t-b-cell-receptor-immune-repertoire-analysis-using-next-generation

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

Immunological memory, the hallmark of adaptive immunity, is orchestrated by T and B lymphocytes. In circulation and different organs, there are billions ...

Sheba Medical Center

Tel Aviv University

tel aviv university

IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD.

Interleukin-10 (IL-10) is an immunoregulatory cytokine that has a central role in suppressing pro-inflammatory responses. Patients with deleterious mutations in IL-10 or IL-10 receptor (IL-10R) genes develop severe colitis and perianal disease in the first months of life. Whether IL-10R expression and signaling in pediatric- or adult-onset Crohn's disease (CD) are altered is unknown. The objective of this study was to characterize IL-10R expression and IL-10R-mediated suppression in patients with CD.

Impaired IL-10 Receptor Mediated Suppression in Monocyte from Patients with Crohn's Disease.

Advances in genomics have facilitated the discovery of monogenic disorders in patients with unique gastro-intestinal phenotypes. Syndromic diarrhea, also called tricho-hepato-enteric (THE) syndrome, results from deleterious mutations in SKIV2L or TTC37 genes. The main features of this disorder are intractable diarrhea, abnormal hair, facial dysmorphism, immunodeficiency and liver disease.

Genetic and Structural Analysis of a SKIV2L Mutation Causing Tricho-hepato-enteric Syndrome.

Inflammatory bowel diseases (IBD) are a multifactorial disorder. Our understanding of the role of bacteria in the pathogenesis of IBD has increased substantially; however, only scarce data exist regarding the role of commensal fungi in maintaining intestinal homeostasis and triggering IBD. Candida albicans (C. albicans) is a member of the intestinal mycobiome and proposed to contribute to IBD pathogenesis. We aimed to investigate the influence of the two morphologies of C. albicans, yeast and hypha, on epithelial cells and T cells from IBD patients versus healthy controls. We found that C. albicans was recognized by both epithelial cells lines and T cells. In the intestinal epithelial cell line, Caco-2, response to hypha was different than to yeast cells, and this was mimicked by synthetic β-glucans and Pam3CSK4. Unstimulated T cells exhibited increased activation and pro-inflammatory cytokine secretion upon exposure, while there was no effect on apoptosis or proliferation. In contrast, C. albicans-challenged CD3-stimulated T-cells exhibited decreased activation, cytokine secretion, apoptosis, and proliferation, suggesting reciprocal responsiveness to C. albicans. Glycans alone did not mimic abovementioned influences on T cells, suggesting alternative modes of recognition. In conclusion, we provide evidence for glycan dependent and independent recognition of C. albicans by epithelial cells and T cells.

Intestinal epithelial cells and T cells differentially recognize and respond to Candida albicans yeast and hypha.

This study aimed to characterize the clinical phenotype, genetic basis, and consequent immunological phenotype of a boy with severe infantile-onset colitis and eosinophilic gastrointestinal disease, and no evidence of recurrent or severe infections.

A Unique Presentation of Infantile-Onset Colitis and Eosinophilic Disease without Recurrent Infections Resulting from a Novel Homozygous CARMIL2 Variant.

There are limited data on fetal and early life development of human intestinal immunity. Using mass cytometry (CyTOF) and next-generation sequencing of B and T cell receptor (BCR and TCR) repertoires, we demonstrate complex intestinal immunity from 16 weeks' gestational age (GA). Both BCR and TCR repertoires are diverse with CDRH and CDR3β length increasing with advancing GA. The difference-from-germline, CDR insertions and/or deletions, similarly occur in utero for TCR but not BCR, suggesting earlier mucosal T than B cell maturity. Innate immunity is dominated by macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer (NK) cells. Follicular and transitional B cells are enriched in fetuses while CD69IgM B cells are abundant in infants. Both CD4 and CD8 T cells are abundant, capable of secreting cytokines and are phenotypically of the tissue resident memory state in utero. Our data provide the foundation for a 2nd trimester and infant intestinal immune atlas and suggest that a complex innate and adaptive immune landscape exists significantly earlier than previously reported.

Maturation of the Human Intestinal Immune System Occurs Early in Fetal Development.

More than 50 different monogenic disorders causing inflammatory bowel disease (IBD) have been identified. Our goal was to characterize the clinical phenotype, genetic workup, and immunologic alterations in an Ashkenazi Jewish patient that presented during infancy with ulcerative colitis and unique clinical manifestations.

An RTEL1 Mutation Links to Infantile-Onset Ulcerative Colitis and Severe Immunodeficiency.

Serological markers can assist in accurate differentiation between Crohn's disease (CD) and ulcerative colitis (UC). One such marker is anti-glycoprotein 2 (anti-GP2) which was shown to be a specific marker for CD in adult patients. The aim of our study was to assess the utility of anti-GP2 and GP2 as biomarkers for pediatric CD, and determine whether they correlate with disease activity.

Antibodies Against Glycoprotein 2 Are Specific Biomarkers for Pediatric Crohn's Disease.

Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions, including nutrient metabolism, intestinal barrier integrity, and immune regulation. Recent studies suggest that education of intestinal immunity may be ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial, and whether microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially derived metabolites can be detected in second trimester human intestinal samples. Although we were unable to amplify bacterial DNA from fetal intestines, we report a fetal metabolomic intestinal profile with an abundance of bacterially derived and host-derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial-associated metabolites either come from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that were below our detection threshold.

In utero human intestine harbors unique metabolome, including bacterial metabolites.

 More than 50 different monogenic disorders have been identified as directly causing inflammatory bowel diseases, typically manifesting in the first years of life. We present the clinical course and immunological work-up of an adult patient who presented in adolescent years with an atypical gastrointestinal phenotype and was diagnosed more than two decades later with a monogenic disorder with important therapeutic implications.  Whole exome sequencing was performed in a 37-years-old patient with a history of diarrhea since adolescence. Sanger sequencing was used to validate the suspected variant. Mass cytometry (CyTOF) and flow cytometry were conducted on peripheral blood mononuclear cells for deep immunophenotyping. Next-generation sequencing of the  and  was performed for global immune repertoire analysis of circulating lymphocytes.  We identified a novel deleterious c.1455C>A (p.Y485X) mutation in . CyTOF studies demonstrated significant changes in immune landscape in the LRBA-deficient patient, including an increase in myeloid derived suppressor cells and double-negative T cells, decreased B cells, low ratio of naïve:memory T cells, and reduced capacity of T cells to secrete various cytokines following stimulation, including tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In addition, this patient exhibited low frequency of regulatory T cells, with a reduction in their CTLA4 expression and interleukin (IL)-10 secretion. Finally, we show marked oligoclonal expansion of specific B- and T-cell clones in the peripheral blood of the LRBA-deficient patient.  LRBA deficiency is characterized by marked immunological changes in innate and adaptive immune cells. This case highlights the importance of advanced genetic studies in patients with a unique phenotype, regardless of their age at presentation.

Monogenic Inflammatory Bowel Disease: It's Never Too Late to Make a Diagnosis.

The integrin α4β7 is highly expressed on activated T cells and is thought to direct homing of lymphocytes to the intestine. Since ulcerative colitis (UC) and Crohn's disease (CD) are characterized by mucosal oligoclonal T cells' expansion, we aimed to assess whether similar repertoire features are identified in circulating gut-specific memory T cells.

Circulating α4β7 Memory T Cells in Pediatric IBD Patients Express a Polyclonal T Cell Receptor Repertoire.

Hyaline fibromatosis syndrome (HFS), resulting from  mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious  mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9  knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or  organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.

Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy.

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, ‎poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases.

Distinct extracellular-matrix remodeling events precede symptoms of inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine. We aimed to assess IL-10 and IL-10 receptor (IL-10R) expression in the gut, and determine whether these patterns are altered in patients with ulcerative colitis (UC). Formalin-fixed paraffin-embedded rectal and transverse colon sections were collected from three groups of patients: (a) control subjects with normal colonoscopy and without history of inflammatory bowel disease; (b) UC patients with extensive colitis or pancolitis (E3/E4 phenotype); and (c) UC patients with limited distal disease (E1/E2 phenotype; n = 8-10 subjects per group). Immunohistochemistry (IHC) was performed to assess expression patterns of IL-10, IL-10R1 and IL-10R2, and was correlated with clinical, endoscopic and histologic severity indices among patients. A trend towards increased IL-10 expression was noted in rectal biopsies of patients with active UC, compared with controls. Moreover, IL-10 levels were significantly increased in transverse colon biopsies of patients with extensive/pancolitis, compared with control subjects and patients with limited distal disease. Rectal IL-10R1 and IL-10R2 levels were comparable between control subject and patients with active UC. However, transverse colon IL-10R1 levels were significantly higher in patients with E3/E4 colitis, compared with controls. Finally, we found no correlation between clinical, endoscopic and histologic severity of inflammation among UC patients and IL-10, IL-10R1 or IL-10R2 expression in rectal sections. Mucosal expression patterns of IL-10 and IL-10R, evaluated by IHC, were overall similar between control subjects and patients with active UC. Given IL-10's anti-inflammatory properties, additional studies are required to determine whether signalling through the IL-10R is altered among these patients.

Article	Year
Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD. Inflammatory bowel diseases\| PubMed ID: 29023267	2017
Impaired IL-10 Receptor Mediated Suppression in Monocyte from Patients with Crohn's Disease. Journal of pediatric gastroenterology and nutrition\| PubMed ID: 29045353	2017
Genetic and Structural Analysis of a SKIV2L Mutation Causing Tricho-hepato-enteric Syndrome. Digestive diseases and sciences\| PubMed ID: 29484573	2018
Intestinal epithelial cells and T cells differentially recognize and respond to Candida albicans yeast and hypha. European journal of immunology\| PubMed ID: 30118145	2018
A Unique Presentation of Infantile-Onset Colitis and Eosinophilic Disease without Recurrent Infections Resulting from a Novel Homozygous CARMIL2 Variant. Journal of clinical immunology\| PubMed ID: 31079270	2019
Maturation of the Human Intestinal Immune System Occurs Early in Fetal Development. Developmental cell\| PubMed ID: 31607651	2019
An RTEL1 Mutation Links to Infantile-Onset Ulcerative Colitis and Severe Immunodeficiency. Journal of clinical immunology\| PubMed ID: 32710398	2020
Antibodies Against Glycoprotein 2 Are Specific Biomarkers for Pediatric Crohn's Disease. Digestive diseases and sciences\| PubMed ID: 32886311	2020
In utero human intestine harbors unique metabolome, including bacterial metabolites. JCI insight\| PubMed ID: 33001863	2020
Monogenic Inflammatory Bowel Disease: It's Never Too Late to Make a Diagnosis. Frontiers in immunology\| PubMed ID: 33013830	2020
Circulating α4β7 Memory T Cells in Pediatric IBD Patients Express a Polyclonal T Cell Receptor Repertoire. Clinical and experimental gastroenterology\| PubMed ID: 33061522	2020
Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy. International journal of molecular sciences\| PubMed ID: 33147779	2020
Distinct extracellular-matrix remodeling events precede symptoms of inflammation. Matrix biology : journal of the International Society for Matrix Biology\| PubMed ID: 33246101	2020
Mucosal IL-10 and IL-10 receptor expression patterns in paediatric patients with ulcerative colitis. International journal of experimental pathology\| PubMed ID: 33405352	2021

Lael Werner

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