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BACKGROUND: Breast cancer (BC) is the most common malignancy in women. Immunotherapy has revolutionized treatment options in many malignancies, and the introduction of immune checkpoint inhibition yielded beneficial results also in BC. However, many BC patients are ineligible for this T cell-based therapy, others do not respond or only briefly. Thus, there remains a high medical need for new therapies, particularly for triple-negative BC. CD276 (B7-H3) is overexpressed in several tumors on both tumor cells and tumor vessels, constituting a promising target for immunotherapy. METHODS: We analyzed tumor samples of 25 patients using immunohistochemistry to assess CD276 levels. The potential of CC-3, a novel bispecific CD276xCD3 antibody, for BC treatment was evaluated using various functional in vitro assays. RESULTS: Pronounced expression of CD276 was observed in all analyzed tumor samples including triple negative BC. In analyses with BC cells, CC-3 induced profound T cell activation, proliferation, and T cell memory subset formation. Moreover, treatment with CC-3 induced cytokine secretion and potent tumor cell lysis. CONCLUSION: Our findings characterize CD276 as promising target and preclinically document the therapeutic potential of CC-3 for BC treatment, providing a strong rationale for evaluation of CC-3 in BC patients in a clinical trial for which the recruitment has recently started.
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Antígenos B7 , Neoplasias da Mama , Imunoterapia , Linfócitos T , Humanos , Feminino , Antígenos B7/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Neoplasias da Mama/patologia , Imunoterapia/métodos , Linfócitos T/imunologia , Linhagem Celular Tumoral , Pessoa de Meia-Idade , Ativação Linfocitária/imunologia , Proliferação de Células , Idoso , Citocinas/metabolismo , AdultoRESUMO
Acute myeloid leukemia (AML) remains a therapeutic challenge despite recent therapeutic advances. Although monoclonal antibodies (mAbs) engaging natural killer (NK) cells via antibody-dependent cellular cytotoxicity (ADCC) hold promise in cancer therapy, almost none have received clinical approval for AML, so far. Recently, CD276 (B7-H3) has emerged as a promising target for AML immunotherapy, due to its high expression on leukemic blasts of AML patients. Here, we present the preclinical development of the Fc-optimized CD276 mAb 8H8_SDIE with enhanced CD16 affinity. We demonstrate that 8H8_SDIE specifically binds to CD276 on AML cell lines and primary AML cells and induces pronounced NK cell activation and degranulation as measured by CD69, CD25, and CD107a. Secretion of IFNγ, TNF, granzyme B, granulysin, and perforin, which mediate NK cell effector functions, was induced by 8H8_SDIE. A pronounced target cell-restricted lysis of AML cell lines and primary AML cells was observed in cytotoxicity assays using 8H8_SDIE. Finally, xenograft models with 8H8_SDIE did not cause off-target immune activation and effectively inhibited leukemia growth in vivo. We here present a novel attractive immunotherapeutic compound that potently induces anti-leukemic NK cell reactivity in vitro and in vivo as treatment option for AML.
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Células Matadoras Naturais , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos B7/metabolismo , Antígenos B7/farmacologiaRESUMO
Pancreatic cancer is a highly lethal disease with limited treatment options. Hence, there is a considerable medical need for novel treatment strategies. Monoclonal antibodies (mAbs) have significantly improved cancer therapy, primarily due to their ability to stimulate antibody-dependent cellular cytotoxicity (ADCC), which plays a crucial role in their therapeutic efficacy. As a result, significant effort has been focused on improving this critical function by engineering mAbs with Fc regions that have increased affinity for the Fc receptor CD16 expressed on natural killer (NK) cells, the major cell population that mediates ADCC in humans. Here we report on the preclinical characterization of a mAb directed to the target antigen B7-H3 (CD276) containing an Fc part with the amino acid substitutions S239D/I332E to increase affinity for CD16 (B7-H3-SDIE) for the treatment of pancreatic cancer. B7-H3 (CD276) is highly expressed in many tumor entities, whereas expression on healthy tissues is more limited. Our findings confirm high expression of B7-H3 on pancreatic cancer cells. Furthermore, our study shows that B7-H3-SDIE effectively activates NK cells against pancreatic cancer cells in an antigen-dependent manner, as demonstrated by the analysis of NK cell activation, degranulation and cytokine release. The activation of NK cells resulted in significant tumor cell lysis in both short-term and long-term cytotoxicity assays. In conclusion, B7-H3-SDIE constitutes a promising agent for the treatment of pancreatic cancer.
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Imunoterapia , Neoplasias Pancreáticas , Humanos , Imunoterapia/métodos , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Monoclonais , Células Matadoras Naturais , Neoplasias Pancreáticas/metabolismo , Antígenos B7/metabolismoRESUMO
T-cell immunity is central for control of COVID-19, particularly in patients incapable of mounting antibody responses. CoVac-1 is a peptide-based T-cell activator composed of SARS-CoV-2 epitopes with documented favorable safety profile and efficacy in terms of SARS-CoV-2-specific T-cell response. We here report a Phase I/II open-label trial (NCT04954469) in 54 patients with congenital or acquired B-cell deficiency receiving one subcutaneous CoVac-1 dose. Immunogenicity in terms of CoVac-1-induced T-cell responses and safety are the primary and secondary endpoints, respectively. No serious or grade 4 CoVac-1-related adverse events have been observed. Expected local granuloma formation has been observed in 94% of study subjects, whereas systemic reactogenicity has been mild or absent. SARS-CoV-2-specific T-cell responses have been induced in 86% of patients and are directed to multiple CoVac-1 peptides, not affected by any current Omicron variants and mediated by multifunctional T-helper 1 CD4+ T cells. CoVac-1-induced T-cell responses have exceeded those directed to the spike protein after mRNA-based vaccination of B-cell deficient patients and immunocompetent COVID-19 convalescents with and without seroconversion. Overall, our data show that CoVac-1 induces broad and potent T-cell responses in patients with B-cell/antibody deficiency with a favorable safety profile, which warrants advancement to pivotal Phase III safety and efficacy evaluation. ClinicalTrials.gov identifier NCT04954469.
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Agamaglobulinemia , COVID-19 , Humanos , SARS-CoV-2 , Linfócitos T , Peptídeos/uso terapêuticoRESUMO
T cell-based immunotherapy has significantly improved treatment options for many malignancies. However, despite these and other therapeutic improvements over the last decades, gastrointestinal cancers, in particular pancreatic, hepatic and gastric cancer, are still characterized by high relapse rates and dismal prognosis, with an accordingly high unmet medical need for novel treatment strategies. We here report on the preclinical characterization of a novel bispecific antibody in an IgG-based format termed CC-3 with B7-H3xCD3 specificity. In many cancer entities including pancreatic, hepatic and gastric cancers, B7-H3 (CD276) is overexpressed on tumor cells and also on the tumor vasculature, the latter allowing for improved access of immune effector cells into the tumor site upon therapeutic targeting. We demonstrate that CC-3 induces profound T cell reactivity against various pancreatic, hepatic and gastric cancer cell lines as revealed by analysis of activation, degranulation and secretion of IL2, IFNγ as well as perforin, resulting in potent target cell lysis. Moreover, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. Together, our results emphasize the potential of CC-3, which is presently being GMP-produced to enable clinical evaluation for treatment of pancreatic, hepatic and gastric cancer.
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Neoplasias Gástricas , Humanos , Neoplasias Gástricas/terapia , Imunoglobulina G , Recidiva Local de Neoplasia , Linfócitos T , Imunoterapia/métodos , Antígenos B7/metabolismoRESUMO
T cell-based immunotherapy has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions remain rare, particularly in gastrointestinal cancers like colorectal cancer (CRC). B7-H3 is overexpressed in multiple cancer entities including CRC on both tumor cells and tumor vasculature, the latter facilitating influx of effector cells into the tumor site upon therapeutic targeting. We generated a panel of T cell-recruiting B7-H3xCD3 bispecific antibodies (bsAbs) and show that targeting a membrane-proximal B7-H3 epitope allows for a 100-fold reduction of CD3 affinity. In vitro, our lead compound CC-3 showed superior tumor cell killing, T cell activation, proliferation, and memory formation, whereas undesired cytokine release was reduced. In vivo, CC-3 mediated potent antitumor activity in three independent models using immunocompromised mice adoptively transferred with human effector cells with regard to prevention of lung metastasis and flank tumor growth as well as elimination of large established tumors. Thus, fine-tuning of both target and CD3 affinities as well as binding epitopes allowed for the generation of a B7-H3xCD3 bsAbs with promising therapeutic activity. CC-3 is presently undergoing good manufacturing practice (GMP) production to enable evaluation in a clinical "first-in-human" study in CRC.
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Anticorpos Biespecíficos , Neoplasias Gastrointestinais , Humanos , Camundongos , Animais , Imunoglobulina G , Linfócitos T , Neoplasias Gastrointestinais/terapia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Imunoterapia , Linhagem Celular TumoralRESUMO
In conventional T cells, OX40 has been identified as a major costimulating receptor augmenting survival and clonal expansion of effector and memory T cell populations. In regulatory T cells, (Treg) OX40 signaling suppresses cellular activity and differentiation. However, clinical trials investigating OX40 agonists to enhance anti-tumor immunity, showed only limited success so far. Here we show that platelets from breast cancer patients express relevant levels of OX40L and platelet OX40L (pOX40L) inversely correlates with platelet-expressed immune checkpoint molecules GITRL (pGITRL) and TACI (pTACI). While high expression of pOX40L correlates with T and NK cell activation, elevated pOX40L levels identify patients with higher tumor grades, the occurrence of metastases, and shorter recurrence-free survival (RFS). Of note, OX40 mRNA levels in breast cancer correlate with enhanced expression of anti-apoptotic, immune-suppressive, and tumor-promoting mRNA gene signatures. Our data suggest that OX40L on platelets might play counteracting roles in cancer and anti-tumor immunity. Since pOX40L reflects disease relapse better than the routinely used predictive markers CA15-3, CEA, and LDH, it could serve as a novel biomarker for refractory disease in breast cancer.
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T cell-based immunotherapy, for example, with T cell-recruiting bispecific antibody (bsAb), has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions are still rare. Several tumor immune evasion mechanisms have been reported to counteract efficiency of T cell-engaging therapeutics. Platelets largely affect cancer pathophysiology by mediating tumor invasion, metastasis, and immune evasion. On treatment of patients in a clinical trial with a PSMA×CD3 bsAb (NCT04104607), we observed profound treatment-associated platelet activation, mirrored by a decrease of total platelet count. On modeling the treatment setting, we found that platelet activation significantly reduced bsAb-mediated CD4+ and CD8+ T-cell reactivity as revealed by impaired T-cell degranulation, secretion of perforin, and ultimately, inhibition of target cell lysis. This effect occurred in a transforming growth factor beta (TGF-ß)-dependent manner and was not restricted to PSMA×CD3 bsAb, but rather observed with various CD3-directed bispecific constructs, including the approved CD19×CD3 bsAb blinatumomab. BsAb-mediated T-cell reactivity could be restored by platelet inhibition and specifically by blocking the TGF-ß axis. Together, our findings demonstrate that platelets undermine the efficacy of T cell-recruiting bsAb and identify modulation of platelet function as a means to reinforce the effectiveness of bsAb treatment.
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Anticorpos Biespecíficos/metabolismo , Plaquetas/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Linfócitos T/metabolismo , Idoso , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , MasculinoRESUMO
Targeting costimulatory receptors of the tumor necrosis factor superfamily (TNFSF) to activate T-cells and promote anti-tumor T-cell function have emerged as a promising strategy in cancer immunotherapy. Previous studies have shown that combining two different members of the TNFSF resulted in dual-acting costimulatory molecules with the ability to activate two different receptors either on the same cell or on different cell types. To achieve prolonged plasma half-life and extended drug disposition, we have developed novel dual-acting molecules by fusing single-chain ligands of the TNFSF to heterodimerizing Fc chains (scDuokine-Fc, scDk-Fc). Incorporating costimulatory ligands of the TNF superfamily into a scDk-Fc molecule resulted in enhanced T-cell proliferation translating in an increased anti-tumor activity in combination with a primary T-cell-activating bispecific antibody. Our data show that the scDk-Fc molecules are potent immune-stimulatory molecules that are able to enhance T-cell mediated anti-tumor responses.
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Anticorpos Biespecíficos , Neoplasias , Anticorpos Biespecíficos/uso terapêutico , Humanos , Imunoterapia/métodos , Ligantes , Neoplasias/tratamento farmacológico , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
Immune-checkpoint inhibitors (ICI) have transformed oncological therapy. Up to 20% of all non-small cell lung cancers (NSCLCs) show durable responses upon treatment with ICI, however, robust markers to predict therapy response are missing. Here we show that blood platelets interact with lung cancer cells and that PD-L1 protein is transferred from tumor cells to platelets in a fibronectin 1, integrin α5ß1 and GPIbα-dependent manner. Platelets from NSCLC patients are found to express PD-L1 and platelet PD-L1 possess the ability to inhibit CD4 and CD8 T-cells. An algorithm is developed to calculate the activation independent adjusted PD-L1 payload of platelets (pPD-L1Adj.), which is found to be superior in predicting the response towards ICI as compared to standard histological PD-L1 quantification on tumor biopsies. Our data suggest that platelet PD-L1 reflects the collective tumor PD-L1 expression, plays important roles in tumor immune evasion and overcomes limitations of histological quantification of often heterogeneous intratumoral PD-L1 expression.
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Antígeno B7-H1/metabolismo , Plaquetas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia , Neoplasias Pulmonares/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/genética , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Fatores Imunológicos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T , Adulto JovemRESUMO
Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.
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Complexo CD3/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Receptores de IgG/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Sarcoma/tratamento farmacológico , Adulto , Idoso , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Complexo CD3/metabolismo , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Cultura Primária de Células , Domínios Proteicos/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Sarcoma/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto JovemRESUMO
In recent decades, antibody-dependent cellular cytotoxicity (ADCC)-inducing monoclonal antibodies (mAbs) have revolutionized cancer immunotherapy, and Fc engineering strategies have been utilized to further improve efficacy. A promising option is to enhance the affinity of an antibody's Fc-part to the Fc-receptor CD16 by altering the amino acid sequence. Herein, we characterized an S239D/I332E-modified CD133 mAb termed 293C3-SDIE for treatment of B cell acute lymphoblastic leukemia (B-ALL). Flow cytometric analysis revealed CD133 expression on B-ALL cell lines and leukemic cells of 50% (14 of 28) B-ALL patients. 293C3-SDIE potently induced NK cell reactivity against the B-ALL cell lines SEM and RS4;11, as well as leukemic cells of B-ALL patients in a target antigen-dependent manner, as revealed by analysis of NK cell activation, degranulation, and cytotoxicity. Of note, CD133 expression did not correlate with BCR-ABL, CD19, CD20, or CD22, which are presently used as therapeutic targets in B-ALL, which revealed CD133 as an independent target for B-ALL treatment. Increased CD133 expression was also observed in MLL-AF4-rearranged B-ALL, indicating that 293C3-SDIE may constitute a particularly suitable treatment option in this hard-to-treat subpopulation. Taken together, our results identify 293C3-SDIE as a promising therapeutic agent for the treatment of B-ALL.
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Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism by which antitumor antibodies mediate therapeutic efficacy. At present, we evaluate an Fc-optimized (amino acid substitutions S239D/I332E) FLT3 antibody termed 4G8-SDIEM (FLYSYN) in patients with acute myeloid leukemia (NCT02789254). Here we studied the possibility to induce NK cell ADCC against B-cell acute lymphoblastic leukemia (B-ALL) by Fc-optimized FLT3 antibody treatment. Flow cytometric analysis confirmed that FLT3 is widely expressed on B-ALL cell lines and leukemic cells of B-ALL patients. FLT3 expression did not correlate with that of CD20, which is targeted by Rituximab, a therapeutic monoclonal antibody (mAb) employed in B-ALL treatment regimens. Our FLT3 mAb with enhanced affinity to the Fc receptor CD16a termed 4G8-SDIE potently induced NK cell reactivity against FLT3-transfectants, the B-ALL cell line SEM and primary leukemic cells of adult B-ALL patients in a target-antigen dependent manner as revealed by analyses of NK cell activation and degranulation. This was mirrored by potent 4G8-SDIE mediated NK cell ADCC in experiments with FLT3-transfectants, the cell line SEM and primary cells as target cells. Taken together, the findings presented in this study provide evidence that 4G8-SDIE may be a promising agent for the treatment of B-ALL, particularly in CD20-negative cases.
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A key weakness in current Earth System Models is the representation of thermal acclimation of photosynthesis in response to changes in growth temperatures. Previous studies in boreal and temperate ecosystems have shown leaf-scale photosynthetic capacity parameters, the maximum rates of carboxylation (Vcmax ) and electron transport (Jmax ), to be positively correlated with foliar nitrogen (N) content at a given reference temperature. It is also known that Vcmax and Jmax exhibit temperature optima that are affected by various environmental factors and, further, that N partitioning among the foliar photosynthetic pools is affected by N availability. However, despite the strong recent anthropogenic influence on atmospheric temperatures and N deposition to forests, little is known about the role of foliar N contents in controlling the photosynthetic temperature responses. In this study, we investigated the temperature dependencies of Vcmax and Jmax in 1-year-old needles of mature boreal Pinus sylvestris (Scots pine) trees growing under low and high N availabilities in northern Sweden. We found that needle N status did not significantly affect the temperature responses of Vcmax or Jmax when the responses were fitted to a peaked function. If such N insensitivity is a common tree trait it will simplify the interpretation of the results from gradient and multi-species studies, which commonly use sites with differing N availabilities, on temperature acclimation of photosynthetic capacity. Moreover, it will simplify modeling efforts aimed at understanding future carbon uptake by precluding the need to adjust the shape of the temperature response curves to variation in N availability.
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Nitrogênio/metabolismo , Fotossíntese/fisiologia , Pinus sylvestris/fisiologia , Folhas de Planta/fisiologia , Temperatura , Aclimatação/fisiologia , Algoritmos , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Ecossistema , Transporte de Elétrons , Fertilizantes , Pinus sylvestris/metabolismo , Folhas de Planta/metabolismo , Estações do Ano , SuéciaRESUMO
Numerous studies have shown that temperate and boreal forests are limited by nitrogen (N) availability. However, few studies have provided a detailed account of how carbon (C) acquisition of such forests reacts to increasing N supply. We combined measurements of needle-scale biochemical photosynthetic capacities and continuous observations of shoot-scale photosynthetic performance from several canopy positions with simple mechanistic modeling to evaluate the photosynthetic responses of mature N-poor boreal Pinus sylvestris to N fertilization. The measurements were carried out in August 2013 on 90-year-old pine trees growing at Rosinedalsheden research site in northern Sweden. In spite of a nearly doubling of needle N content in response to the fertilization, no effect on the long-term shoot-scale C uptake was recorded. This lack of N-effect was due to strong light limitation of photosynthesis in all investigated canopy positions. The effect of greater N availability on needle photosynthetic capacities was also constrained by development of foliar phosphorus (P) deficiency following N addition. Thus, P deficiency and accumulation of N in arginine appeared to contribute toward lower shoot-scale nitrogen-use efficiency in the fertilized trees, thereby additionally constraining tree-scale responses to increasing N availability. On the whole our study suggests that the C uptake response of the studied N-poor boreal P. sylvestris stand to enhanced N availability is constrained by the efficiency with which the additional N is utilized. This efficiency, in turn, depends on the ability of the trees to use the greater N availability for additional light capture. For stands that have not reached canopy closure, increase in leaf area following N fertilization would be the most effective way for improving light capture and C uptake while for mature stands an increased leaf area may have a rather limited effect on light capture owing to increased self-shading. This raises the question if N limitation in boreal forests acts primarily by constraining growth of young stands while the commonly recorded increase in stem growth of mature stands following N addition is primarily the result of altered allocation and only to a limited extent the result of increased stand C-capture.
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An important ecosystem service provided by urban trees is the cooling effect caused by their transpiration. The aim of this study was to quantify the magnitude of daytime and night-time transpiration of common urban tree species in a high latitude city (Gothenburg, Sweden), to analyse the influence of weather conditions and surface permeability on the tree transpiration, and to find out whether tree transpiration contributed to daytime or nocturnal cooling. Stomatal conductance and leaf transpiration at day and night were measured on mature street and park trees of seven common tree species in Gothenburg: Tilia europaea, Quercus robur, Betula pendula, Acer platanoides, Aesculus hippocastanum, Fagus sylvatica and Prunus serrulata. Transpiration increased with vapour pressure deficit and photosynthetically active radiation. Midday rates of sunlit leaves ranged from less than 1 mmol m(-2) s(-1) (B. pendula) to over 3 mmol m(-2) s(-1) (Q. robur). Daytime stomatal conductance was positively related to the fraction of permeable surfaces within the vertically projected crown area. A simple estimate of available rainwater, comprising of precipitation sum and fractional surface permeability within the crown area, was found to explain 68% of variation in midday stomatal conductance. Night-time transpiration was observed in all studied species and amounted to 7 and 20% of midday transpiration of sunlit and shaded leaves, respectively. With an estimated night-time latent heat flux of 24 W m(-2), tree transpiration significantly increased the cooling rate around and shortly after sunset, but not later in the night. Despite a strong midday latent heat flux of 206 W m(-2), a cooling effect of tree transpiration was not observed during the day.