Origin and evolutionary malleability of T cell receptor α diversity.

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

IgG exacerbates genital chlamydial pathology in females by enhancing pathogenic CD8+ T cell responses.

Chlamydia trachomatis infections are an important sexually transmitted infection that can lead to inflammation, scarring and hydrosalpinx/infertility. However, infections are commonly clinically asymptomatic and do not receive treatment. The underlying cause of asymptomatic immunopathology remains unknown. Here, we demonstrate that IgG produced during male infection enhanced the incidence of immunopathology and infertility in females. Human endocervical cells expressing the neonatal Fc Receptor (FcRn) increased translocation of human IgG-opsonized C. trachomatis. Using total IgG purified from infected male mice, we opsonized C. muridarum and then infected female mice, mimicking sexual transmission. Following infection, IgG-opsonized Chlamydia was found to transcytose the epithelial barrier in the uterus, where it was phagocytosed by antigen-presenting cells (APCs) and trafficked to the draining lymph nodes. APCs then expanded both CD4+ and CD8+ T cell populations and caused significantly more infertility in female mice infected with non-opsonized Chlamydia. Enhanced phagocytosis of IgG-opsonized Chlamydia significantly increased pro-inflammatory signalling and T cell proliferation. As IgG is transcytosed by FcRn, we utilized FcRn-/- mice and observed that shedding kinetics of Chlamydia were only affected in FcRn-/- mice infected with IgG-opsonized Chlamydia. Depletion of CD8+ T cells in FcRn-/- mice lead to a significant reduction in the incidence of infertility. Taken together, these data demonstrate that IgG seroconversion during male infection can amplify female immunopathology, dependent on FcRn transcytosis, APC differentiation and enhanced CD8 T cell responses.

Flubendazole inhibits PD-1 and suppresses melanoma growth in immunocompetent mice.

BACKGROUND: Immune checkpoint inhibitor therapy has revolutionized the clinical management of a diverse range of cancer types, including advanced cutaneous melanoma. While immunotherapy targeting the PD-1/PD-L1 system has become standard of care, overall response rates remain unsatisfactory for most patients and there are no approved small molecule inhibitors of the PD-1/PD-L1 system. Flubendazole (FLU) is an anthelmintic that has been used to treat worm infections in humans and animals for decades. METHODS: Here we tested the anti-cancer activity of systemically delivered FLU with suppression of PD-1 in immunocompetent mice. RESULTS: In C57BL/6J mice bearing subcutaneous B16F10 melanoma, FLU reduced both tumor growth and PD-1 protein levels without affecting levels of PD-L1. FLU's suppression of PD-1 was accompanied by increased CD3+ T cell infiltration. Western blotting with extracts from human Jurkat T cells showed that FLU inhibited PD-1 protein expression, findings confirmed by flow cytometry. To gain mechanistic insights on FLU's ability to suppress PD-1 protein levels, we performed bulk RNA sequencing on extracts of Jurkat T cells exposed to the benzimidazole for 4 h. From a pool of 14,475 genes there were 1218 differentially-expressed genes; 687 with increased expression and 531 with decreased expression. Among the genes induced by FLU was the AP-1 family member, JUN and surprisingly, pdcd1. KEGG pathway analysis showed FLU up-regulated genes over-represented in multiple pathways (p < 0.01), the top hit being amoebiasis. FLU also affected the expression of genes in cancer-associated pathways, both through down-regulation and up-regulation. Gene set enrichment analysis revealed a large number of immunological signature gene sets correlated with FLU treatment, including gene sets associated with T cell differentiation, proliferation and function. The AP-1 inhibitor T5224 rescued PD-1 protein expression from inhibition by FLU. CONCLUSION: This study is the first to show that FLU can inhibit melanoma growth with PD-1 suppression in immunocompetent mice.

Pervasive changes of mRNA splicing in upf1-deficient zebrafish identify rpl10a as a regulator of T cell development.

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.

Immune Checkpoint Inhibitors, Small-Molecule Immunotherapies and the Emerging Role of Neutrophil Extracellular Traps in Therapeutic Strategies for Head and Neck Cancer.

Immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of many cancer types, including head and neck cancers (HNC). When checkpoint and partner proteins bind, these send an "off" signal to T cells, which prevents the immune system from destroying tumor cells. However, in HNC, and indeed many other cancers, more people do not respond and/or suffer from toxic effects than those who do respond. Hence, newer, more effective approaches are needed. The challenge to durable therapy lies in a deeper understanding of the complex interactions between immune cells, tumor cells and the tumor microenvironment. This will help develop therapies that promote lasting tumorlysis by overcoming T-cell exhaustion. Here we explore the strengths and limitations of current ICI therapy in head and neck squamous cell carcinoma (HNSCC). We also review emerging small-molecule immunotherapies and the growing promise of neutrophil extracellular traps in controlling tumor progression and metastasis.

Chlamydia-infected macrophages are resistant to azithromycin treatment and are associated with chronic oviduct inflammation and hydrosalpinx development.

Chlamydia infection remains the leading sexually-transmitted bacterial infection worldwide, causing damaging sequelae such as tubal scarring, infertility and ectopic pregnancy. As infection is often asymptomatic, prevention via vaccination is the optimal strategy for disease control. Vaccination strategies aimed at preventing bacterial infection have shown some promise, although these strategies often fail to prevent damaging inflammatory pathology when Chlamydia is encountered. Using a murine model of Chlamydia muridarum genital infection, we employed two established independent models to compare immune responses underpinning pathologic development of genital Chlamydia infection. Model one uses antibiotic treatment during infection, with only early treatment preventing pathology. Model two uses a plasmid-cured variant strain of C. muridarum that does not cause pathologic outcomes like the plasmid-containing wild-type counterpart. Using these infection models, contrasted by the development of pathology, we identified an unexpected role for macrophages. We observed that mice showing signs of pathology had greater numbers of activated macrophages present in the oviducts. This may have been due to early differences in macrophage activation and proinflammatory signaling leading to persistent or enhanced infection. These results provide valuable insight into the cellular mechanisms driving pathology in Chlamydia infection and contribute to the design and development of more effective vaccine strategies for protection against the deleterious sequelae of Chlamydia infection of the female reproductive tract.

Extracellular histones induce erythrocyte fragility and anemia.

Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients.

Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections.

Immunoglobulin A is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intra-epithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant secretory IgA (SIgA) we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra- and intra-epithelial stages of infection. We developed an in vitro model using polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model using pIgR(-/-) mice. Secretory IgA targeting the extra-epithelial chlamydial antigen, the major outer membrane protein, significantly reduced infection in vitro by 24% and in vivo by 44%. Conversely, pIgR-mediated delivery of IgA targeting the intra-epithelial inclusion membrane protein A bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intra-epithelial IgA targeting the secreted protease Chlamydia protease-like activity factor also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra-epithelial, but not intra-epithelial, chlamydial antigens for protection against a genital tract infection.

Divergent outcomes following transcytosis of IgG targeting intracellular and extracellular chlamydial antigens.

Antibodies can have a protective but non-essential role in natural chlamydial infections dependent on antigen specificity and antibody isotype. IgG is the dominant antibody in both male and female reproductive tract mucosal secretions, and is bi-directionally trafficked across epithelia by the neonatal Fc receptor (FcRn). Using pH-polarized epididymal epithelia grown on Transwells, IgG specifically targeted at an extracellular chlamydial antigen; the major outer membrane protein (MOMP), enhanced uptake and translocation of infection at pH 6-6.5 but not at neutral pH. This was dependent on FcRn expression. Conversely, FcRn-mediated transport of IgG targeting the intracellular chlamydial inclusion membrane protein A (IncA), induced aberrant inclusion morphology, recruited autophagic proteins independent of lysosomes and significantly reduced infection. Challenge of female mice with MOMP-specific IgG-opsonized Chlamydia muridarum delayed infection clearance but exacerbated oviduct occlusion. In male mice, MOMP-IgG elicited by immunization afforded no protection against testicular chlamydial infection, whereas the transcytosis of IncA-IgG significantly reduced testicular chlamydial burden. Together these data show that the protective and pathological effects of IgG are dependent on FcRn-mediated transport as well as the specificity of IgG for intracellular or extracellular antigens.

Immunity against a Chlamydia infection and disease may be determined by a balance of IL-17 signaling.

Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG-oligodeoxynucleotide-CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.

Targeted therapy, immunotherapy, and small molecules and peptidomimetics as emerging immunoregulatory agents for melanoma.

Primary cutaneous melanoma is the most lethal of all skin neoplasms and its incidence is increasing. Clinical management of advanced melanoma in the last decade has been revolutionised by the availability of immunotherapies and targeted therapies, used alone and in combination. This article summarizes advances in the treatment of late-stage melanoma including use of protein kinase inhibitors, antibody-based immune checkpoint inhibitors, adoptive immunotherapy, vaccines and more recently, small molecules and peptidomimetics as emerging immunoregulatory agents.

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish.

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Plasma dynamics of neutrophil extracellular traps and cell-free DNA in septic and non-septic vasoplegic shock: a prospective comparative observational cohort study.

BACKGROUND: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 hours after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or non-infectious (following cardiac surgery, CARDIAC) origin. METHODS: Prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H and 48H in SEPSIS and CARDIAC patients. The Vasopressor Inotropic Score (VIS), the Sequential Organ Failure Assessment (SOFA) score and time spent with invasive ventilation, in ICU and in hospital were recorded. Associations between NETs/cfDNA and VIS and SOFA were analysed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalisation times by generalised linear regression. RESULTS: Both NETs and cfDNA remained elevated over 48 hours in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], p = 0.005; cfDNA median difference 0.48 [0.20-1.02], p < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, p < 0.01, rho = 0.36-0.57 in CARDIAC, p ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, p < 0.01, rho = 0.38-0.47 in CARDIAC, p < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalisation times. CONCLUSION: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 hours in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or non-infectious etiology.

Personalised Medicine and the Potential Role of Electrospinning for Targeted Immunotherapeutics in Head and Neck Cancer.

Advanced head and neck cancer (HNC) is functionally and aesthetically destructive, and despite significant advances in therapy, overall survival is poor, financial toxicity is high, and treatment commonly exacerbates tissue damage. Although response and durability concerns remain, antibody-based immunotherapies have heralded a paradigm shift in systemic treatment. To overcome limitations associated with antibody-based immunotherapies, exploration into de novo and repurposed small molecule immunotherapies is expanding at a rapid rate. Small molecule immunotherapies also have the capacity for chelation to biodegradable, bioadherent, electrospun scaffolds. This article focuses on the novel concept of targeted, sustained release immunotherapies and their potential to improve outcomes in poorly accessible and risk for positive margin HNC cases.

Stage-specific and cell type-specific requirements of ikzf1 during haematopoietic differentiation in zebrafish.

The zinc finger transcription factor Ikaros1 (Ikzf1) is required for lymphoid development in mammals. Four zinc fingers constitute its DNA binding domain and two zinc fingers are present in the C-terminal protein interaction module. We describe the phenotypes of zebrafish homozygous for two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain. An intact ikzf1 gene is required for larval T cell development, whereas low levels of adult lymphoid development recover in the mutants. By contrast, the mutants exhibit a signature of increased myelopoiesis at larval and adult stages. Both mutations stimulate erythroid differentiation in larvae, indicating that the C-terminal zinc fingers negatively regulate the extent of red blood cell production. An unexpected differential effect of the two mutants on adult erythropoiesis suggests a direct requirement of an intact DNA binding domain for entry of progenitors into the red blood cell lineage. Collectively, our results reinforce the biological differences between larval and adult haematopoiesis, indicate a stage-specific function of ikzf1 in regulating the hierarchical bifurcations of differentiation, and assign distinct functions to the DNA binding domain and the C-terminal zinc fingers.

Synchronous sinonasal and respiratory papilloma: could long-term positive pressure ventilation be the cause? A rare case report.

This case report describes a rare presentation of synchronous pathologies-sinonasal inverted papilloma (SIP) and recurrent respiratory papillomatosis (RRP)-in a 47-year-old man using continuous positive airway pressure (CPAP) ventilation for progressive obstructive sleep apnoea. As far as we know, this is the first case of concurrent SIP and RRP disease described in the literature. The patient initially presented for management of chronic rhinosinusitis symptoms. He was found to have an extensive nasal lesion on flexible nasendoscopy, for which surgical management was recommended. However, during anaesthetic induction, he obstructed unexpectedly and was found to have an occlusive supraglottic lesion that required expedient ENT airway management. Diagnosis was made clinically and was supported with histopathology of excised tissue. Management involved multiple staged procedures for excision of sinonasal and glottic lesions and regular follow-up and imaging.

Beware the respiratory epithelial adenomatoid hamartoma-a malignant masquerador.

Respiratory epithelial adenomatoid hamartoma (REAH) is a rare benign tumour, which can masquerade as a sinonasal malignancy. Commonly arising from the posterior nasal septum, we present the second described case of a lateral nasal cavity wall REAH in a 68-year-old male with a 2-year history of progressive left nasal obstruction. Clinical and radiological assessment predicted malignancy; however, histopathology identified a benign pathology. He was subsequently treated with narrow local excision under general anaesthetic with no evidence of recurrence at post-operative intervals.

Genetic landscape of T cells identifies synthetic lethality for T-ALL.

To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.

Antigen receptor repertoires of one of the smallest known vertebrates.

The rules underlying the structure of antigen receptor repertoires are not yet fully defined, despite their enormous importance for the understanding of adaptive immunity. With current technology, the large antigen receptor repertoires of mice and humans cannot be comprehensively studied. To circumvent the problems associated with incomplete sampling, we have studied the immunogenetic features of one of the smallest known vertebrates, the cyprinid fish Paedocypris sp. "Singkep" ("minifish"). Despite its small size, minifish has the key genetic facilities characterizing the principal vertebrate lymphocyte lineages. As described for mammals, the frequency distributions of immunoglobulin and T cell receptor clonotypes exhibit the features of fractal systems, demonstrating that self-similarity is a fundamental property of antigen receptor repertoires of vertebrates, irrespective of body size. Hence, minifish achieve immunocompetence via a few thousand lymphocytes organized in robust scale-free networks, thereby ensuring immune reactivity even when cells are lost or clone sizes fluctuate during immune responses.