MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Programmed death ligand 1 intracellular interactions with STAT3 and focal adhesion protein Paxillin facilitate lymphatic endothelial cell remodeling.

Lymphatic endothelial cells (LECs) comprise lymphatic capillaries and vessels that guide immune cells to lymph nodes (LNs) and form the subcapsular sinus and cortical and medullary lymphatic structures of the LN. During an active immune response, the lymphatics remodel to accommodate the influx of immune cells from the tissue, but factors involved in remodeling are unclear. Here, we determined that a TSS motif within the cytoplasmic domain of programmed death ligand 1 (PD-L1), expressed by LECs in the LN, participates in lymphatic remodeling. Mutation of the TSS motif to AAA does not affect surface expression of PD-L1, but instead causes defects in LN cortical and medullary lymphatic organization following immunostimulant, Poly I:C, administration in vivo. Supporting this observation, in vitro treatment of the LEC cell line, SVEC4-10, with cytokines TNFα and IFNα significantly impeded SVEC4-10 movement in the presence of the TSS-AAA cytoplasmic mutation. The cellular movement defects coincided with reduced F-actin polymerization, consistent with differences previously found in dendritic cells. Here, in addition to loss of actin polymerization, we define STAT3 and Paxillin as important PD-L1 binding partners. STAT3 and Paxillin were previously demonstrated to be important at focal adhesions for cellular motility. We further demonstrate the PD-L1 TSS-AAA motif mutation reduced the amount of pSTAT3 and Paxillin bound to PD-L1 both before and after exposure to TNFα and IFNα. Together, these findings highlight PD-L1 as an important component of a membrane complex that is involved in cellular motility, which leads to defects in lymphatic organization.

Haemopedia RNA-seq: a database of gene expression during haematopoiesis in mice and humans.

During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.

Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.

Lymph node (LN) expansion during an immune response is a complex process that involves the relaxation of the fibroblastic network, germinal center formation, and lymphatic vessel growth. These processes require the stromal cell network of the LN to act deliberately to accommodate the influx of immune cells to the LN. The molecular drivers of these processes are not well understood. Therefore, we asked whether the immediate cytokines type 1 IFN produced during viral infection influence the lymphatic network of the LN in mice. We found that following an IFN-inducing stimulus such as viral infection or polyI:C, programmed cell death ligand 1 (PD-L1) expression is dynamically upregulated on lymphatic endothelial cells (LECs). We found that reception of type 1 IFN by LECs is important for the upregulation of PD-L1 of mouse and human LECs and the inhibition of LEC expansion in the LN. Expression of PD-L1 by LECs is also important for the regulation of LN expansion and contraction after an IFN-inducing stimulus. We demonstrate a direct role for both type 1 IFN and PD-L1 in inhibiting LEC division and in promoting LEC survival. Together, these data reveal a novel mechanism for the coordination of type 1 IFN and PD-L1 in manipulating LEC expansion and survival during an inflammatory immune response.

Type I Interferon Counters or Promotes Coxiella burnetii Replication Dependent on Tissue.

Coxiella burnetii is an intracellular pathogen and the cause of Q fever. Gamma interferon (IFN-γ) is critical for host protection from infection, but a role for type I IFN in C. burnetii infection has not been determined. Type I IFN supports host protection from a related pathogen, Legionella pneumophila, and we hypothesized that it would be similarly protective in C. burnetii infection. In contrast to our prediction, IFN-α receptor-deficient (IFNAR(-/-)) mice were protected from C. burnetii-induced infection. Therefore, the role of type I IFN in C. burnetii infection was distinct from that in L. pneumophila Mice treated with a double-stranded-RNA mimetic were protected from C. burnetii-induced weight loss through an IFNAR-independent pathway. We next treated mice with recombinant IFN-α (rIFN-α). When rIFN-α was injected by the intraperitoneal route during infection, disease-induced weight loss was exacerbated. Mice that received rIFN-α by this route had dampened interleukin 1ß (IL-1ß) expression in bronchoalveolar lavage fluids. However, when rIFN-α was delivered to the lung, bacterial replication was decreased in all tissues. Thus, the presence of type I IFN in the lung protected from infection, but when delivered to the periphery, type I IFN enhanced disease, potentially by dampening inflammatory cytokines. To better characterize the capacity for type I IFN induction by C. burnetii, we assessed expression of IFN-ß transcripts by human macrophages following stimulation with lipopolysaccharide (LPS) from C. burnetii Understanding innate responses in C. burnetii infection will support the discovery of novel therapies that may be alternative or complementary to the current antibiotic treatment.

Quantitative MRI in the evaluation of articular cartilage health: reproducibility and variability with a focus on T2 mapping.

PURPOSE: Early diagnosis of cartilage degeneration and longitudinal tracking of cartilage health including repair following surgical intervention would benefit from the ability to detect and monitor changes of the articular cartilage non-invasively and before gross morphological alterations appear. METHODS: Quantitative MR imaging has shown promising results with various imaging biomarkers such as T2 mapping, T1 rho and dGEMRIC demonstrating sensitivity in the detection of biochemical alterations within tissues of interest. However, acquiring accurate and clinically valuable quantitative data has proven challenging, and the reproducibility of the quantitative mapping technique and its values are essential. Although T2 mapping has been the focus in this discussion, all quantitative mapping techniques are subject to the same issues including variability in the imaging protocol, unloading and exercise, analysis, scanner and coil, calculation methods, and segmentation and registration concerns. RESULTS: The causes for variability between time points longitudinally in a patient, among patients, and among centres need to be understood further and the issues addressed. CONCLUSIONS: The potential clinical applications of quantitative mapping are vast, but, before the clinical community can take full advantage of this tool, it must be automated, standardized, validated, and have proven reproducibility prior to its implementation into the standard clinical care routine.

T2 values of articular cartilage in clinically relevant subregions of the asymptomatic knee.

PURPOSE: In order for T2 mapping to become more clinically applicable, reproducible subregions and standardized T2 parameters must be defined. This study sought to: (1) define clinically relevant subregions of knee cartilage using bone landmarks identifiable on both MR images and during arthroscopy and (2) determine healthy T2 values and T2 texture parameters within these subregions. METHODS: Twenty-five asymptomatic volunteers (age 18-35) were evaluated with a sagittal T2 mapping sequence. Manual segmentation was performed by three raters, and cartilage was divided into twenty-one subregions modified from the International Cartilage Repair Society Articular Cartilage Mapping System. Mean T2 values and texture parameters (entropy, variance, contrast, homogeneity) were recorded for each subregion, and inter-rater and intra-rater reliability was assessed. RESULTS: The central regions of the condyles had significantly higher T2 values than the posterior regions (P < 0.05) and higher variance than the posterior region on the medial side (P < 0.001). The central trochlea had significantly greater T2 values than the anterior and posterior condyles. The central lateral plateau had lower T2 values, lower variance, higher homogeneity, and lower contrast than nearly all subregions in the tibia. The central patellar regions had higher entropy than the superior and inferior regions (each P ≤ 0.001). Repeatability was good to excellent for all subregions. CONCLUSION: Significant differences in mean T2 values and texture parameters were found between subregions in this carefully selected asymptomatic population, which suggest that there is normal variation of T2 values within the knee joint. The clinically relevant subregions were found to be robust as demonstrated by the overall high repeatability.

Stool Gluten Peptide Detection Is Superior to Urinary Analysis, Coeliac Serology, Dietary Adherence Scores and Symptoms in the Detection of Intermittent Gluten Exposure in Coeliac Disease: A Randomised, Placebo-Controlled, Low-Dose Gluten Challenge Study.

Monitoring adherence to a gluten-free diet is an important goal of coeliac disease management. Urine and stool gluten immunogenic peptide (GIP) assays provide an objective readout of gluten ingestion, with the former favoured due to its convenience and acceptability. This study assessed stool GIP excretion after low-dose gluten challenge designed to mimic accidental gluten exposure. A total of 52 coeliac participants undertook a randomised, double-blind gluten (50-1000 mg) or placebo challenge. Stool and urinary GIP, serology, dietary adherence and symptoms were assessed. Stool GIP was 100% sensitive for gluten intake ≥250 mg and 71% for 50 mg. Peak GIP detection was 12-36 h after gluten exposure. The mean stool GIP after 1000 mg gluten ingestion remained above the limit of quantification for 5 days. Urine GIP assessment had poor sensitivity for GIP excretion compared to stool. Serology, dietary adherence score and symptoms did not correlate with gluten excretion during lead-in. We conclude that stool GIP detection is highly sensitive, with levels related to gluten dose and time from ingestion. Weekly or bi-weekly testing will detect low-level exposure more effectively than urine GIP assessments or traditional methods. In this seronegative, apparently well-treated cohort, a high frequency of baseline-positive GIP suggests ongoing gluten exposure, but the assessment of patient behaviour and assay specificity is needed.

Circulating KLRG1+ long-lived effector memory T cells retain the flexibility to become tissue resident.

KLRG1+ CD8 T cells persist for months after clearance of acute infections and maintain high levels of effector molecules, contributing protective immunity against systemic pathogens. Upon secondary infection, these long-lived effector cells (LLECs) are incapable of forming other circulating KLRG1- memory subsets such as central and effector memory T cells. Thus, KLRG1+ memory T cells are frequently referred to as a terminally differentiated population that is relatively short lived. Here, we show that after viral infection of mice, effector cells derived from LLECs rapidly enter nonlymphoid tissues and reduce pathogen burden but are largely dependent on receiving antigen cues from vascular endothelial cells. Single-cell RNA sequencing reveals that secondary memory cells in nonlymphoid tissues arising from either KLRG1+ or KLRG1- memory precursors develop a similar resident memory transcriptional signature. Thus, although LLECs cannot differentiate into other circulating memory populations, they still retain the flexibility to enter tissues and establish residency.

Cytomegalovirus drives Vδ1+ γδ T cell expansion and clonality in common variable immunodeficiency.

The function and phenotype of γδ T cells in the context of common variable immunodeficiency (CVID) has not been explored. CVID is a primary immunodeficiency disorder characterized by impaired antibody responses resulting in increased susceptibility to infections. γδ T cells are a subset of unconventional T cells that play crucial roles in host defence against infections. In this study, we aim to determine the roles and functions of γδ T cells in CVID. We observe a higher frequency of Vδ1+ γδ T cells compared to healthy controls, particularly in older patients. We also find a higher proportion of effector-memory Vδ1+ γδ T cells and a more clonal T cell receptor (TCR) repertoire in CVID. The most significant driver of the Vδ1+ γδ T cell expansion and phenotype in CVID patients is persistent cytomegalovirus (CMV) viremia. These findings provide valuable insights into γδ T cell biology and their contribution to immune defence in CVID.

Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection.

Antigens from viruses or immunizations can persist or are archived in lymph node stromal cells such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration resulted in cross-presentation of archived antigens and boosted memory CD8 + T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8 + T cells specific to the archived antigen from the immunization were significantly higher than memory CD8 + T cells of a different antigen specificity. Finally, the boosted memory CD8 + T cells resulted in increased protection against Listeria monocytogenes expressing the antigen from the immunization, but only for the duration that the antigen was archived. These findings outline an important mechanism by which lymph node stromal cell archived antigens, in addition to bystander activation, can augment memory CD8 + T cell responses during repeated inflammatory insults.

Vaccine-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection.

Viral and vaccine antigens persist or are archived in lymph node stromal cells (LNSC) such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration boosted memory CD8+ T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8+ T cells specific to the vaccine archived antigen were significantly higher than memory CD8+ T cells of a different antigen specificity. Finally, the boosted memory CD8+ T cells resulted in increased protection against Listeria monocytogenes expressing the vaccine antigen, but only for the duration that the vaccine antigen was archived. These findings outline a novel mechanism by which LNSC archived antigens, in addition to bystander activation, can augment memory CD8+ T cell responses during repeated inflammatory insults.

Islet Lymphocytes Maintain a Stable Regulatory Phenotype Under Homeostatic Conditions and Metabolic Stress.

T cells and B cells have been identified in human and murine islets, but the phenotype and role of islet lymphocytes is unknown. Resident immune populations set the stage for responses to inflammation in the islets during homeostasis and diabetes. Thus, we sought to identify the phenotype and effector function of islet lymphocytes to better understand their role in normal islets and in islets under metabolic stress. Lymphocytes were located in the islet parenchyma, and were comprised of a mix of naïve, activated, and memory T cell and B cell subsets, with an enrichment for regulatory B cell subsets. Use of a Nur77 reporter indicated that CD8 T cells and B cells both received local antigen stimulus, indicating that they responded to antigens present in the islets. Analysis of effector function showed that islet T cells and B cells produced the regulatory cytokine IL-10. The regulatory phenotype of islet T cells and B cells and their response to local antigenic stimuli remained stable under conditions of metabolic stress in the diet induced obesity (DIO) model. T cells present in human islets retained a similar activated and memory phenotype in non-diabetic and T2D donors. Under steady-state conditions, islet T cells and B cells have a regulatory phenotype, and thus may play a protective role in maintaining tissue homeostasis.

MicroRNA-21 is immunosuppressive and pro-metastatic via separate mechanisms.

MiR-21 was identified as a gene whose expression correlated with the extent of metastasis of murine mammary tumours. Since miR-21 is recognised as being associated with poor prognosis in cancer, we investigated its contribution to mammary tumour growth and metastasis in tumours with capacity for spontaneous metastasis. Unexpectedly, we found that suppression of miR-21 activity in highly metastatic tumours resulted in regression of primary tumour growth in immunocompetent mice but did not impede growth in immunocompromised mice. Analysis of the immune infiltrate of the primary tumours at the time when the tumours started to regress revealed an influx of both CD4+ and CD8+ activated T cells and a reduction in PD-L1+ infiltrating monocytes, providing an explanation for the observed tumour regression. Loss of anti-tumour immune suppression caused by decreased miR-21 activity was confirmed by transcriptomic analysis of primary tumours. This analysis also revealed reduced expression of genes associated with cell cycle progression upon loss of miR-21 activity. A second activity of miR-21 was the promotion of metastasis as shown by the loss of metastatic capacity of miR-21 knockdown tumours established in immunocompromised mice, despite no impact on primary tumour growth. A proteomic analysis of tumour cells with altered miR-21 activity revealed deregulation of proteins known to be associated with tumour progression. The development of therapies targeting miR-21, possibly via targeted delivery to tumour cells, could be an effective therapy to combat primary tumour growth and suppress the development of metastatic disease.

Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

The lymphatic system is a network of ducts that transports fluid, proteins, and immune cells from different organs around the body. Lymph nodes provide pit stops at hundreds of points along this network where immune cells reside, and lymph fluid can be filtered and cleaned. When pathogens, such as viruses or bacteria, enter the body during an infection, fragments of their proteins can get swept into the lymph nodes. These pathogenic proteins or protein fragments activate resident immune cells and kickstart the immune response. Vaccines are designed to mimic this process by introducing isolated pathogenic proteins in a controlled way to stimulate similar immune reactions in lymph nodes. Once an infection has been cleared by the immune system, or a vaccination has triggered the immune system, most pathogenic proteins get cleared away. However, a small number of pathogenic proteins remain in the lymph nodes to enable immune cells to respond more strongly and quickly the next time they see the same pathogen. Yet it is largely unclear how much protein remains for training and how or where it is all stored. Current techniques are not sensitive or long-lived enough to accurately detect and track these small protein deposits over time. Walsh, Sheridan, Lucas, et al. have addressed this problem by developing biological tags that can be attached to the pathogenic proteins so they can be traced. These tags were designed so the body cannot easily break them down, helping them last as long as the proteins they are attached to. Walsh, Sheridan, Lucas et al. tested whether vaccinating mice with the tagged proteins allowed the proteins to be tracked. The method they used was designed to identify individual cell types based on their genetic information along with the tag. This allowed them to accurately map the complex network of cells involved in storing and retrieving archived protein fragments, as well as those involved in training new immune cells to recognize them. These results provide important insights into the protein archiving system that is involved in enhancing immune memory. This may help guide the development of new vaccination strategies that can manipulate how proteins are archived to establish more durable immune protection. The biological tags developed could also be used to track therapeutic proteins, allowing scientists to determine how long cancer drugs, antibody therapies or COVID19 anti-viral agents remain in the body. This information could then be used by doctors to plan specific and personalized treatment timetables for patients.

Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes.

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes.

PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.

Although the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding the intracellular domain of PD-L1. Here, we outline a major role for PD-L1 intracellular signaling in the control of dendritic cell (DC) migration from the skin to the draining lymph node (dLN). Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. The TSS motif proves critical for chemokine-mediated DC migration to the dLN during inflammation. This loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. Finally, the TSS motif is required for chemokine receptor signaling downstream of the Gα subunit of the heterotrimeric G protein complex, ERK phosphorylation, and actin polymerization in DCs.

Lymph Node Lymphatic Endothelial Cell Expansion and Contraction and the Programming of the Immune Response.

Lymphatic endothelial cells (LECs) form the structure of the lymphatic vessels and the sinuses of the lymph nodes, positioning them to be key players in many different aspects of the immune response. Following an inflammatory stimulus, LECs produce chemokines that recruit immune cells to the lymph nodes. The recruitment of immune cells aids in the coordination of both LEC and lymph node expansion and contraction. More recent data has demonstrated that to coordinate LEC division and death, cell surface molecules, such as PD-L1 and interferon receptors, are required. During homeostasis, LECs use PD-L1 to maintain peripheral tolerance by presenting specific peripheral tissue antigens in order to eliminate tissue specific responses. LECs also have the capacity to acquire, present, and exchange foreign antigens following viral infection or immunization. Here we will review how lymph node LECs require immune cells to expand and contract in response to an immune stimulus, the factors involved and how direct LEC-immune cell interactions are important for programming immunity.