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Abstract Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3’ and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8+ T cell-mediated inhibition of HIV replication in CD4+ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6–13 days), purity of CD4 cultures [isolated CD4+ T cells or CD8+ depleted peripheral blood mononuclear cells (PBMCs)], HIV strains (laboratory strains, isolates, reporter viruses) and read-outs of virus inhibition (p24 ELISA, intracellular measurement of p24, luciferase reporter expression, and viral gag RNA) have been reported.Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence intervals. We identify methodological and analysis changes that could be incorporated into other protocols to improve assay reproducibility. We found that in people living with HIV (PLWH) on antiretroviral therapy (ART), CD8 T cell virus inhibition was largely stable over time, supporting the use of this assay and/or analysis methods to examine therapeutic interventions.Graphic abstract:

Summary: The archetypal T cell-dependent antigen is sheep red blood cells (SRBCs), which have defined much of what we know about humoral immunity. Early studies using solubilized or sonicated SRBCs argued that the intact structure of SRBCs was important for optimal antibody responses. However, the reason for the requirement of intact SRBCs for the response to polyvalent protein antigen remained unknown. Here, we report that the immune response to SRBCs is driven by cytosolic recognition of SRBC RNA through the RIG-I-like receptor (RLR)-mitochondrial anti-viral signaling adaptor (MAVS) pathway. Following the uptake of SRBCs by antigen-presenting cells, the MAVS signaling complex governs the differentiation of both T follicular cells and antibody-producing B cells. Importantly, the involvement of the RLR-MAVS pathway precedes that of endosomal Toll-like receptor pathways, yet both are required for optimal effect. : Immunization with sheep red blood cells (SRBCs) has been used for almost a century to study antibody generation. Loetsch et al. now show that, after engulfment, SRBC RNA accesses antigen-presenting cell RNA-sensing pathways to stimulate the immune system.

IL-21 is central to follicular helper T cell function and germinal centre responses. Here the authors show that IL-21 signalling directly inhibits T follicular regulatory cells by limiting Bcl-6-dependent IL-2 receptor expression.

Summary: The development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackievirus (CV), but how they induce T1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from β cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the β cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of β cell mass. These findings show an important role for β cells and, specifically, lack of β cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D. : Lalwani et al. describe a role for β cell hypoxia-inducible factor-1α (HIF1a) in determining whether β cell injury is followed by resolution and normal function or ongoing injury, autoimmunity, and type 1 diabetes. Keywords: type 1 diabetes, coxsackievirus, beta-cell, apoptosis, autoimmunity, streptozotocin, inflammation

CD4+ T helper (Th) cells that express the gut homing chemokine receptor CCR9 are increased in the peripheral blood of patients with inflammatory bowel disease and Sjögren's syndrome and in the inflamed lesions of autoimmune diseases that affect the accessory organs of the digestive system. However, despite the important role of the GIT in both immunity and autoimmunity, the nature of CCR9-expressing cells in GIT lymphoid organs and their role in chronic inflammatory diseases remains unknown. In this study, we analyzed the characteristics of CCR9+ Th and T follicular helper (Tfh) cells in GIT associated lymphoid tissues in health, chronic inflammation and autoimmunity. Our findings reveal an association between the transcriptome and phenotype of CCR9+ Th in the pancreas and CCR9+ Tfh cells from GIT-associated lymphoid tissues. GIT CCR9+ Tfh cells exhibited characteristics, including a Th17-like transcriptome and production of effector cytokines, which indicated a microenvironment-specific signature. Both CCR9+ Tfh cells and CCR9+ Th cells from GIT-associated lymphoid tissues migrated to the pancreas. The expression of CCR9 was important for migration of both subsets to the pancreas, but Tfh cells that accumulated in the pancreas had downmodulated expression of CXCR5. Taken together, the findings provide evidence that CCR9+ Tfh cells and Th cells from the GIT exhibit plasticity and can accumulate in distal accessory organs of the digestive system where they may participate in autoimmunity.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 x C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization. A combination of an FFPE nuclei preparation protocol and a probe-based transcriptomic profiling technique enables snRNA-seq characterization of archival human FFPE tissues, holding promise for retrospective studies involving aged clinical cohorts. [ABSTRACT FROM AUTHOR]

Background Persistence of HIV-1 in reservoirs necessitates life-long antiretroviral therapy (ART). There are conflicting data using genetic analysis on whether persistence includes an actively replicating reservoir with strong evidence arguing against replication. Methods We investigated the possibility of ongoing viral evolution during suppressive therapy by comparing near full-length viral genomic sequences using phylogenetic analysis of viral RNA in plasma before therapy initiation early after infection and from virus induced to grow from the latent reservoir after a period of suppressive ART. We also focused our analysis on evidence of selective pressure by drugs in the treatment regimen and at sites of selective pressure by the adaptive immune response. Results Viral genomes induced to grow from the latent reservoir from 10 participants with up to 9 years on suppressive ART were highly similar to the nearly homogeneous sequences in plasma taken early after infection at ART initiation. This finding was consistent across the entire genome and when the analysis focused on sites targeted by the drug regimen and by host selective pressure of antibody and cytotoxic T cells. The lack of viral evolution away from pretherapy sequences in spite of demonstrated selective pressure is most consistent with a lack of viral replication during reservoir persistence. Conclusions These results do not support ongoing viral replication as a mechanism of HIV-1 persistence during suppressive ART. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation. Methods: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation–sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme. Results: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets. Conclusions/interpretation: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes. [ABSTRACT FROM AUTHOR]

Digital markets now fundamentally intertwine with our social and economic lives. International enforcement actions--the United States (U.S.) and European Union (E.U.) Google cases in particular--demonstrate from a behavioral economic perspective how digital platforms may be beginning to implicate antitrust's two most fundamental doctrinal components--conduct and market power--in nuanced ways. In short, the regulatory and policy landscape showcases that we may be moving closer towards an antitrust world whereby firms can manipulate consumers' psychological shortcomings to foreclose competition--a new form of nefarious conduct that might appropriately be termed "cognitive foreclosure." Yet as a demand-side market failure, one should be cautious about categorizing behavioral market failures as antitrust issues. The behavioral deviation from perfect competition, then, would need to be "substantial" and "sustainable" if such market failures are to justifiably attract antitrust scrutiny. [ABSTRACT FROM AUTHOR]

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5–8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8–14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR–Cas9-mediated deletion of a transposable element—namely the LINE-1 retrotransposon Lx9c11—in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11−/− mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.Experiments in mice show that a LINE-1 transposable element, Lx9c11, has a functional role in immunity by negatively regulating the response to viral infection to protect the host from an over-reactive immune response. [ABSTRACT FROM AUTHOR]

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3 I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3 I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3 I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility. [ABSTRACT FROM AUTHOR]

In its en banc decision in LePage's Inc. v. 3M, the Third Circuit held that a 3M loyalty rebate program, which provided above-cost price discounts to customers who purchased multiple 3M product lines, violated section 2 of the Sherman Act. Prior to this decision, many practitioners and scholars understood the antitrust case law to hold that a strategic pricing scheme would not violate section 2 so long as the discounted prices remained above cost. The Third Circuit found that this test applies only to predatory pricing cases, and ruled that claims alleging exclusionary conduct other than predatory pricing--as it characterized 3M's loyalty rebate program--are cognizable under section 2 even without a showing of below-cost pricing. The Supreme Court recently denied certiorari in LePage's, leaving the issue in the hands of the lower courts. In this Comment, Joanna Warren criticizes the Third Circuit's decision as lacking sufficient economic analysis of the rebate scheme and providing unclear guidance for addressing future claims. She argues for the adoption of a test that would recognize above-cost pricing as generally legitimate while invalidating schemes that threaten to eliminate equally efficient competitors from the marketplace. [ABSTRACT FROM AUTHOR]

The relative contributions of climate versus interspecific interactions in shaping species distributions have important implications for closely related species at contact zones. When hybridization occurs within a contact zone, these factors regulate hybrid zone location and movement. While a hybrid zone's position may depend on both climate and interactions between the hybridizing species, little is known about how these factors interact to affect hybrid zone dynamics. Here, we utilize SDM (species distribution modeling) both to characterize the factors affecting the current location of a moving North American avian hybrid zone and to predict potential direct and indirect effects of climate change on future distributions. We focus on two passerine species that hybridize where their ranges meet, the Black-capped ( Poecile atricapillus) and Carolina ( P. carolinensis) chickadee. Our contemporary climate models predict the occurrence of climatically suitable habitat extending beyond the hybrid zone for P. atricapillus only, suggesting that interspecific interactions primarily regulate this range boundary in P. atricapillus, while climatic factors regulate P. carolinensis. Year 2050 climate models predict a drastic northward shift in suitable habitat for P. carolinensis. Because of the greater importance of interspecific interactions for regulating the southern range limit of P. atricapillus, these climate-mediated shifts in the distribution of P. carolinensis may indirectly lead to a range retraction in P. atricapillus. Together, our results highlight the ways climate change can both directly and indirectly affect species distributions and hybrid zone location. In addition, our study lends support to the longstanding hypothesis that abiotic factors regulate species' poleward range limits, while biotic factors shape equatorial range limits. [ABSTRACT FROM AUTHOR]

Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6'-sulfo-sLeX (Neu5Aca23[6-SO4] Gal14[Fuca13]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6'-sulfo-sLeX. Secondary analysis showed that related structures, sialyl-Lewis X (sLeX) and 6-sulfo-sLeX containing 6-GlcNAc-SO4 showed much lower binding avidity, indicating significant contribution of 6-Gal-SO4 on Siglec-F binding to 6'-sulfo-sLex. The lectin activity of Siglec-F on mouse eosinophils was masked by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6'-sulfo-sLeX structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6'-sulfo-sLeX. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6'-sulfo-sLeX ligand in eosinophil biology. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE. Several strains of gene-targeted mice develop systemic lupus erythematosus (SLE). Analysis of these strains demonstrates that the genetic background profoundly influences the development of autoimmunity. [ABSTRACT FROM AUTHOR]

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants. [ABSTRACT FROM AUTHOR]

I. INTRODUCTION A bundled discount, also known as a "loyalty discount" or "bundled rebate," is created when a seller offers a buyer a reduction in price that is contingent upon [...]