Local fibroblast development and densities influence organ health and disease, although it remains unclear how tissue fibroblast topography is controlled in situ. Here, we defined Group 2 innate lymphoid cells (ILC2s) as key regulators of fibroblast homeostasis in the pancreas. ILC2s colocalized with fibroblasts expressing the genes Pi16+Dpp4+Ly6c+ in an interstitial niche of the exocrine pancreas, which encapsulates the organ parenchyma. ILC2s specifically regulated the expansion of Pi16+Dpp4+Ly6c+ fibroblasts, which have progenitor capacity, while restraining differentiated intraparenchymal Col15a1+ fibroblasts during inflammation. These circuits reinforced fibroblast numbers after injury and set an inflammatory threshold. The ILC2 and Pi16+Dpp4+Ly6c+ fibroblast progenitor niche expanded around tumors and controlled cancer-associated fibroblast ontogeny and density. Hence, ILC2-fibroblast dialogue represents a regulatory node that locally orchestrates tissue homeostasis and pathology.

Natural killer (NK) cells are cytotoxic innate lymphoid cells which directly kill tumor cells, thus represent an attractive target for immunotherapy. However, NK cells face immunosuppression in the tumor microenvironment (TME), rendering them dysfunctional. While cancer-associated fibroblasts (CAFs) represent an abundant, heterogeneous component of pancreatic ductal adenocarcinoma (PDAC), their interplay with NK cells is largely understudied. Analyzing human samples and employing mouse models of PDAC and functional assays, we observed that intratumoral NK cells are immature, and TGF-β driven myofibroblastic (my)CAFs are strong NK suppressors, in contrast to inflammatory (i)CAF. Furthermore, myCAF-enriched tumor areas excluded NK cells, consistent with their reduced capacity to attract NK cells. Pancreatic CAFs in general reduced NK cell cytotoxicity by direct contact and via soluble factors, including prostaglandin E2 (PGE2). This work reveals distinct and overlapping roles of CAF subpopulations on NK cell functions, suggesting that overcoming CAF-imposed barriers to NK cytotoxicity and tumor infiltration is essential to unleash their anti-tumoral properties.

Immersing the brain in a solution containing formaldehyde is a commonly used method for preserving the structure of human brain tissue in brain banking. However, there are questions about the quality of preservation using this method, as formaldehyde takes a relatively long period of time to penetrate a large organ such as the human brain. As a result, there is a critical need to determine whether immersion fixation is an adequate initial preservation method. To address this, we present exploratory histologic findings from our brain bank following the immersion fixation of hemi-sectioned brain specimens under refrigeration. Using light microscopy, we found that there was no significant change in the size of pericellular or perivascular rarefaction areas based on the postmortem interval (PMI) or on the progression from the outer (frontal cortex) to the inner (striatum) brain regions. Additionally, we did not identify any significant number of ghost cells - a state of late-stage cellular necrosis - in the light micrographs analyzed. Using transmission electron microscopy of tissue from the frontal cortex, we found that synapses could still be visualized, but there was vacuolization and variable degrees of myelin disbanding identified. Using serial section transmission electron microscopy, we found that identified synapses could be traced from one section to the next. Using serial block face scanning electron microscopy, we also found that myelinated axons on 2D images can be traced with high fidelity from one image to the next, even at PMIs of up to 27 hours. Collectively, our data corroborate previous findings that immersion fixation is effective for prevention of cellular necrosis and for visualizing many ultrastructural features in at least the surface areas of the brain. However, how structural preservation quality should best be assessed in brain banking is an open question that depends on the intended research applications.

ABSTRACT Background Alarmin cytokine IL‐25 promotes type 2 inflammatory responses in disorders such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) and known targets include ILC2 and Th2 cells. However, other cellular targets for IL‐25 remain poorly defined. Objective To investigate induction and expression of IL‐25 receptor (IL‐17RB) by B cells and evaluate responsiveness of IL‐17RB‐expressing B cells to IL‐25 in vitro. Methods IL‐17RB expression, regulation and function on B cells were evaluated in peripheral blood‐derived B cells by flow cytometry and RT‐PCR, including in response to IgE‐inducing stimuli (anti‐CD40 mAb and IL‐4). Single‐cell RNA sequencing was used to compare IL‐17RB+ and IL‐17RB‐activated peripheral blood‐derived B cells. To evaluate B cell IL‐17RB expression within type 2 inflamed tissue, B cells were compared from nasal polyps, control turbinate tissue and matched peripheral blood. Results Activation of B cells with anti‐CD40 and IL‐4 increased IL‐17RB expression at both protein and mRNA level, which was further upregulated by IL‐25. B cells induced to express IL‐17RB responded to IL‐25 with enhanced antibody production. Single‐cell RNA‐sequencing showed that IL17RB+ activated B cells expressed higher levels of IGHE, CCL17 and CCL22 compared to IL17RB‐ B cells. B cells from nasal polyp tissue expressed higher levels of surface IL‐17RB compared with control tissue, correlating with patient‐reported CRSwNP severity (SNOT‐22). Conclusion Peripheral blood B cells activated under IgE‐inducing conditions express surface IL‐17RB, and tissue IL‐17RB+ B cells are increased in type 2 inflammation. IL‐17RB+ cells have a distinct transcriptional profile and respond to IL‐25 with enhanced antibody production, highlighting the IL‐25/IL‐17RB pathway as a potential therapeutic target for CRSwNP and other type 2 inflammatory disorders.

Regulatory T cells (T regs ) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident T regs and group 2 innate lymphoid cells (ILC2s); however, how T regs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of T reg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and T regs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3 high T regs , which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-T reg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3 high T regs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-T reg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.

Regulatory T cells are fundamentally important for maintaining immune homeostasis, and their potent immune-suppressive roles make them attractive immunotherapeutic targets in cancer. Recent work suggests potential functions of tissue-resident Tregs (trTregs) in tissue-repair and epithelial cell homeostasis. Here, we describe a rare population of trTreg in the exocrine pancreas. We show that these cells share common features of trTregs, including expression of the IL-33 receptor ST2 and production of the epithelial growth factor Amphiregulin, and display an oligoclonal T cell receptor repertoire. Using a mouse model of acute pancreatitis, we show that pancreatic Tregs rapidly expand upon release of IL-33 by fibroblasts. Moreover, depletion of Tregs after initiation of pancreatic injury impairs the regeneration of the exocrine parenchyma. This effect is due, in part, through a direct effect of Tregs on acinar cell proliferation. Finally, we show that transient Treg depletion in established orthotopic pancreatic tumours leads to tumour rejection yet provokes long-lasting damages to surrounding exocrine parenchyma. In all, our results demonstrate the tissue-repair capacity of pancreatic Tregs, and highlight a dualistic role of these cells in the pancreatic tumour ecosystem, with their harmful immune-suppressive function in the tumour coupled to a beneficial tissue-repair function in the surrounding tissue.

T helper 2 (TH2) cells orchestrate type 2 immunity during protective antihelminth immunity and help restore tissue homoeostasis. Their misdirected activities against innocuous substances also underlie atopic diseases, such as asthma and allergy. Recent technological advances are uncovering novel insights into the molecular mechanisms governing TH2 cell differentiation and function.

Innate lymphoid cells (ILCs) and adaptive T lymphocytes promote tissue homeostasis and protective immune responses. Their production depends on the transcription factor GATA3, which is further elevated specifically in ILC2s and T helper 2 cells to drive type-2 immunity during tissue repair, allergic disorders, and anti-helminth immunity. The control of this crucial up-regulation is poorly understood. Using CRISPR screens in ILCs we identified previously unappreciated myocyte-specific enhancer factor 2d (Mef2d)-mediated regulation of GATA3-dependent type-2 lymphocyte differentiation. Mef2d-deletion from ILC2s and/or T cells specifically protected against an allergen lung challenge. Mef2d repressed Regnase-1 endonuclease expression to enhance IL-33 receptor production and IL-33 signaling and acted downstream of calcium-mediated signaling to translocate NFAT1 to the nucleus to promote type-2 cytokine-mediated immunity.

Migration and homing of immune cells are critical for immune surveillance. Trafficking is mediated by combinations of adhesion and chemokine receptors that guide immune cells, in response to chemokine signals, to specific locations within tissues and the lymphatic system to support tissue-localized immune reactions and systemic immunity1,2. Here we show that disruption of leukaemia inhibitory factor (LIF) production from group 2 innate lymphoid cells (ILC2s) prevents immune cells leaving the lungs to migrate to the lymph nodes (LNs). In the absence of LIF, viral infection leads to plasmacytoid dendritic cells (pDCs) becoming retained in the lungs where they improve tissue-localized, antiviral immunity, whereas chronic pulmonary allergen challenge leads to marked immune cell accumulation and the formation of tertiary lymphoid structures in the lung. In both cases immune cells fail to migrate to the lymphatics, leading to highly compromised LN reactions. Mechanistically, ILC2-derived LIF induces the production of the chemokine CCL21 from lymphatic endothelial cells lining the pulmonary lymphatic vessels, thus licensing the homing of CCR7+ immune cells (including dendritic cells) to LNs. Consequently, ILC2-derived LIF dictates the egress of immune cells from the lungs to regulate tissue-localized versus systemic immunity and the balance between allergen and viral responsiveness in the lungs.

Fluid preservation is nearly universally used in brain banking to store fixed tissue specimens for future research applications. However, the effects of long-term immersion on neural circuitry and biomolecules are not well characterized. As a result, there is a need to synthesize studies investigating fluid preservation of brain tissue. We searched PubMed and other databases to identify studies measuring the effects of fluid preservation in nervous system tissue. We categorized studies based on the fluid preservative used: formaldehyde solutions, buffer solutions, alcohol solutions, storage after tissue clearing, and cryoprotectant solutions. We identified 91 studies containing 197 independent observations of the effects of long-term storage on cellular morphology. Most studies did not report any significant alterations due to long-term storage. When present, the most frequent alteration was decreased antigenicity, commonly attributed to progressive crosslinking by aldehydes that renders biomolecules increasingly inaccessible over time. To build a mechanistic understanding, we discuss biochemical aspects of long-term fluid preservation. A subset of lipids appears to be chemical altered or extracted over time due to incomplete retention in the crosslinked gel. Alternative storage fluids mitigate the problem of antigen masking but have not been extensively characterized and may have other downsides. We also compare fluid preservation to cryopreservation, paraffin embedding, and resin embedding. Overall, existing evidence suggests that fluid preservation provides maintenance of neural architecture for decades, including precise structural details. However, to avoid the well-established problem of overfixation caused by storage in high concentration formaldehyde solutions, fluid preservation procedures can use an initial fixation step followed by an alternative long-term storage fluid. Further research is warranted on optimizing protocols and characterizing the generalizability of the storage artifacts that have been identified.

Innate lymphoid cells (ILCs) are critical in maintaining tissue homeostasis, and during infection and inflammation. Here we identify, by using combinatorial reporter mice, a rare ILC progenitor (ILCP) population, resident to the small intestinal lamina propria (siLP) in adult mice. Transfer of siLP-ILCP into recipients generates group 1 ILCs (including ILC1 and NK cells), ILC2s and ILC3s within the intestinal microenvironment, but almost exclusively group 1 ILCs in the liver, lung and spleen. Single cell gene expression analysis and high dimensional spectral cytometry analysis of the siLP-ILCPs and ILC progeny indicate that the phenotype of the group 1 ILC progeny is also influenced by the tissue microenvironment. Thus, a local pool of siLP-ILCP can contribute to pan-ILC generation in the intestinal microenvironment but has more restricted potential in other tissues, with a greater propensity than bone marrow-derived ILCPs to favour ILC1 and ILC3 production. Therefore, ILCP potential is influenced by both tissue of origin and the microenvironment during development. This may provide additional flexibility during the tuning of immune reactions. The small intestinal lamina propria is rich in innate lymphoid cells, which are important contributors of mucosal immunity. Here authors identify a small progenitor cell population that can develop into all types of innate lymphoid cells in their intestinal microenvironment but their developmental potential becomes more restricted when placed into other tissues, such as liver, lung or spleen.

Drug sensitivity of NSCLC specimens to HDAC inhibitors and combination with standards of care.

HGF expression in CAFS and MSCs.

Growth of NSCLC specimens in the 3D-TGA.

A schematic of the patient derived 3D-TGA method.

The neuropathological hallmarks of Alzheimer’s disease (AD), including amyloid plaques and neurofibrillary tangles, also accumulate in the brains of aging individuals. However, some individuals are able to age successfully without significant degenerative neuropathology or cognitive impairment, a phenomenon referred to as “successful aging”. The predictors of successful “neuropathologic” and “cognitive” aging are not fully understood, creating a critical knowledge gap. Factors such as resistance, resilience, and reserve are believed to play important roles. Furthermore, the relationship between neuropathological changes, such as tau pathology and amyloid pathology, and cognitive impairment in the oldest old population remains unclear, and the concept of amyloid plaques causing tau pathology may not be universally valid. The association between amyloid plaques and dementia also continues to be debated. In this study, we utilized a collection of 414 post-mortem brain tissues (171 female, 241 male) from aged individuals (average age 90, range 80-108) with neuropathological and clinical data (215 without cognitive impairment and 161 with cognitive impairment) to perform a cross-sectional analysis. Our cohort included subjects who had cognitive assessments available and included those with no amyloid plaque pathology or mild burden based on neuropathological criteria. Variables (including new traits derived from A.I. based feature extraction) associated with the cognitive measures were determined using multivariable logistic regression. Our results revealed that age of death and Braak neurofibrillary tangle stage were significant predictors of cognitive impairment (p<0.05), while amyloid positivity was not. These findings suggest that tau pathology may have a stronger association with cognitive impairment in the oldest old population compared to amyloid plaques. Understanding the neuropathological factors associated with successful brain and cognitive aging will contribute to our knowledge of the underlying mechanisms and may pave the way for the development of interventions to promote successful aging and prevent cognitive decline in old age. Further research in this area is warranted to elucidate the complex relationship between neuropathology, cognitive function, and aging in the oldest old population.

Group 2 innate lymphoid cells (ILC2s) adapt to tissue physiology and contribute to immunity, inflammatory pathology and metabolism. We show that mouse uterine ILC2s have a heightened type-2 gene signature and expand during pregnancy. Indeed, maternal ILC2s promote fetal growth and protect against fetal mortality upon systemic endotoxin challenge. Absence of ILC2s leads to utero-placental abnormalities, including poor vascular remodelling, increased Il1b and decreased Il4, Il5 , and Il13 gene expression, and reduced alternative activation of dendritic cells (DCs) and macrophages. Placentas exhibit signs of adaptation to stress, including larger maternal blood spaces and increased expression of nutrient transporter genes. Endotoxin induces the expansion of IL-1β-producing uterine DCs and, in response, more uterine ILC2s produce IL-4, IL-5 and IL-13. In a protective feedback mechanism, these cytokines suppress IL-1β-producing DCs, in line with a protective role of uILC2s against endotoxin-induced abortion. Uterine ILC2s emerge as pivotal for both normal and complicated pregnancies.

Type 2 immune responses are critical in tissue homeostasis, anti-helminth immunity, and allergy. T helper 2 (Th2) cells produce interleukin-4 (IL-4), IL-5, and IL-13 from the type 2 gene cluster under regulation by transcription factors (TFs) including GATA3. To better understand transcriptional regulation of Th2 cell differentiation, we performed CRISPR-Cas9 screens targeting 1,131 TFs. We discovered that activity-dependent neuroprotector homeobox protein (ADNP) was indispensable for immune reactions to allergen. Mechanistically, ADNP performed a previously unappreciated role in gene activation, forming a critical bridge in the transition from pioneer TFs to chromatin remodeling by recruiting the helicase CHD4 and ATPase BRG1. Although GATA3 and AP-1 bound the type 2 cytokine locus in the absence of ADNP, they were unable to initiate histone acetylation or DNA accessibility, resulting in highly impaired type 2 cytokine expression. Our results demonstrate an important role for ADNP in promoting immune cell specialization.

Achievable peak serum concentration of chemotherapies in humans and associated references.

Brain cell structure is a key determinant of neural function that is frequently altered in neurobiological disorders. Following the global loss of blood flow to the brain that initiates the postmortem interval (PMI), cells rapidly become depleted of energy and begin to decompose. To ensure that our methods for studying the brain using autopsy tissue are robust and reproducible, there is a critical need to delineate the expected changes in brain cell morphometry during the PMI. We searched multiple databases to identify studies measuring the effects of PMI on the morphometry (i.e. external dimensions) of brain cells. We screened 2119 abstracts, 361 full texts, and included 172 studies. Mechanistically, fluid shifts causing cell volume alterations and vacuolization are an early event in the PMI, while the loss of the ability to visualize cell membranes altogether is a later event. Decomposition rates are highly heterogenous and depend on the methods for visualization, the structural feature of interest, and modifying variables such as the storage temperature or the species. Geometrically, deformations of cell membranes are common early events that initiate within minutes. On the other hand, topological relationships between cellular features appear to remain intact for more extended periods. Taken together, there is an uncertain period of time, usually ranging from several hours to several days, over which cell membrane structure is progressively lost. This review may be helpful for investigators studying human postmortem brain tissue, wherein the PMI is an unavoidable aspect of the research.