep waters and other frontier regions, while conservation-related legislation and planning is often lacking. Conservation challenges arising from offshore hydrocarbon development are wide-ranging. These challenges include threats to ecosystems and marine species from oil spills, negative impacts on native biodiversity from invasive species colonizing drilling infrastructure, and increased political conflicts that can delay conservation actions. With mounting offshore operations, conservationists need to urgently consider some possible opportunities that could be leveraged for conservation. Leveraging options, as part of multi-billion dollar marine hydrocarbon operations, include the use of facilities and costly equipment of the deep and ultra-deep hydrocarbon industry for deep-sea conservation research and monitoring and establishing new conservation research, practice, and monitoring funds and environmental offsetting schemes. The conservation community, including conservation scientists, should become more involved in the earliest planning and exploration phases and remain involved throughout the operations so as to influence decision making and promote continuous monitoring of biodiversity and ecosystems. A prompt response by conservation professionals to offshore oil and gas developments can mitigate impacts of future decisions and actions of the industry and governments. New environmental decision support tools can be used to explicitly incorporate the impacts of hydrocarbon operations on biodiversity into marine spatial and conservation plans and thus allow for optimum trade-offs among multiple objectives, costs, and risks.

Back-extrapolating a land use regression model for estimating past exposures to traffic-related air pollution.

Land use regression (LUR) models rely on air pollutant measurements for their development, and are therefore limited to recent periods where such measurements are available. Here we propose an approach to overcome this gap and calculate LUR models several decades before measurements were available. We first developed a LUR model for NOx using annual averages of NOx at all available air quality monitoring sites in Israel between 1991 and 2011 with time as one of the independent variables. We then reconstructed historical spatial data (e.g., road network) from historical topographic maps to apply the model's prediction to each year from 1961 to 2011. The model's predictions were then validated against independent estimates about the national annual NOx emissions from on-road vehicles in a top-down approach. The model's cross validated R2 was 0.74, and the correlation between the model's annual averages and the national annual NOx emissions between 1965 and 2011 was 0.75. Information about the road network and population are persistent predictors in many LUR models. The use of available historical data about these predictors to resolve the spatial variability of air pollutants together with complementary national estimates on the change in pollution levels over time enable historical reconstruction of exposures.

Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment. METHODS: We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand. RESULTS: We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models. CONCLUSIONS: Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation. TRIAL REGISTRATION NUMBER: NCT00068003, NCT01174121, and NCT03412877.

Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers.

Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ T cells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCRPBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

Between-country collaboration and consideration of costs increase conservation planning efficiency in the Mediterranean Basin.

The importance of global and regional coordination in conservation is growing, although currently, the majority of conservation programs are applied at national and subnational scales. Nevertheless, multinational programs incur transaction costs and resources beyond what is required in national programs. Given the need to maximize returns on investment within limited conservation budgets, it is crucial to quantify how much more biodiversity can be protected by coordinating multinational conservation efforts when resources are fungible. Previous studies that compared different scales of conservation decision-making mostly ignored spatial variability in biodiversity threats and the cost of actions. Here, we developed a simple integrating metric, taking into account both the cost of conservation and threats to biodiversity. We examined the Mediterranean Basin biodiversity hotspot, which encompasses over 20 countries. We discovered that for vertebrates to achieve similar conservation benefits, one would need substantially more money and area if each country were to act independently as compared to fully coordinated action across the Basin. A fully coordinated conservation plan is expected to save approximately US$67 billion, 45% of total cost, compared with the uncoordinated plan; and if implemented over a 10-year period, the plan would cost approximately 0.1% of the gross national income of all European Union (EU) countries annually. The initiative declared in the recent Paris Summit for the Mediterranean provides a political basis for such complex coordination. Surprisingly, because many conservation priority areas selected are located in EU countries, a partly coordinated solution incorporating only EU-Mediterranean countries is almost as efficient as the fully coordinated scenario.

From the Levant to Gibraltar: a regional perspective for marine conservation in the Mediterranean Sea.

Marine protected areas (MPAs) are critical to the well-being of threatened ecosystems and thus can be highly beneficial to humans, especially to those residing nearby. We explore the qualities of 117 MPAs in the Mediterranean basin and develop a taxonomy of their characteristics. We relate the spatial distribution of the MPAs to the various characteristics of the taxonomy (size, distance from shore, protection levels, management regimes, etc.) and to areas of high human impact and influence levels. To do this we use information on biogeographic regions and information from two different human influence models; one model developed for the marine environment and one covering the littoral terrestrial environment. Our analysis provides insights to planners and managers working in a regional capacity and trying to build MPA networks. Generally, current MPAs have not been established in high impact areas despite their being close to shores containing intense human activity. Decision-makers wishing to design and establish new MPAs may seek out areas of high cumulative human impacts (near the marine-terrestrial interface) or avoid them depending on marine conservation objectives, including the desire to vary types of MPAs within a network. Limitations of our analysis and methodology indicate areas for further research.

Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo-expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53-reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02-restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919.

Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

Spatial and temporal assessment of oil spills in the Mediterranean Sea.

Ship-generated oil pollution is a significant threat to the Mediterranean Sea. We present a geostatistical analysis of oil spills using three databases for the Mediterranean Sea: REMPEC (1977-2000) with 385 spills (17/year), ITOPF (1970-2018) with 167 spills (3.5/year) and EMSA (2015-2017) with 2066 detections (688/year). It was found that 88% of spills reported by REMPEC occurred near coastline areas, while 65% of the spills detected by EMSA occurred within a range of 22-100 km from the coastline. At the Exclusive Economic Zone (EEZ) level, EMSA oil spills densities were positively correlated with shipping and port activity. We conclude that there is a need to develop an open-access database of oil spills that will be based on both reports and remote sensing acquisition methods. Such a database will facilitate more efficient enforcement of international conventions in offshore areas and will increase the likelihood of effective response.

The Intracellular Domain of CD40 is a Potent Costimulatory Element in Chimeric Antigen Receptors.

The costimulatory domains incorporated into second-generation and third-generation chimeric antigen receptors (CARs) strongly influence CAR-T-cell function. Here, we explored second-generation and third-generation CARs harboring the signaling domain of the CD40 receptor as a new costimulatory element in comparison with similar CARs carrying the 4-1BB domain. In CARs of both generations, CD40 was more potent than 4-1BB in triggering the NF-κB signaling pathway. In human T cells from 2 donors, CD40 was comparable to 4-1BB in upregulating costimulatory and activation markers, inducing proinflammatory cytokine secretion and mediating target cell killing. Interestingly, differences in the response pattern of T cells from the 2 donors with respect to CD40 and 4-1BB were evident. We conclude that in human T cells, the CD40 signaling domain is a potent costimulatory element in both second-generation and third-generation CARs.