Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease.

The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21) specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn's disease, ulcerative colitis (UC) and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL) for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner.

MAGI2 Mutations Cause Congenital Nephrotic Syndrome.

Steroid-resistant nephrotic syndrome (SRNS), a heterogeneous disorder of the renal glomerular filtration barrier, results in impairment of glomerular permselectivity. Inheritance of genetic SRNS may be autosomal dominant or recessive, with a subset of autosomal recessive SRNS presenting as congenital nephrotic syndrome (CNS). Mutations in 53 genes are associated with human SRNS, but these mutations explain ≤30% of patients with hereditary cases and only 20% of patients with sporadic cases. The proteins encoded by these genes are expressed in podocytes, and malfunction of these proteins leads to a universal end point of podocyte injury, glomerular filtration barrier disruption, and SRNS. Here, we identified novel disease-causing mutations in membrane-associated guanylate kinase, WW, and PDZ domain-containing 2 (MAGI2) through whole-exome sequencing of a deeply phenotyped cohort of patients with congenital, childhood-onset SRNS. Although MAGI2 has been shown to interact with nephrin and regulate podocyte cytoskeleton and slit diaphragm dynamics, MAGI2 mutations have not been described in human SRNS. We detected two unique frameshift mutations and one duplication in three patients (two families); two siblings shared the same homozygous frameshift mutation, whereas one individual with sporadic SRNS exhibited compound heterozygosity. Two mutations were predicted to introduce premature stop codons, and one was predicted to result in read through of the normal translational termination codon. Immunohistochemistry in kidney sections from these patients revealed that mutations resulted in lack of or diminished podocyte MAGI2 expression. Our data support the finding that mutations in the MAGI2 gene are causal for congenital SRNS.

Genomic and clinical profiling of a national nephrotic syndrome cohort advocates a precision medicine approach to disease management.

Steroid Resistant Nephrotic Syndrome (SRNS) in children and young adults has differing etiologies with monogenic disease accounting for 2.9-30% in selected series. Using whole exome sequencing we sought to stratify a national population of children with SRNS into monogenic and non-monogenic forms, and further define those groups by detailed phenotypic analysis. Pediatric patients with SRNS were identified via a national United Kingdom Renal Registry. Whole exome sequencing was performed on 187 patients, of which 12% have a positive family history with a focus on the 53 genes currently known to be associated with nephrotic syndrome. Genetic findings were correlated with individual case disease characteristics. Disease causing variants were detected in 26.2% of patients. Most often this occurred in the three most common SRNS-associated genes: NPHS1, NPHS2, and WT1 but also in 14 other genes. The genotype did not always correlate with expected phenotype since mutations in OCRL, COL4A3, and DGKE associated with specific syndromes were detected in patients with isolated renal disease. Analysis by primary/presumed compared with secondary steroid resistance found 30.8% monogenic disease in primary compared with none in secondary SRNS permitting further mechanistic stratification. Genetic SRNS progressed faster to end stage renal failure, with no documented disease recurrence post-transplantation within this cohort. Primary steroid resistance in which no gene mutation was identified had a 47.8% risk of recurrence. In this unbiased pediatric population, whole exome sequencing allowed screening of all current candidate genes. Thus, deep phenotyping combined with whole exome sequencing is an effective tool for early identification of SRNS etiology, yielding an evidence-based algorithm for clinical management.

Monocytes control natural killer cell differentiation to effector phenotypes.

Natural killer (NK) cells play a major role in immunologic surveillance of cancer. Whether NK-cell subsets have specific roles during antitumor responses and what the signals are that drive their terminal maturation remain unclear. Using an in vivo model of tumor immunity, we show here that CD11b(hi)CD27(low) NK cells migrate to the tumor site to reject major histocompatibility complex class I negative tumors, a response that is severely impaired in Txb21(-/-) mice. The phenotypical analysis of Txb21-deficient mice shows that, in the absence of Txb21, NK-cell differentiation is arrested specifically at the CD11b(hi)CD27(hi) stage, resulting in the complete absence of terminally differentiated CD11b(hi)CD27(low) NK cells. Adoptive transfer experiments and radiation bone marrow chimera reveal that a Txb21(+/+) environment rescues the CD11b(hi)CD27(hi) to CD11b(hi)CD27(low) transition of Txb21(-/-) NK cells. Furthermore, in vivo depletion of myeloid cells and in vitro coculture experiments demonstrate that spleen monocytes mediate the terminal differentiation of peripheral NK cells in a Txb21- and IL-15Rα-dependent manner. Together, these data reveal a novel, unrecognized role for Txb21 expression in monocytes in promoting NK-cell development and help appreciate how various NK-cell subsets are generated and participate in antitumor immunity.

Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses.

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.

Genes and podocytes - new insights into mechanisms of podocytopathy.

After decades of primarily morphological study, positional cloning of the NPHS1 gene was the landmark event that established aberrant podocyte genetics as a pivotal cause of malfunction of the glomerular filter. This ended any uncertainty whether genetic mutation plays a significant role in hereditary nephrotic syndromes (NS) and confirmed podocytes as critical players in regulating glomerular protein filtration. Although subsequent sequencing of candidate genes chosen on the basis of podocyte biology had less success, unbiased analysis of genetically informative kindreds and syndromic disease has led to further gene discovery. However, the 45 genes currently associated with human NS explain not more than 20-30% of hereditary and only 10-20% of sporadic cases. It is becoming increasingly clear both from genetic analysis and phenotypic data - including occasional response to immunosuppressive agents and post-transplant disease recurrence in Mendelian disease - that monogenic inheritance of abnormalities in podocyte-specific genes disrupting filter function is only part of the story. Recent advances in genetic screening technology combined with increasingly robust bioinformatics are set to allow identification and characterization of novel disease causing variants and more importantly, disease modifying genes. Emerging data also support a significant but incompletely characterized immunoregulatory component.

Strategies for translational research in the United Kingdom.

In the United Kingdom, many foundations and institutions and the government have made substantial investments in translational research. We examine the structures that surround this support and consider some of the results of this prodigious push toward enhancing translational research pursuits and thus improved clinical medicine.