Co-clustering of EphB6 and ephrinB1 in trans restrains cancer cell invasion.

EphB6 is an understudied ephrin receptor tyrosine pseudokinase that is downregulated in multiple types of metastatic cancers. Unlike its kinase-active counterparts which autophosphorylate and transmit signals upon intercellular interaction, little is known about how EphB6 functions in the absence of intrinsic kinase activity. Here, we unveil a molecular mechanism of cell-cell interaction driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and show that in trans interaction of EphB6 with ephrinB1 on neighboring cells leads to the formation of large co-clusters at the plasma membrane. These co-clusters exhibit a decreased propensity towards endocytosis, suggesting a unique characteristic for this type of cell-cell interaction. Using lattice light-sheet microscopy, 3D structured illumination microscopy and cryo-electron tomography techniques, we show that co-clustering of EphB6 and ephrinB1 promotes the formation of double-membrane tubular structures between cells. Importantly, we also demonstrate that these intercellular structures stabilize cell-cell adhesion, leading to a reduction in the invasive behavior of cancer cells. Our findings rationalize a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans.

Development of adenoviral vectors that transduce Purkinje cells and other cerebellar cell-types in the cerebellum of a humanized mouse model.

Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19.

The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1ß, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1ß after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19.

The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1ß, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1ß after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.

Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease. Key features Virus-mediated genetic perturbation in neonatal and young adult mice. Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.

Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery.

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri­lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri­lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase.

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

CD103 and periplakin are potential biomarkers for response of metastatic melanoma to pembrolizumab.

This study was designed to screen for preliminary evidence of predictive markers of melanoma response to PD-1 blockade. We hypothesized that the following immune markers would be positive predictors of response: increased densities of CD103 + CD8 + T cells or Th1 lineage T-bet + T cells, high expression of CXCL9-11 and presence of tertiary lymphoid structures. Conversely, we hypothesized that the high expression of barrier molecules would be a negative predictor of response. Patients with advanced melanoma treated with pembrolizumab were identified, and clinical response as well as overall survival data were collected. Tumor samples were evaluated by multiplex immunofluorescence histology. All statistical analyses were performed in R Studio and Microsoft Excel using the Mann-Whitney U test, chi-square test, Spearman's rank correlation and Kaplan-Meier survival curves. Sixty-five advanced melanoma patients were identified, of whom 46 met inclusion criteria and were included in this study. Increased densities ( P = 0.04) and proportions ( P = 0.02) of CD8 + T cells expressing CD103 + were associated with complete response (CR) to pembrolizumab. Improved survival was associated with increased proportions of CD8 + cells expressing CD103 ( P = 0.0085) as well as decreased density of periplakin + cells ( P = 0.012) and periplakin + SOX10 + cells ( P = 0.0012). The density and proportion of CD8 + T cells expressing CD103 + positively correlated with PD-L1 expression, though PD-L1 expression was not significantly correlated with outcomes. This screening study found that increased density and proportion of CD8 + T cells expressing CD103 and decreased density of periplakin were associated with positive outcomes in patients with melanoma metastases treated with pembrolizumab and may warrant further study.

VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production.

Recombinant viral vectors have become integral tools for basic in vivo research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression in vitro and in vivo. The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either loxP or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.

Proliferating CD8+ T Cell Infiltrates Are Associated with Improved Survival in Glioblastoma.

BACKGROUND: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. METHODS: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. RESULTS: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). CONCLUSIONS: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts.

Tumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-α1ß2. Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.

Heterogeneity in tertiary lymphoid structure B-cells correlates with patient survival in metastatic melanoma.

BACKGROUND: Tertiary lymphoid structures (TLSs) are immune aggregates in peripheral tissues that may support adaptive immune responses. Their presence has been associated with clinical response to checkpoint blockade therapy (CBT), but it is unknown whether TLS have prognostic significance independent of CBT in melanoma. We hypothesized that TLS in melanoma metastases would be associated with increased intratumoral lymphocyte infiltration, but that the intra-TLS immunological milieu would be distinct from the intratumoral immunological milieu. We also hypothesized that the presence of TLS would be associated with improved survival, and that TLS maturation or intra-TLS lymphocyte activity would also correlate with survival. METHODS: Cutaneous melanoma metastases (CMM) from 64 patients were evaluated by multiplex immunofluorescence for the presence and maturation status of TLS. Intra-TLS lymphocyte density, proliferation and B-cell Ig somatic hypermutation (AID+) were analyzed, as were markers of T-cell exhaustion and Th1/Tc1 differentiation. Associations between TLS maturation and intra-TLS immunologic activity were assessed, as well as associations with intratumoral immune cell infiltration. Independent associations with overall survival (OS) were assessed using log-rank tests and Cox proportional hazards models. RESULTS: TLS were identified in 30 (47%) of 64 CMM (TLS+) and were associated with increased intratumoral lymphocyte infiltration. However, proliferation of intra-TLS lymphocytes did not correlate with intratumoral lymphocyte proliferation. Most were early TLS; however, subsets of primary or secondary follicle-like TLS were also present. TLS+ lesions were associated with lower risk of tumor recurrence after metastasectomy and with improved OS in multivariate analyses (HR 0.51, p=0.04). OS was longer for TLS with low fractions of CD21+ B-cells (HR 0.29, p=0.02) and shorter for those with low AID+ fraction of B-cells (HR 2.74, p=0.03). CONCLUSIONS: The presence of TLS in CMMs is associated with improved OS in patients treated with surgery before CBT, but TLS vary widely in maturation state, in proportions of proliferating T and B cells, and in markers of B cell function, including AID and CD21. Importantly, these features have additional prognostic significance, which suggest that some TLS may have regulatory function, while others functioning to support antigen-driven immune responses, depending on the cellular composition and activation status.

SMCHD1's ubiquitin-like domain is required for N-terminal dimerization and chromatin localization.

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains - the N-terminal ATPase and C-terminal SMC hinge - suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing.

Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-Associated Tertiary Lymphoid Structures.

The presence of tertiary lymphoid structures (TLS) is correlated with prolonged patient survival in a variety of solid cancers, including melanoma. However, few methods have been described that could enable a more comprehensive understanding of the organization and functionality of TLS in solid cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying TLS and intra-tumoral immune infiltrates in melanoma. The described methods are not limited to melanoma alone and could be used to evaluate tertiary lymphoid structures in a wide variety of human cancers.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).

Incomplete Freund's adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment.

BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).