Functional enhancement of mesothelin-targeted TRuC-T cells by a PD1-CD28 chimeric switch receptor.

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Distinct subsets of neutrophils crosstalk with cytokines and metabolites in patients with sepsis.

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in patients with sepsis. Our findings identify differentially expressed mature and immature neutrophil subsets in patients with sepsis. These subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in patients with sepsis. These results enabled the construction of a statistical model based on weighted multi-omics linear regression analysis for sepsis biomarker identification. These findings could help inform early patient stratification and treatment options, and facilitate further mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.

Tropical wetlands and land use changes: The case of oil palm in neotropical riverine floodplains.

Oil palm plantations are expanding in Latin America due to the global demand for food and biofuels, and much of this expansion has occurred at expense of important tropical ecosystems. Nevertheless, there is limited knowledge about effects on aquatic ecosystems near to oil palm-dominated landscapes. In this study, we used Landsat 7 ETM+, Landsat 8 OLI imagery and high-resolution images in Google Earth to map the current extent of oil palm plantations and determined prior land use land cover (LULC) in the Usumacinta River Basin as a case-study site. In addition, we assess the proximity of the crop with aquatic ecosystems distributed in the Usumacinta floodplains and their potential effects. Based on our findings, the most significant change was characterized by the expansion of oil palm crop areas mainly at expenses of regional rainforest and previously intervened lands (e.g. secondary vegetation and agriculture). Although aquatic ecosystem class (e.g. rivers, lagoons and channels) decreased in surface around 3% during the study period (2001-2017), the change was not due to the expansion of oil palm lands. However, we find that more than 50% of oil palm cultivations are near (between 500 and 3000 m) to aquatic ecosystems and this could have significant environmental impacts on sediment and water quality. Oil palm crops tend to spatially concentrate in the Upper Usumacinta ecoregion (Guatemala), which is recognized as an area of important fish endemism. We argue that the basic information generated in this study is essential to have better land use decision-making in a region that is relative newcomer to oil palm boom.

Vaccine Hyporesponse Induced by Individual Antibiotic Treatment in Mice and Non-Human Primates Is Diminished upon Recovery of the Gut Microbiome.

Emerging evidence demonstrates a connection between microbiome composition and suboptimal response to vaccines (vaccine hyporesponse). Harnessing the interaction between microbes and the immune system could provide novel therapeutic strategies for improving vaccine response. Currently we do not fully understand the mechanisms and dynamics by which the microbiome influences vaccine response. Using both mouse and non-human primate models, we report that short-term oral treatment with a single antibiotic (vancomycin) results in the disruption of the gut microbiome and this correlates with a decrease in systemic levels of antigen-specific IgG upon subsequent parenteral vaccination. We further show that recovery of microbial diversity before vaccination prevents antibiotic-induced vaccine hyporesponse, and that the antigen specific IgG response correlates with the recovery of microbiome diversity. RNA sequencing analysis of small intestine, spleen, whole blood, and secondary lymphoid organs from antibiotic treated mice revealed a dramatic impact on the immune system, and a muted inflammatory signature is correlated with loss of bacteria from Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. These results suggest that microbially modulated immune pathways may be leveraged to promote vaccine response and will inform future vaccine design and development strategies.

Primary Human Dendritic Cells and Whole-Blood Based Assays to Evaluate Immuno-Modulatory Properties of Heat-Killed Commensal Bacteria.

There is mounting evidence that the microbiome plays a critical role in training and maturation of the host immune system. Pre-clinical and clinical studies have shown that microbiome perturbation is correlated with sub-optimal host responses to vaccines and cancer immunotherapy. As such, identifying species of commensal bacteria capable of modulating immunological outcomes is of considerable interest. Currently, the lack of reliable primary immune cell-based assays capable of differentiating immuno-modulatory properties of various commensal bacteria is a major limitation. Here, we demonstrate that primary human monocyte-derived dendritic cells (MoDC) are capable of stratifying different strains of live and heat-killed commensal bacteria in an in vitro culture system. Specifically, heat-killed bacterial strains were able to differentially modulate co-stimulation/maturation markers CD80, CD83, and HLA-DR, as well as cytokine/chemokine signatures, such as IL-1b, MIP-1a, and TNFa in primary human MoDC. We further validated our observations using the TruCulture® (Myriad RBM, Inc., Austin, TX, USA) whole-blood ex vivo culture system. Using this ex vivo system allowed us to measure immune-altering effects of commensal bacteria in primary human whole-blood. As such, we report that both these primary in vitro and ex vivo systems are robust and enable identification, stratification, and differentiation of various commensal bacteria as potential modulators of host immunity.

Spatial analysis, local people's perception and economic valuation of wetland ecosystem services in the Usumacinta floodplain, Southern Mexico.

The Usumacinta floodplain is an exceptional area for biodiversity with important ecosystem services for local people. The main objective of this paper was to estimate reference values and define local perceptions of ecosystem services provided by wetlands and overlapping them with spatially explicit socioeconomic and biodiversity indicators. We used the Usumacinta floodplain as an example of a territory where high dependence of rural people on ecosystem services is confronted with development projects that threat the flow of ecosystem services, thus affecting rural people well-being. With a combination of data from remote sensing, global databases of ecosystem service values, local perception of ecosystem services and socioeconomic and biodiversity richness indicators in a spatially explicit framework, we develop a policy-oriented approach for rapid assessment to manage wetlands and maintain people's livelihoods. Regulating and provisioning services are identified as the most relevant ecosystem services in terms of their monetary value and local perceived importance. In a spatially explicit manner, this approach highlights the most valuable wetlands and identifies rural societies that are highly dependent on ecosystem services. Our approach can be replicated elsewhere and could provide valuable information for policymakers to design policies that can contribute to conserve wetland ecosystems where under threat of development.

Mechanisms of diversity maintenance in dung beetle assemblages in a heterogeneous tropical landscape.

BACKGROUND: Anthropized landscapes play a crucial role in biodiversity conservation, as they encompass about 90% of the remaining tropical forest. Effective conservation strategies require a deep understanding of how anthropic disturbances determine diversity patterns across these landscapes. Here, we evaluated how attributes and assembly mechanisms of dung beetle communities vary across the Selva El Ocote Biosphere Reserve (REBISO) landscape. METHODS: Community attributes (species diversity, abundance, and biomass) were assessed at the landscape scale, using spatial windows and vegetation classes. Windows were categorized as intact, variegated, or fragmented based on their percent cover of tropical forest. The vegetation classes analyzed were tropical forest, second-growth forest, and pastures. RESULTS: We collected 15,457 individuals and 55 species. Variegated windows, tropical forests, and second-growth forests showed the highest diversity values, while the lowest values were found in intact windows and pastures. Landscape fragmentation was positively and strongly related to dung beetle diversity and negatively related to their abundance; biomass was positively associated with forest cover. Beta diversity was the primary driver of the high dung beetle diversity in the landscape analyzed. DISCUSSION: The landscape heterogeneity and its biodiversity-friendly matrix facilitate the complementarity of dung beetle assemblages in the Selva El Ocote Biosphere Reserve. Random processes govern beta diversity patterns in intact and variegated windows. Therefore, vegetation cover in the region is sufficient to maintain a continuous flow of dung beetles between forested landscape segments. However, intense anthropic disturbances acted as deterministic environmental filters in fragmented windows and pastures sites, leading to biotic homogenization processes. Our results suggest that increasing habitat variegation in highly fragmented sites is an effective strategy to prevent or buffer homogenization processes in the REBISO landscape.

Impaired insulin signaling in the B10.D2-Hc0 H2d H2-T18c/oSnJ mouse model of complement factor 5 deficiency.

Given the chemoattractant potential of complement factor 5 (C5) and its increased expression in adipose tissue (AT) of obese mice, we determined whether this protein of the innate immune system impacts insulin action. C5 control (C5cont) and spontaneously C5-deficient (C5def, B10.D2-Hc0 H2d H2-T18c/oSnJ) mice were placed on low- and high-fat diets to investigate their inflammatory and metabolic phenotypes. Adenoviral delivery was used to evaluate the effects of exogenous C5 on systemic metabolism. C5def mice gained less weight than controls while fed a high-fat diet, accompanied by reduced AT inflammation, liver mass, and liver triglyceride content. Despite these beneficial metabolic effects, C5def mice demonstrated severe glucose intolerance and systemic insulin resistance, as well as impaired insulin signaling in liver and AT. C5def mice also exhibited decreased expression of insulin receptor (INSR) gene and protein, as well as improper processing of pro-INSR. These changes were not due to the C5 deficiency alone as other C5-deficient models did not recapitulate the INSR processing defect; rather, in addition to the mutation in the C5 gene, whole genome sequencing revealed an intronic 31-bp deletion in the Insr gene in the B10.D2-Hc0 H2d H2-T18c/oSnJ model. Irrespective of the genetic defect, adenoviral delivery of C5 improved insulin sensitivity in both C5cont and C5def mice, indicating an insulin-sensitizing function of C5.

Evaluating and supporting conservation action in agricultural landscapes of the Usumacinta River Basin.

There is increasing recognition that ecosystems and their services need to be managed at landscape scale and greater. The development of landscape-scale conservation strategies need to incorporate information from multiple sources. In this study, we combine various research tools to link landscape patterns with production units and systems in the Usumacinta River Basin, and inform the discussion of key questions around decision-making related to conservation action and policy in Southern Mexico. A typology based on policy-relevant farmer characteristics (land tenure, farm size, source of income, farming system) differentiated between farmers (traditional vs. cattle ranching) with different motivations that determine how management affects landscape configuration. Five main types of traditional farming systems were identified that combine different forms of land use and vary in their degree of land intensification. Major fragmentation and decrease in connectivity coincided spatially with floodplains dominated by large-scale commercial farms that specialize in livestock production. Traditional practices within large units with low-sloped high quality land were also seen to be intensive; however the presence of trees was notable throughout these units. Policies that promote livestock farming are among the principle causes motivating deforestation. Land intensification by traditional farmers decreased as the landscape became increasingly rugged. Traditional farmers are the focus of initiatives developed by the Biological Corridor project which seeks to increase forest cover and landscape connectivity. These initiatives have shown high levels of rural participation (10,010 farmers benefited from 27,778 projects involving 95,374 ha of land) and acceptance (producers carried out more than one project and several types of projects during the first eight years of work). Strong action is still required to take on the segment of large-scale ranchers. Changes in the structure of land tenure over the past decade are highlighted that could have a profound impact on conservation policies and programs.

Hematopoietic Kit Deficiency, rather than Lack of Mast Cells, Protects Mice from Obesity and Insulin Resistance.

Obesity, insulin resistance, and related pathologies are associated with immune-mediated chronic inflammation. Kit mutant mice are protected from diet-induced obesity and associated co-morbidities, and this phenotype has previously been attributed to their lack of mast cells. We performed a comprehensive metabolic analysis of Kit-dependent Kit(W/Wv) and Kit-independent Cpa3(Cre/+) mast-cell-deficient mouse strains, employing diet-induced or genetic (Lep(Ob/Ob) background) models of obesity. Our results show that mast cell deficiency, in the absence of Kit mutations, plays no role in the regulation of weight gain or insulin resistance. Moreover, we provide evidence that the metabolic phenotype observed in Kit mutant mice, while independent of mast cells, is immune regulated. Our data underscore the value of definitive mast cell deficiency models to conclusively test the involvement of this enigmatic cell in immune-mediated pathologies and identify Kit as a key hematopoietic factor in the pathogenesis of metabolic syndrome.

CCR2 deficiency leads to increased eosinophils, alternative macrophage activation, and type 2 cytokine expression in adipose tissue.

Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL-4/IL-13-dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2(-/-) AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2(-/-) mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2(-/-) bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2(-/-) mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2(-/-) mice, an increased eosinophil number positively correlated with M2-like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation.

Type 1 diabetes in NOD mice unaffected by mast cell deficiency.

Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4(+) and CD8(+) T cells, in the regulation of T-cell and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization, all of which could significantly impact the immune response against self-antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell-deficient nonobese diabetic (NOD) mice, NOD.Cpa3(Cre/+) (Heidelberg) and NOD.Kit(W-sh/W-sh) (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process nonessential and excluding them as potential therapeutic targets.

A sting in the tale of TH2 immunity.

Allergies are widely considered misguided Th2 cell responses. In this issue, Palm et al. (2013) and Marichal et al. (2013) show that mice mount anti-venom Th2 cell responses that share components of the "allergic" response but confer protection.

Weight cycling increases T-cell accumulation in adipose tissue and impairs systemic glucose tolerance.

Obesity is one of the leading causes of morbidity in the U.S. Accumulation of proinflammatory immune cells in adipose tissue (AT) contributes to the development of obesity-associated disorders. Weight loss is the ideal method to counteract the negative consequences of obesity; however, losses are rarely maintained, leading to bouts of weight cycling. Fluctuations in weight have been associated with worsened metabolic and cardiovascular outcomes; yet, the mechanisms explaining this potential correlation are not known. For determination of whether weight cycling modulates AT immune cell populations, inflammation, and insulin resistance, mice were subjected to a diet-switch protocol designed to induce weight cycling. Weight-cycled mice displayed decreased systemic glucose tolerance and impaired AT insulin sensitivity when compared with mice that gained weight but did not cycle. AT macrophage number and polarization were not modulated by weight cycling. However, weight cycling did increase the number of CD4(+) and CD8(+) T cells in AT. Expression of multiple T helper 1-associated cytokines was also elevated subsequent to weight cycling. Additionally, CD8(+) effector memory T cells were present in AT of both obese and weight-cycled mice. These studies indicate that an exaggerated adaptive immune response in AT may contribute to metabolic dysfunction during weight cycling.

Impact of macrophage inflammatory protein-1α deficiency on atherosclerotic lesion formation, hepatic steatosis, and adipose tissue expansion.

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR(-/-)) mice were transplanted with bone marrow from CCL3(-/-) or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3(-/-) marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3(-/-) recipients after 6 weeks and in male CCL3(-/-) recipients after 12 weeks of WD feeding (P<0.05). Surprisingly, male CCL3(-/-) recipients had a 50% decrease in adipose tissue mass after WD-feeding, and plasma insulin, and leptin levels were also significantly lower. These results were specific to CCL3, as LDLR(-/-) recipients of monocyte chemoattractant protein(-/-) (CCL2) marrow were not protected from the metabolic consequences of high fat feeding. Despite these improvements in LDLR(-/-) recipients of CCL3(-/-) marrow in the bone marrow transplantation (BMT) model, double knockout mice, globally deficient in both proteins, did not have decreased body weight, plasma lipids, or atherosclerosis compared with LDLR(-/-) controls. Finally, there were no differences in myeloid progenitors or leukocyte populations, indicating that changes in body weight and plasma lipids in CCL3(-/-) recipients was not due to differences in hematopoiesis. Taken together, these data implicate a role for CCL3 in lipid metabolism in hyperlipidemic mice following hematopoietic reconstitution.

Haematopoietic leptin receptor deficiency does not affect macrophage accumulation in adipose tissue or systemic insulin sensitivity.

The adipokine leptin is primarily produced by white adipose tissue (AT) and is a potent monocyte/macrophage chemoattractant in vitro. The long form of the leptin receptor (LepR) is required for monocyte/macrophage chemotaxis towards leptin. In this study, we examined the effects of haematopoietic LepR as well as LepR with C-C chemokine receptor 2 (CCR2) deficiency (double knockout (DKO)) on macrophage recruitment to AT after two different periods of high fat diet (HFD) feeding. Briefly, 8-week-old C57BL/6 mice were transplanted with bone marrow (BM) from Lepr(+/+), Lepr(-/-) or DKO donors (groups named BM-Lepr(+/+), BM-Lepr(-/-) and BM-DKO respectively), and were placed on an HFD for 6 or 12 weeks. At the end of the study, macrophage infiltration and the inflammatory state of AT were evaluated by real-time RT-PCR, histology and flow cytometry. In addition, glucose and insulin tolerance were assessed at both time points. Our results showed no differences in macrophage accumulation or AT inflammatory state between the BM-Lepr(+/+) and BM-Lepr(-/-) mice after 6 or 12 weeks of HFD feeding; any effects observed in the BM-DKO were attributed to the haematopoietic deficiency of CCR2. In addition, no changes in glucose or insulin tolerance were observed between groups after either period of HFD feeding. Our findings suggest that although leptin is a potent chemoattractant in vitro, haematopoietic LepR deficiency does not affect macrophage accumulation in AT in early to moderate stages of diet-induced obesity.

Aberrant accumulation of undifferentiated myeloid cells in the adipose tissue of CCR2-deficient mice delays improvements in insulin sensitivity.

OBJECTIVE: Mice with CCR2 deficiency are protected from insulin resistance but only after long periods of high-fat diet (HFD) feeding, despite the virtual absence of circulating inflammatory monocytes. We performed a time course study in mice with hematopoietic and global CCR2 deficiency to determine adipose tissue-specific mechanisms for the delayed impact of CCR2 deficiency on insulin resistance. RESEARCH DESIGN AND METHODS: Mice with global or hematopoietic CCR2 deficiency (CCR2(-/-) and BM-CCR2(-/-), respectively) and wild-type controls (CCR2(+/+) and BM-CCR2(+/+), respectively) were placed on an HFD for 6, 12, and 20 weeks. Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed. RESULTS: Flow cytometry analysis showed that two different populations of F4/80(+) myeloid cells (CD11b(lo)F4/80(lo) and CD11b(hi)F4/80(hi)) accumulated in the adipose tissue of CCR2(-/-) and BM-CCR2(-/-) mice after 6 and 12 weeks of HFD feeding, whereas only the CD11b(hi)F4/80(hi) population was detected in the CCR2(+/+) and BM-CCR2(+/+) controls. After 20 weeks of HFD feeding, the CD11b(lo)F4/80(lo) cells were no longer present in the adipose tissue of CCR2(-/-) mice, and only then were improvements in adipose tissue inflammation detected. Gene expression and histological analysis of the CD11b(lo)F4/80(lo) cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11b(lo)F4/80(lo) cells are transiently found in wild-type mice, but CCR2 deficiency leads to the aberrant accumulation of these cells in adipose tissue. CONCLUSIONS: The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

Adipose tissue recruitment of leukocytes.

PURPOSE OF REVIEW: In December of 2003, two seminal articles describing the presence of macrophages in obese adipose tissue were published. These adipose tissue macrophages (ATMs) are inflammatory and promote local and systemic insulin resistance. Due to the continuing rise in obesity around the world, understanding how these ATMs contribute to metabolic disorders is of much interest. RECENT FINDINGS: Chemokines have been extensively studied for their role in ATM recruitment. Deficiency or antagonism of chemokine receptors that interact with multiple chemokine ligands reduces ATM accumulation. ATMs are now defined as either classically (M1) or alternatively (M2) activated. Peroxisome proliferator-activated receptor activation and adiponectin promote an M2-polarized state resulting in improved insulin sensitivity. Finally, recent studies have provided evidence that T lymphocytes, natural killer T cells, mast cells, and B cells also enter adipose tissue and may interact with macrophages and adipocytes. SUMMARY: Literature published during the past year has shown that macrophage recruitment to adipose tissue is only one of the important mediators of obesity-related insulin resistance. The phenotype of ATMs and recruitment of other immune cells to the adipose tissue play key roles in the overall contribution of adipose tissue to systemic metabolic outcomes of obesity.