Paneth Cells: tales of a gutsy cell

The focus of this thesis is the study of Paneth cells and their role in the intestinal stem cell niche in homeostasis, injury and tissue repair and cancer. Paneth cells are niche cells for the rapidly dividing Lgr5+ intestinal stem cells. In chapter 2 we describe a method designed to elucidate and dissect genetic and/or biochemical modifications in Lgr5+ intestinal stem cells or Paneth cells. We used the striking ability of these cells to attach to each other and form organoids or 3D “mini-guts” ex-vivo. The developing of this method was essential for the rest of the discoveries elucidated in this thesis. In chapter 3 the role of the secretory phospholipase A2 group IIa (Pla2g2a ) or Mom1 in Paneth cells is studied. The study elucidated the role of phospholipases in homeostasis to downregulate canonical Wnt signalling in Paneth cells by upregulating yes associated protein 1 (Yap ). Under tissue injury conditions Pla2g2a was highly secreted and acted in opposite fashion thus by upregulating canonical Wnt signaling. In Chapter 4, the metabolism of the intestinal niche and stem cells is elucidated. Strikingly Paneth cells were found to be highly glycolytic whereas Lgr5+ stem cells used oxidative phosphorylation for their metabolic needs. The work done elucidated that lactate, the end product of glycolysis, secreted by Paneth cells was taken up by Lgr5+ stem cells and used by stem cells to fuel their metabolism. In other terms Paneth cells provide a metabolic niche for intestinal stem cells. In chapter 5 of this thesis another important role of Paneth cells is described. Under severe tissue injury conditions these cells can de-differentiate and regenerate the small intestine, giving rise to all intestinal cell lineages. In Chapter 4, the metabolism of the intestinal niche and stem cells is elucidated. Strikingly Paneth cells were found to be highly glycolytic whereas Lgr5+ stem cells used oxidative phosphorylation for their metabolic needs. The work done elucidated that lactate, the end product of glycolysis, secreted by Paneth cells was taken up by Lgr5+ stem cells and used by stem cells to fuel their metabolism. In other terms Paneth cells provide a metabolic niche for intestinal stem cells. In chapter 5 of this thesis another important role of Paneth cells is described. Under severe tissue injury conditions these cells can de-differentiate and regenerate the small intestine, giving rise to all intestinal cell lineages

RSPO3 expands intestinal stem cell and niche compartments and drives tumorigenesis

Objective The gross majority of colorectal cancer cases results from aberrant Wnt/ß-catenin signalling through adenomatous polyposis coli (APC) or CTNNB1 mutations. However, a subset of human colon tumours harbour, mutually exclusive with APC and CTNNB1 mutations, gene fusions in RSPO2 or RSPO3, leading to enhanced expression of these R-spondin genes. This suggested that RSPO activation can substitute for the most common mutations as an alternative driver for intestinal cancer. Involvement of RSPO3 in tumour growth was recently shown in RSPO3-fusion-positive xenograft models. The current study determines the extent into which solely a gain in RSPO3 actually functions as a driver of intestinal cancer in a direct, causal fashion, and addresses the in vivo activities of RSPO3 in parallel. Design We generated a conditional Rspo3 transgenic mouse model in which the Rspo3 transgene is expressed upon Cre activity. Cre is provided by cross-breeding with Lgr5-GFP-CreERT2 mice. Results Upon in vivo Rspo3 expression, mice rapidly developed extensive hyperplastic, adenomatous and adenocarcinomatous lesions throughout the intestine. RSPO3 induced the expansion of Lgr5+ stem cells, Paneth cells, non-Paneth cell label-retaining cells and Lgr4+ cells, thus promoting both intestinal stem cell and niche compartments. Wnt/ß-catenin signalling was modestly increased upon Rspo3 expression and mutant Kras synergised with Rspo3 in hyperplastic growth. Conclusions We provide in vivo evidence that RSPO3 stimulates the crypt stem cell and niche compartments and drives rapid intestinal tumorigenesis. This establishes RSPO3 as a potent driver of intestinal cancer and proposes RSPO3 as a candidate target for therapy in patients with colorectal cancer harbouring RSPO3 fusions

Differential climate impacts for policy-relevant limits to global warming: The case of 1.5 °c and 2 °c

Robust appraisals of climate impacts at different levels of global-mean temperature increase are vital to guide assessments of dangerous anthropogenic interference with the climate system. The 2015 Paris Agreement includes a two-headed temperature goal: "holding the increase in the global average temperature to well below 2°C above pre-industrial levels and pursuing efforts to limit the temperature increase to 1.5°C". Despite the prominence of these two temperature limits, a comprehensive overview of the differences in climate impacts at these levels is still missing. Here we provide an assessment of key impacts of climate change at warming levels of 1.5°C and 2°C, including extreme weather events, water availability, agricultural yields, sea-level rise and risk of coral reef loss. Our results reveal substantial differences in impacts between a 1.5°C and 2°C warming that are highly relevant for the assessment of dangerous anthropogenic interference with the climate system. For heat-related extremes, the additional 0.5°C increase in global-mean temperature marks the difference between events at the upper limit of present-day natural variability and a new climate regime, particularly in tropical regions. Similarly, this warming difference is likely to be decisive for the future of tropical coral reefs. In a scenario with an end-of-century warming of 2°C, virtually all tropical coral reefs are projected to be at risk of severe degradation due to temperature-induced bleaching from 2050 onwards. This fraction is reduced to about 90% in 2050 and projected to decline to 70% by 2100 for a 1.5°C scenario. Analyses of precipitation-related impacts reveal distinct regional differences and hot-spots of change emerge. Regional reduction in median water availability for the Mediterranean is found to nearly double from 9% to 17% between 1.5°C and 2°C, and the projected lengthening of regional dry spells increases from 7 to 11%. Projections for agricultural yields differ between crop types as well as world regions. While some (in particular high-latitude) regions may benefit, tropical regions like West Africa, South-East Asia, as well as Central and northern South America are projected to face substantial local yield reductions, particularly for wheat and maize. Best estimate sea-level rise projections based on two illustrative scenarios indicate a 50cm rise by 2100 relative to year 2000-levels for a 2°C scenario, and about 10 cm lower levels for a 1.5°C scenario. In a 1.5°C scenario, the rate of sea-level rise in 2100 would be reduced by about 30% compared to a 2°C scenario. Our findings highlight the importance of regional differentiation to assess both future climate risks and different vulnerabilities to incremental increases in global-mean temperature. The article provides a consistent and comprehensive assessment of existing projections and a good basis for future work on refining our understanding of the difference between impacts at 1.5°C and 2°C warming

Dual checkpoint blockade of CD47 and LILRB1 enhances CD20 antibody-dependent phagocytosis of lymphoma cells by macrophages

Antibody-dependent cellular phagocytosis (ADCP) by macrophages, an important effector function of tumor targeting antibodies, is hampered by ‘Don´t Eat Me!’ signals such as CD47 expressed by cancer cells. Yet, human leukocyte antigen (HLA) class I expression may also impair ADCP by engaging leukocyte immunoglobulin-like receptor subfamily B (LILRB) member 1 (LILRB1) or LILRB2. Analysis of different lymphoma cell lines revealed that the ratio of CD20 to HLA class I cell surface molecules determined the sensitivity to ADCP by the combination of rituximab and an Fc-silent variant of the CD47 antibody magrolimab (CD47-IgGσ). To boost ADCP, Fc-silent antibodies against LILRB1 and LILRB2 were generated (LILRB1-IgGσ and LILRB2-IgGσ, respectively). While LILRB2-IgGσ was not effective, LILRB1-IgGσ significantly enhanced ADCP of lymphoma cell lines when combined with both rituximab and CD47-IgGσ. LILRB1-IgGσ promoted serial engulfment of lymphoma cells and potentiated ADCP by non-polarized M0 as well as polarized M1 and M2 macrophages, but required CD47 co-blockade and the presence of the CD20 antibody. Importantly, complementing rituximab and CD47-IgGσ, LILRB1-IgGσ increased ADCP of chronic lymphocytic leukemia (CLL) or lymphoma cells isolated from patients. Thus, dual checkpoint blockade of CD47 and LILRB1 may be promising to improve antibody therapy of CLL and lymphomas through enhancing ADCP by macrophages

Venetoclax enhances the efficacy of therapeutic antibodies in B-cell malignancies by augmenting tumor cell phagocytosis

Immunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with "double-hit" diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies

Myeloid checkpoint blockade improves killing of T-acute lymphoblastic leukemia cells by an IgA2 variant of daratumumab

Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all- trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy

Global ensemble projections reveal trophic amplification of ocean biomass declines with climate change

While the physical dimensions of climate change are now routinely assessed through multimodel intercomparisons, projected impacts on the global ocean ecosystem generally rely on individual models with a specific set of assumptions. To address these single-model limitations, we present standardized ensemble projections from six global marine ecosystem models forced with two Earth system models and four emission scenarios with and without fishing. We derive average biomass trends and associated uncertainties across the marine food web. Without fishing, mean global animal biomass decreased by 5% (±4% SD) under low emissions and 17% (±11% SD) under high emissions by 2100, with an average 5% decline for every 1 °C of warming. Projected biomass declines were primarily driven by increasing temperature and decreasing primary production, and were more pronounced at higher trophic levels, a process known as trophic amplification. Fishing did not substantially alter the effects of climate change. Considerable regional variation featured strong biomass increases at high latitudes and decreases at middle to low latitudes, with good model agreement on the direction of change but variable magnitude. Uncertainties due to variations in marine ecosystem and Earth system models were similar. Ensemble projections performed well compared with empirical data, emphasizing the benefits of multimodel inference to project future outcomes. Our results indicate that global ocean animal biomass consistently declines with climate change, and that these impacts are amplified at higher trophic levels. Next steps for model development include dynamic scenarios of fishing, cumulative human impacts, and the effects of management measures on future ocean biomass trends

A framework for ensemble modelling of climate change impacts on lakes worldwide : the ISIMIP Lake Sector

Empirical evidence demonstrates that lakes and reservoirs are warming across the globe. Consequently, there is an increased need to project future changes in lake thermal structure and resulting changes in lake biogeochemistry in order to plan for the likely impacts. Previous studies of the impacts of climate change on lakes have often relied on a single model forced with limited scenario-driven projections of future climate for a relatively small number of lakes. As a result, our understanding of the effects of climate change on lakes is fragmentary, based on scattered studies using different data sources and modelling protocols, and mainly focused on individual lakes or lake regions. This has precluded identification of the main impacts of climate change on lakes at global and regional scales and has likely contributed to the lack of lake water quality considerations in policy-relevant documents, such as the Assessment Reports of the Intergovernmental Panel on Climate Change (IPCC). Here, we describe a simulation protocol developed by the Lake Sector of the Inter-Sectoral Impact Model Intercomparison Project (ISIMIP) for simulating climate change impacts on lakes using an ensemble of lake models and climate change scenarios for ISIMIP phases 2 and 3. The protocol prescribes lake simulations driven by climate forcing from gridded observations and different Earth system models under various representative greenhouse gas concentration pathways (RCPs), all consistently bias-corrected on a 0.5 degrees x 0.5 degrees global grid. In ISIMIP phase 2, 11 lake models were forced with these data to project the thermal structure of 62 well-studied lakes where data were available for calibration under historical conditions, and using uncalibrated models for 17 500 lakes defined for all global grid cells containing lakes. In ISIMIP phase 3, this approach was expanded to consider more lakes, more models, and more processes. The ISIMIP Lake Sector is the largest international effort to project future water temperature, thermal structure, and ice phenology of lakes at local and global scales and paves the way for future simulations of the impacts of climate change on water quality and biogeochemistry in lakes.Peer reviewe

Scenario set-up and forcing data for impact model evaluation and impact attribution within the third round of the Inter-Sectoral Model Intercomparison Project (ISIMIP3a)

This paper describes the rationale and the protocol of the first component of the third simulation round of the Inter-Sectoral Impact Model Intercomparison Project (ISIMIP3a, www.isimip.org) and the associated set of climate-related and direct human forcing data (CRF and DHF, respectively). The observation-based climate-related forcings for the first time include high-resolution observational climate forcings derived by orographic downscaling, monthly to hourly coastal water levels, and wind fields associated with historical tropical cyclones. The DHFs include land use patterns, population densities, information about water and agricultural management, and fishing intensities. The ISIMIP3a impact model simulations driven by these observation-based climate-related and direct human forcings are designed to test to what degree the impact models can explain observed changes in natural and human systems. In a second set of ISIMIP3a experiments the participating impact models are forced by the same DHFs but a counterfactual set of atmospheric forcings and coastal water levels where observed trends have been removed. These experiments are designed to allow for the attribution of observed changes in natural, human and managed systems to climate change, rising CH4 and CO2 concentrations, and sea level rise according to the definition of the Working Group II contribution to the IPCC AR6