Highlight of 2023: Thymic B cells-important players in the establishment of T-cell tolerance.

In this article for the Highlights of 2023 Series, we discuss four recent articles that investigated thymic B cells, in both mice and humans. These studies provide important novel insights into the biology of this unique B-cell population, from their activation and differentiation to their role in promoting the negative selection of thymocytes and the generation of regulatory T cells.

Highlight of 2023: From fundamental studies to clinical trials, the importance of NK cells against cancer.

In this article for the Highlights of 2023 Series, we discuss how various factors affect the ability of natural killer (NK) cells to fight tumors. For instance, tumor cells can hinder NK cell function by reducing surface protrusions or increasing HLA-E expression via platelets. Lower UTX protein levels in male NK cells also decrease their cytotoxicity compared with females. Fortunately, recent advancements in therapeutic approaches have emerged, including the development of a comprehensive atlas of NK cell heterogeneity within the tumor microenvironment, as well as a trispecific engager molecule that has shown promise in enhancing the anti-tumor functions of NK cells.

A genetic mouse model of lean-NAFLD unveils sexual dimorphism in the liver-heart axis.

Lean patients with NAFLD may develop cardiac complications independently of pre-existent metabolic disruptions and comorbidities. To address the underlying mechanisms independent of the development of obesity, we used a murine model of hepatic mitochondrial deficiency. The liver-heart axis was studied as these mice develop microvesicular steatosis without obesity. Our results unveil a sex-dependent phenotypic remodeling beyond liver damage. Males, more than females, show fasting hypoglycemia and increased insulin sensitivity. They exhibit diastolic dysfunction, remodeling of the circulating lipoproteins and cardiac lipidome. Conversely, females do not manifest cardiac dysfunction but exhibit cardiometabolic impairments supported by impaired mitochondrial integrity and ß-oxidation, remodeling of circulating lipoproteins and intracardiac accumulation of deleterious triglycerides. This study underscores metabolic defects in the liver resulting in significant sex-dependent cardiac abnormalities independent of obesity. This experimental model may prove useful to better understand the sex-related variability, notably in the heart, involved in the progression of lean-NAFLD.

Modified host defence peptide GF19 slows TNT-mediated spread of corneal herpes simplex virus serotype I infection.

Corneal HSV-1 infections are a leading cause of infectious blindness globally by triggering tissue damage due to the intense inflammation. HSV-1 infections are treated mainly with antiviral drugs that clear the infections but are inefficient as prophylactics. The body produces innate cationic host defence peptides (cHDP), such as the cathelicidin LL37. Various epithelia, including the corneal epithelium, express LL37. cHDPs can cause disintegration of pathogen membranes, stimulate chemokine production, and attract immune cells. Here, we selected GF17, a peptide containing the LL37 fragment with bioactivity but with minimal cytotoxicity, and added two cell-penetrating amino acids to enhance its activity. The resulting GF19 was relatively cell-friendly, inducing only partial activation of antigen presenting immune cells in vitro. We showed that HSV-1 spreads by tunneling nanotubes in cultured human corneal epithelial cells. GF19 given before infection was able to block infection, most likely by blocking viral entry. When cells were sequentially  exposed to viruses and GF19,  the infection was attenuated but not arrested, supporting the contention that the GF19 mode of action was to block viral entry. Encapsulation into silica nanoparticles allowed a more sustained release of GF19, enhancing its activity. GF19 is most likely suitable as a prevention rather than a virucidal treatment.

Armstrong strain lymphocytic choriomeningitis virus infection after accidental laboratory exposure.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) is a human pathogen naturally present in wild rodents. In addition, LCMV is routinely used in immunology research as a model of viral infection in mice. The Armstrong common laboratory strain and the Clone-13 variant induce acute and chronic infections in mice, respectively. The frequent use of this virus in laboratory settings is associated with a risk of human infection for laboratory personnel. In contrast to LCMV Clone-13, few human laboratory infections with LCMV Armstrong have been reported, leading to a poor understanding of symptoms related to infection with this specific LCMV strain. CASE PRESENTATION: A researcher accidentally infected herself percutaneously with LCMV Armstrong. Symptoms including headaches, dizziness, eye pain and nausea appeared seven days post-exposure and lasted ten days. LCMV-IgM antibodies were detected at 28 days post-infection and IgG seroconversion was observed later. Complete recovery was confirmed three months post exposure. CONCLUSIONS: Research involving live viruses comes with the risk of infection for research personnel. This case is the first reported accidental human infection with LCMV Armstrong. The symptoms differed from reported infections with LCMV Clone-13, by the absence of fever and vomiting, and presence of leg numbness. This report will therefore help clinicians and public health authorities to recognize the symptoms associated with LCMV Armstrong infections and to offer appropriate counselling to individuals who accidentally expose themselves.

Uncoupling of Natural Killer cell functional maturation and cytolytic function in NOD mice.

NK cells are innate immune cells that target infected and tumor cells. Mature NK (mNK) cells undergo functional maturation characterized by four distinct stages, during which they acquire their cytotoxic properties. mNK cells from non-obese diabetic (NOD) mice exhibit a defect in functional maturation and have impaired cytotoxic functions. Hence, we tested whether the impaired cytotoxic function observed in mNK cells from NOD mice can be explained by their defect in functional maturation. By comparing the function of mNK cells from B6, B6g7 and NOD mice, we show that the expression of granzyme B is severely impaired in mNK cells from NOD mice, agreeing with their inability to control tumor growth in vivo. The low level of granzyme B expression in mNK cells from NOD mice is found at all stages of functional maturation and is therefore independent of their functional maturation defect. Consequently, this study demonstrates that phenotypic functional maturation of mNK cells can be uncoupled from the acquisition of cytotoxic functions.

Phosphorylcholine and KR12-Containing Corneal Implants in HSV-1-Infected Rabbit Corneas.

Severe HSV-1 infection can cause blindness due to tissue damage from severe inflammation. Due to the high risk of graft failure in HSV-1-infected individuals, cornea transplantation to restore vision is often contraindicated. We tested the capacity for cell-free biosynthetic implants made from recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) to suppress inflammation and promote tissue regeneration in the damaged corneas. To block viral reactivation, we incorporated silica dioxide nanoparticles releasing KR12, the small bioactive core fragment of LL37, an innate cationic host defense peptide produced by corneal cells. KR12 is more reactive and smaller than LL37, so more KR12 molecules can be incorporated into nanoparticles for delivery. Unlike LL37, which was cytotoxic, KR12 was cell-friendly and showed little cytotoxicity at doses that blocked HSV-1 activity in vitro, instead enabling rapid wound closure in cultures of human epithelial cells. Composite implants released KR12 for up to 3 weeks in vitro. The implant was also tested in vivo on HSV-1-infected rabbit corneas where it was grafted by anterior lamellar keratoplasty. Adding KR12 to RHCIII-MPC did not reduce HSV-1 viral loads or the inflammation resulting in neovascularization. Nevertheless, the composite implants reduced viral spread sufficiently to allow stable corneal epithelium, stroma, and nerve regeneration over a 6-month observation period.

Association of low-density neutrophils with lung function and disease progression in adult cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) neutrophils fail to eradicate infection despite their massive recruitment into the lung. While studies mostly focus on pathogen clearance by normal density neutrophils in CF, the contribution of low-density neutrophil (LDNs) subpopulations to disease pathogenesis remains unclear. METHODS: LDNs were isolated from whole blood donations of clinically stable adult CF patients and from healthy donors. LDN proportion and immunophenotype was assessed by flow cytometry. Associations of LDNs with clinical parameters were determined. RESULTS: LDN proportion was increased in CF patients' circulation compared with healthy donors. LDNs are a heterogeneous population of both mature and immature cells in CF and in healthy individuals. Moreover, a higher proportion of mature LDN correlates with a gradual decline in lung function and repeated pulmonary exacerbations in CF patients. CONCLUSIONS: Collectively, our observations suggest that low-density neutrophils are linked to CF pathogenesis and underscore the potential clinical relevance of neutrophil subpopulations in CF.

Immature and mature bone marrow-derived dendritic cells exhibit distinct intracellular mechanical properties.

Dendritic cells (DCs) patrol the organism at an immature stage to detect the presence of pathogens. Once activated, these mature DCs reach the lymph nodes to activate antigen-specific T lymphocytes and thus initiate an adaptative immune response to control the pathogen. The migration of both immature and mature DCs is a key process for their optimal function. DC migration requires transit through narrow constrictions that is allowed by their high local and global deformation capabilities. In addition to cytoplasmic changes, the nucleus mechanical properties also have a major impact for cellular migration and motility. Yet, nucleus intracellular mobility of dendritic cells or its variation upon maturation have not been investigated. Our study defines the biophysical phenotypic variations of dendritic cells upon maturation using interferometric deformability cytometry. This method characterizes different cellular mechanical properties, such as elongation and nucleus offset, by assessing the refractive index spatial distribution of shear-induced deformed cells. By using these parameters, our data suggest that in vitro bone marrow derived dendritic cell (BMDC) maturation induces cell stiffening and reduces nucleus mobility, allowing to distinguish immature and mature dendritic cells. Overall, our method provides insights on intracellular mechanical properties of two dendritic cell states.

Activation-induced cytidine deaminase expression by thymic B cells promotes T-cell tolerance and limits autoimmunity.

Elimination of self-reactive T cells in the thymus is critical to establish T-cell tolerance. A growing body of evidence suggests a role for thymic B cells in the elimination of self-reactive thymocytes. To specifically address the role of thymic B cells in central tolerance, we investigated the phenotype of thymic B cells in various mouse strains, including non-obese diabetic (NOD) mice, a model of autoimmune diabetes. We noted that isotype switching of NOD thymic B cells is reduced as compared to other, autoimmune-resistant, mouse strains. To determine the impact of B cell isotype switching on thymocyte selection and tolerance, we generated NOD.AID-/- mice. Diabetes incidence was enhanced in these mice. Moreover, we observed reduced clonal deletion and a resulting increase in self-reactive CD4+ T cells in NOD.AID-/- mice relative to NOD controls. Together, this study reveals that AID expression in thymic B cells contributes to T-cell tolerance.

Serum Lipidomic Screen Identifies Key Metabolites, Pathways, and Disease Classifiers in Crohn's Disease.

BACKGROUND: There is an unmet medical need for biomarkers that capture host and environmental contributions in inflammatory bowel diseases (IBDs). This study aimed at testing the potential of circulating lipids as disease classifiers given their major roles in inflammation. METHODS: We applied a previously validated comprehensive high-resolution liquid chromatography-mass spectrometry-based untargeted lipidomic workflow covering 25 lipid subclasses to serum samples from 100 Crohn's disease (CD) patients and 100 matched control subjects. Findings were replicated and expanded in another 200 CD patients and 200 control subjects. Key metabolites were tested for associations with disease behavior and location, and classification models were built and validated. Their association with disease activity was tested using an independent cohort of 42 CD patients. RESULTS: We identified >70 metabolites with strong association (P < 1 × 10-4, q < 5 × 10-4) to CD. Highly performing classification models (area under the curve > 0.84-0.97) could be built with as few as 5 to 9 different metabolites, representing 6 major correlated lipid clusters. These classifiers included a phosphatidylethanolamine ether (O-16:0/20:4), a sphingomyelin (d18:1/21:0) and a cholesterol ester (14:1), a very long-chain dicarboxylic acid [28:1(OH)] and sitosterol sulfate. These classifiers and correlated lipids indicate a dysregulated metabolism in host cells, notably in peroxisomes, as well as dysbiosis, oxidative stress, compromised inflammation resolution, or intestinal membrane integrity. A subset of these were associated with disease behavior or location. CONCLUSIONS: Untargeted lipidomic analyses uncovered perturbations in the circulating human CD lipidome, likely resulting from multiple pathogenic mechanisms. Models using as few as 5 biomarkers had strong disease classifier characteristics, supporting their potential use in diagnosis or prognosis.

This study reports a comprehensive untargeted lipidomic analysis of 600 serum samples from patients with Crohn's disease and matched control subjects, identified and replicated ~70 metabolites associated with Crohn's disease, and developed highly performing classification models (area under the curve > 0.84-0.97) with as few as 5 metabolites.

Identifying transcript-level differential expression in primary human immune cells.

BACKGROUND: Multipotential hematopoietic stem cells differentiate into a wide variety of immune cells with a diversity of functions, including the ability to respond to a variety of stimuli. Importantly, numerous studies have demonstrated the importance of gene transcription in defining cell identity and functions. While these studies have primarily been performed at the level of the gene, it is known that key immune genes such as CD44 and CD45 generate multiple different transcripts that are differentially expressed across different immune cells, and that encode proteins with different sequences and functions. Prior genomic surveys have shown that the mechanisms for generating diversity in expressed transcripts (alternate splicing, alternate transcription start sites, etc.) are very active in immune cells, but have been lacking in terms of identifying genes with multiple transcripts, that are differentially expressed, and likely to affect cell functions. METHODS: We first identified the set of genes that had at least two transcripts expressed in our RNA sequencing dataset generated from purified populations of neutrophils, monocytes and five lymphocyte populations (B, NK, γδ T, CD4 + T and CD8 + T) from twelve healthy donors. Next, we developed a heuristic approach to identify genes where two or more transcripts have distinct expression patterns across lymphoid and/or myeloid populations. We then focused our annotation and interpretation on differentially expressed transcripts that affect the coding sequence. This process was repeated to identify transcripts that were differentially expressed between monocytes and populations of macrophages and LPS-stimulated macrophages derived from these monocytes in vitro. RESULTS: We found that over 55 % of genes had two or more expressed transcripts, with an average ∼3 transcripts per gene, and that 70 % of these had at least two of the transcripts that encoded proteins with different sequences. As expected, we identified a complex pattern of differential expression for multiple transcripts encoding the CD45 transmembrane protein, but we also found similar evidence for ten other genes (CD300A, FYB1, GPI, LITAF, PSMA1, PTMA, RPL32, SEPTIN9, SH3BP2, SH3KBP1) when comparing the expression patterns of transcripts within myeloid and lymphoid cells. We also identified five genes with differentially expressed transcripts associated with the transition from monocytes to macrophages (FNBP1, KLF6, and SEPTIN9) or between macrophages and LPS-stimulated macrophages (CD44, OAZ2, and SEPTIN9). For the most part, we found that the different transcripts of these genes are expected to impact specific biological functions, for example the different transcripts of SEPTIN9 likely regulate the cytoskeleton in immune cells via their interactions with actins filaments and microtubules. CONCLUSIONS: This analytic approach successfully identified multi-transcript genes that are differentially expressed across immune cells and could be applied to other transcriptomic data. DATA AVAILABILITY STATEMENT: Researchers can request access to the individual-level data from the current study by contacting the Montreal Heart Institute ethics committee at the following institutional email address: cer.icm@icm-mhi.org.

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits.

Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.

The NOD Mouse Beyond Autoimmune Diabetes.

Autoimmune diabetes arises spontaneously in Non-Obese Diabetic (NOD) mice, and the pathophysiology of this disease shares many similarities with human type 1 diabetes. Since its generation in 1980, the NOD mouse, derived from the Cataract Shinogi strain, has represented the gold standard of spontaneous disease models, allowing to investigate autoimmune diabetes disease progression and susceptibility traits, as well as to test a wide array of potential treatments and therapies. Beyond autoimmune diabetes, NOD mice also exhibit polyautoimmunity, presenting with a low incidence of autoimmune thyroiditis and Sjögren's syndrome. Genetic manipulation of the NOD strain has led to the generation of new mouse models facilitating the study of these and other autoimmune pathologies. For instance, following deletion of specific genes or via insertion of resistance alleles at genetic loci, NOD mice can become fully resistant to autoimmune diabetes; yet the newly generated diabetes-resistant NOD strains often show a high incidence of other autoimmune diseases. This suggests that the NOD genetic background is highly autoimmune-prone and that genetic manipulations can shift the autoimmune response from the pancreas to other organs. Overall, multiple NOD variant strains have become invaluable tools for understanding the pathophysiology of and for dissecting the genetic susceptibility of organ-specific autoimmune diseases. An interesting commonality to all autoimmune diseases developing in variant strains of the NOD mice is the presence of autoantibodies. This review will present the NOD mouse as a model for studying autoimmune diseases beyond autoimmune diabetes.

OCA-B does not act as a transcriptional coactivator in T cells.

Pou2af1 encodes for OCA-B, a coactivator of OCT-1/2 transcription factors, which plays a key role in B-cell maturation. The function of OCA-B has also been studied in T cells, where T cells from Pou2af1-/- mice have impaired functions, such as cytokine production and T follicular helper (Tfh) differentiation. Arguably, some of these T-cell phenotypes may result from impaired T-B interactions, secondary to the well-documented B-cell defects in Pou2af1-/- mice. Yet, Pou2af1 is actively transcribed in activated T cells, suggesting a T-cell-intrinsic role. To isolate the T-cell-intrinsic impact of Pou2af1, we generated Pou2af1fl/fl mice with specific genetic disruption of Pou2af1 either in all hematopoietic cells or exclusively in T cells. While we confirm that Pou2af1 is expressed in activated T cells, we surprisingly find that T-cell cytokine production is not impaired in Pou2af1-deficient T cells. Moreover, Pou2af1-sufficient and Pou2af1-deficient T cells have comparable transcriptome profiles, arguing against a T-cell-intrinsic role for Pou2af1. In line with these observations, we demonstrate that Tfh maturation is influenced by T-cell-extrinsic deletion of Pou2af1, as observed both in competitive bone marrow chimeras and in Pou2af1fl/fl mice with specific deletion in B cells. Overall, this study provides strong evidence that Pou2af1 does not act as a transcriptional coactivator in T cells, and conclusively demonstrates that loss of OCA-B in B cells indirectly impacts Tfh differentiation, clarifying the role of OCA-B in the immune system.

The Idd2 Locus Confers Prominent Resistance to Autoimmune Diabetes.

Type 1 diabetes is an autoimmune disease characterized by pancreatic ß cell destruction. It is a complex genetic trait driven by >30 genetic loci with parallels between humans and mice. The NOD mouse spontaneously develops autoimmune diabetes and is widely used to identify insulin-dependent diabetes (Idd) genetic loci linked to diabetes susceptibility. Although many Idd loci have been extensively studied, the impact of the Idd2 locus on autoimmune diabetes susceptibility remains to be defined. To address this, we generated a NOD congenic mouse bearing B10 resistance alleles on chromosome 9 in a locus coinciding with part of the Idd2 locus and found that NOD.B10-Idd2 congenic mice are highly resistant to diabetes. Bone marrow chimera and adoptive transfer experiments showed that the B10 protective alleles provide resistance in an immune cell-intrinsic manner. Although no T cell-intrinsic differences between NOD and NOD.B10-Idd2 mice were observed, we found that the Idd2 resistance alleles limit the formation of spontaneous and induced germinal centers. Comparison of B cell and dendritic cell transcriptome profiles from NOD and NOD.B10-Idd2 mice reveal that resistance alleles at the Idd2 locus affect the expression of specific MHC molecules, a result confirmed by flow cytometry. Altogether, these data demonstrate that resistance alleles at the Idd2 locus impair germinal center formation and influence MHC expression, both of which likely contribute to reduced diabetes incidence.

Serum Analyte Profiles Associated With Crohn's Disease and Disease Location.

BACKGROUND: Crohn's disease (CD) can affect any segment of the digestive tract but is most often localized in the ileal, ileocolonic, and colorectal regions of the intestines. It is believed that the chronic inflammation in CD is a result of an imbalance between the epithelial barrier, the immune system, and the intestinal microbiota. The aim of the study was to identify circulating markers associated with CD and/or disease location in CD patients. METHODS: We tested 49 cytokines, chemokines, and growth factors in serum samples from 300 patients with CD and 300 controls. After quality control, analyte levels were tested for association with CD and disease location. RESULTS: We identified 13 analytes that were higher in CD patients relative to healthy controls and that remained significant after conservative Bonferroni correction (P < 0.0015). In particular, CXCL9, CXCL1, and interleukin IL-6 had the greatest effect and were highly significant (P < 5 × 10-7). We also identified 9 analytes that were associated with disease location, with VEGF, IL-12p70, and IL-6 being elevated in patients with colorectal disease (P < 3 × 10-4). CONCLUSIONS: Multiple serum analytes are elevated in CD. These implicate the involvement of multiple cell types from the immune, epithelial, and endothelial systems, suggesting that circulating analytes reflect the inflammatory processes that are ongoing within the gut. Moreover, the identification of distinct profiles according to disease location supports the existence of a biological difference between ileal and colonic CD, consistent with previous genetic and clinical observations.

The Potential Causes of Cystic Fibrosis-Related Diabetes.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Cystic fibrosis-related diabetes (CFRD) is the most common comorbidity, affecting more than 50% of adult CF patients. Despite this high prevalence, the etiology of CFRD remains incompletely understood. Studies in young CF children show pancreatic islet disorganization, abnormal glucose tolerance, and delayed first-phase insulin secretion suggesting that islet dysfunction is an early feature of CF. Since insulin-producing pancreatic ß-cells express very low levels of CFTR, CFRD likely results from ß-cell extrinsic factors. In the vicinity of ß-cells, CFTR is expressed in both the exocrine pancreas and the immune system. In the exocrine pancreas, CFTR mutations lead to the obstruction of the pancreatic ductal canal, inflammation, and immune cell infiltration, ultimately causing the destruction of the exocrine pancreas and remodeling of islets. Both inflammation and ductal cells have a direct effect on insulin secretion and could participate in CFRD development. CFTR mutations are also associated with inflammatory responses and excessive cytokine production by various immune cells, which infiltrate the pancreas and exert a negative impact on insulin secretion, causing dysregulation of glucose homeostasis in CF adults. In addition, the function of macrophages in shaping pancreatic islet development may be impaired by CFTR mutations, further contributing to the pancreatic islet structural defects as well as impaired first-phase insulin secretion observed in very young children. This review discusses the different factors that may contribute to CFRD.