Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program.

The tissues are the site of many important immunological reactions, yet how the immune system is controlled at these sites remains opaque. Recent studies have identified Foxp3+ regulatory T (Treg) cells in non-lymphoid tissues with unique characteristics compared with lymphoid Treg cells. However, tissue Treg cells have not been considered holistically across tissues. Here, we performed a systematic analysis of the Treg cell population residing in non-lymphoid organs throughout the body, revealing shared phenotypes, transient residency, and common molecular dependencies. Tissue Treg cells from different non-lymphoid organs shared T cell receptor (TCR) sequences, with functional capacity to drive multi-tissue Treg cell entry and were tissue-agnostic on tissue homing. Together, these results demonstrate that the tissue-resident Treg cell pool in most non-lymphoid organs, other than the gut, is largely constituted by broadly self-reactive Treg cells, characterized by transient multi-tissue migration. This work suggests common regulatory mechanisms may allow pan-tissue Treg cells to safeguard homeostasis across the body.

The cellular composition of the human immune system is shaped by age and cohabitation.

Detailed population-level description of the human immune system has recently become achievable. We used a 'systems-level' approach to establish a resource of cellular immune profiles of 670 healthy individuals. We report a high level of interindividual variation, with low longitudinal variation, at the level of cellular subset composition of the immune system. Despite the profound effects of antigen exposure on individual antigen-specific clones, the cellular subset structure proved highly elastic, with transient vaccination-induced changes followed by a return to the individual's unique baseline. Notably, the largest influence on immunological variation identified was cohabitation, with 50% less immunological variation between individuals who share an environment (as parents) than between people in the wider population. These results identify local environmental conditions as a key factor in shaping the human immune system.

Release of miR-29 Target Laminin C2 Improves Skin Repair.

miRNAs are small noncoding RNAs that regulate mRNA targets in a cell-specific manner. miR-29 is expressed in murine and human skin, where it may regulate functions in skin repair. Cutaneous wound healing model in miR-29a/b1 gene knockout mice was used to identify miR-29 targets in the wound matrix, where angiogenesis and maturation of provisional granulation tissue was enhanced in response to genetic deletion of miR-29. Consistently, antisense-mediated inhibition of miR-29 promoted angiogenesis in vitro by autocrine and paracrine mechanisms. These processes are likely mediated by miR-29 target mRNAs released upon removal of miR-29 to improve cell-matrix adhesion. One of these, laminin (Lam)-c2 (also known as laminin γ2), was strongly up-regulated during skin repair in the wound matrix of knockout mice. Unexpectedly, Lamc2 was deposited in the basal membrane of endothelial cells in blood vessels forming in the granulation tissue of knockout mice. New blood vessels showed punctate interactions between Lamc2 and integrin α6 (Itga6) along the length of the proto-vessels, suggesting that greater levels of Lamc2 may contribute to the adhesion of endothelial cells, thus assisting angiogenesis within the wound. These findings may be of translational relevance, as LAMC2 was deposited at the leading edge in human wounds, where it formed a basal membrane for endothelial cells and assisted neovascularization. These results suggest a link between LAMC2, improved angiogenesis, and re-epithelialization.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

CTLA4-Ig Effectively Controls Clinical Deterioration and Immune Condition in a Murine Model of Foxp3 Deficiency.

PURPOSE: FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice. METHOD: We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig. RESULTS: We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process. CONCLUSION: These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

A guide to relocating your laboratory.

Research positions have become increasingly less stable, thus increasing the frequency at which research laboratories need to be relocated. A laboratory relocation can be a positive transition for you and your team; however, it requires careful planning in order to minimize the disruption and mitigate potential harms. Here we discuss key planning steps involved in successfully relocating your laboratory.

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence.

Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

Parallel and Serial Sensory Processing in Developing Primary Somatosensory and Motor Cortex.

It is generally supposed that primary motor cortex (M1) receives somatosensory input predominantly via primary somatosensory cortex (S1). However, a growing body of evidence indicates that M1 also receives direct sensory input from the thalamus, independent of S1; such direct input is particularly evident at early ages before M1 contributes to motor control. Here, recording extracellularly from the forelimb regions of S1 and M1 in unanesthetized rats at postnatal day (P)8 and P12, we compared S1 and M1 responses to self-generated (i.e., reafferent) forelimb movements during active sleep and wake, and to other-generated (i.e., exafferent) forelimb movements. At both ages, reafferent responses were processed in parallel by S1 and M1; in contrast, exafferent responses were processed in parallel at P8 but serially, from S1 to M1, at P12. To further assess this developmental difference in processing, we compared exafferent responses to proprioceptive and tactile stimulation. At both P8 and P12, proprioceptive stimulation evoked parallel responses in S1 and M1, whereas tactile stimulation evoked parallel responses at P8 and serial responses at P12. Independent of the submodality of exafferent stimulation, pairs of S1-M1 units exhibited greater coactivation during active sleep than wake. These results indicate that S1 and M1 independently develop somatotopy before establishing the interactive relationship that typifies their functionality in adults.SIGNIFICANCE STATEMENT Learning any new motor task depends on the ability to use sensory information to update motor outflow. Thus, to understand motor learning, we must also understand how animals process sensory input. Primary somatosensory cortex (S1) and primary motor cortex (M1) are two interdependent structures that process sensory input throughout life. In adults, the functional relationship between S1 and M1 is well established; however, little is known about how S1 and M1 begin to transmit or process sensory information in early life. In this study, we investigate the early development of S1 and M1 as a sensory processing unit. Our findings provide new insights into the fundamental principles of sensory processing and the development of functional connectivity between these important sensorimotor structures.

Sensory Coding of Limb Kinematics in Motor Cortex across a Key Developmental Transition.

Primary motor cortex (M1) undergoes protracted development in mammals, functioning initially as a sensory structure. Throughout the first postnatal week in rats, M1 is strongly activated by self-generated forelimb movements-especially by the twitches that occur during active sleep. Here, we quantify the kinematic features of forelimb movements to reveal receptive-field properties of individual units within the forelimb region of M1. At postnatal day 8 (P8), nearly all units were strongly modulated by movement amplitude, especially during active sleep. By P12, only a minority of units continued to exhibit amplitude tuning, regardless of behavioral state. At both ages, movement direction also modulated M1 activity, though to a lesser extent. Finally, at P12, M1 population-level activity became more sparse and decorrelated, along with a substantial alteration in the statistical distribution of M1 responses to limb movements. These findings reveal a transition toward a more complex and informationally rich representation of movement long before M1 develops its motor functionality.SIGNIFICANCE STATEMENT Primary motor cortex (M1) plays a fundamental role in the generation of voluntary movements and motor learning in adults. In early development, however, M1 functions as a prototypical sensory structure. Here, we demonstrate in infant rats that M1 codes for the kinematics of self-generated limb movements long before M1 develops its capacity to drive movements themselves. Moreover, we identify a key transition during the second postnatal week in which M1 activity becomes more informationally complex. Together, these findings further delineate the complex developmental path by which M1 develops its sensory functions in support of its later-emerging motor capacities.

Competent but complex communication: The phenomena of pheromone-responsive plasmids.

Enterococci are robust gram-positive bacteria that are found in a variety of surroundings and that cause a significant number of healthcare-associated infections. The genus possesses a high-efficiency pheromone-responsive plasmid (PRP) transfer system for genetic exchange that allows antimicrobial-resistance determinants to spread within bacterial populations. The pCF10 plasmid system is the best characterised, and although other PRP systems are structurally similar, they lack exact functional homologues of pCF10-encoded genes. In this review, we provide an overview of the enterococcal PRP systems, incorporating functional details for the less-well-defined systems. We catalogue the virulence-associated elements of the PRPs that have been identified to date, and we argue that this reinforces the requirement for elucidation of the less studied systems.

Enterococcal biofilm-A nidus for antibiotic resistance transfer?

Enterococci, which are on the WHO list of priority pathogens, are commonly encountered in hospital acquired infection and are becoming increasing significant due to the development of strains resistant to multiple antibiotics. Enterococci are also important microorganisms in the environment, and their presence is frequently used as an indicator of faecal pollution. Their success is related to their ability to survive within a broad range of habitats and the ease by which they acquire mobile genetic elements, including plasmids, from other bacteria. The enterococci are frequently present within a bacterial biofilm, which provides stability and protection to the bacterial population along with an opportunity for a variety of bacterial interactions. Enterococci can accept extrachromosomal DNA both from within its own species and from other bacterial species, and this is enhanced by the proximity of the donor and recipient strains. It is this exchange of genetic material that makes the role of biofilms such an important aspect of the success of enterococci. There remain many questions regarding the most suitable model systems to study enterococci in biofilms and regarding the transfer of genetic material including antibiotic resistance in these biofilms. This review focuses on some important aspects of biofilm in the context of horizontal gene transfer (HGT) in enterococci.

CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function.

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

Phenotypic analysis of pyrin-associated autoinflammation with neutrophilic dermatosis patients during treatment.

OBJECTIVE: In 2016 specific heterozygous gain-of-function mutations in the Mediterranean fever gene MEFV were reported as causal for a distinct autoinflammatory disease coined pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). We sought to provide an extended report on clinical manifestations in PAAND patients to date and evaluate the efficacy and safety of treatment with the IL-1-blocking agent anakinra. METHODS: We undertook an open-label pilot study with anakinra. Three patients were recruited in a preliminary phase of the study with the intention to expand the treatment cohort in case of a favourable response. Acute-phase reactants and plasma cytokine levels were monitored throughout. Skin biopsies at baseline and at week 12 were stained for relevant cytokines. Available clinical data on treatment responses were retrospectively collected on additional patients. RESULTS: The three patients from the preliminary phase of the study [patients 1-3 (P1-P3)] demonstrated one failed and two partial treatment responses, where one patient opted to continue treatment with anakinra and the other favoured adalimumab. While a partial systemic response was observed, there was no appreciable effect of anakinra on the prominent cutaneous manifestations, reflected in residual local inflammatory cytokine expression in lesional skin. These observations did not warrant further expansion of the treatment cohort. Clinical data was retrospectively collected on an additional eight patients (P4-P11), highlighting both dominant and recessive inheritance with variable penetrance in PAAND and common gastrointestinal involvement that was not previously appreciated. CONCLUSION: In our experience, while anakinra appears safe, it was not superior to biologicals targeting TNF-α in PAAND despite evidence directly implicating dysregulated IL-1ß signalling.

Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

ATP7B variant penetrance explains differences between genetic and clinical prevalence estimates for Wilson disease.

Wilson disease (WD) is a genetic disorder of copper metabolism caused by variants in the copper transporting P-type ATPase gene ATP7B. Estimates for WD population prevalence vary with 1 in 30,000 generally quoted. However, some genetic studies have reported much higher prevalence rates. The aim of this study was to estimate the population prevalence of WD and the pathogenicity/penetrance of WD variants by determining the frequency of ATP7B variants in a genomic sequence database. A catalogue of WD-associated ATP7B variants was constructed, and then, frequency information for these was extracted from the gnomAD data set. Pathogenicity of variants was assessed by (a) comparing gnomAD allele frequencies against the number of reports for variants in the WD literature and (b) using variant effect prediction algorithms. 231 WD-associated ATP7B variants were identified in the gnomAD data set, giving an initial estimated population prevalence of around 1 in 2400. After exclusion of WD-associated ATP7B variants with predicted low penetrance, the revised estimate showed a prevalence of around 1 in 20,000, with higher rates in the Asian and Ashkenazi Jewish populations. Reanalysis of other recent genetic studies using our penetrance criteria also predicted lower population prevalences for WD in the UK and France than had been reported. Our results suggest that differences in variant penetrance can explain the discrepancy between reported epidemiological and genetic prevalences of WD. They also highlight the challenge in defining penetrance when assigning causality to some ATP7B variants.

IFN-γ and CD25 drive distinct pathologic features during hemophagocytic lymphohistiocytosis.

BACKGROUND: Inflammatory activation of CD8+ T cells can, when left unchecked, drive severe immunopathology. Hyperstimulation of CD8+ T cells through a broad set of triggering signals can precipitate hemophagocytic lymphohistiocytosis (HLH), a life-threatening systemic inflammatory disorder. OBJECTIVE: The mechanism linking CD8+ T-cell hyperactivation to pathology is controversial, with excessive production of IFN-γ and, more recently, excessive consumption of IL-2, which are proposed as competing hypotheses. We formally tested the proximal mechanistic events of each pathway in a mouse model of HLH. METHODS: In addition to reporting a complete autosomal recessive IFN-γ receptor 1-deficient patient with multiple aspects of HLH pathology, we used the mouse model of perforin (Prf1)KO mice infected with lymphocytic choriomeningitis virus to genetically eliminate either IFN-γ production or CD25 expression and assess the immunologic, hematologic, and physiologic disease measurement. RESULTS: We found a striking dichotomy between the mechanistic basis of the hematologic and inflammatory components of CD8+ T cell-mediated pathology. The hematologic features of HLH were completely dependent on IFN-γ production, with complete correction after loss of IFN-γ production without any role for CD8+ T cell-mediated IL-2 consumption. By contrast, the mechanistic contribution of the immunologic features was reversed, with no role for IFN-γ production but substantial correction after reduction of IL-2 consumption by hyperactivated CD8+ T cells. These results were complemented by the characterization of an IFN-γ receptor 1-deficient patients with HLH-like disease, in whom multiple aspects of HLH pathology were observed in the absence of IFN-γ signaling. CONCLUSION: These results synthesize the competing mechanistic models of HLH pathology into a dichotomous pathogenesis driven through discrete pathways. A holistic model provides a new paradigm for understanding HLH and, more broadly, the consequences of CD8+ T-cell hyperactivation, thereby paving the way for clinical intervention based on the features of HLH in individual patients.