Lisa A. Miosge

Identifying the MAGUK Protein Carma-1 as a Central Regulator of Humoral Immune Responses and Atopy by Genome-Wide Mouse Mutagenesis

In a genome-wide ENU mouse mutagenesis screen a recessive mouse mutation, unmodulated, was isolated with profound defects in humoral immune responses, selective deficits in B cell activation by antigen receptors and T cell costimulation by CD28, and gradual development of atopic dermatitis with hyper-IgE. Mutant B cells are specifically defective in forming connections between antigen receptors and two key signaling pathways for immunogenic responses, NF-kappaB and JNK, but signal normally to calcium, NFAT, and ERK. The mutation alters a conserved leucine in the coiled-coil domain of CARMA-1/CARD11, a member of the MAGUK protein family implicated in organizing multimolecular signaling complexes. These results define Carma-1 as a key regulator of the plasticity in antigen receptor signaling that underpins opposing mechanisms of immunity and tolerance.

Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects

BACKGROUND Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. OBJECTIVE We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. METHODS Molecular, immunologic, and functional assays were performed. RESULTS The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. CONCLUSION Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects.

Comparison of predicted and actual consequences of missense mutations

Significance Computational tools applied to any human genome sequence identify hundreds of genetic variants predicted to disrupt the function of individual proteins as the result of a single codon change. These tools have been trained on disease mutations and common polymorphisms but have yet to be tested against an unbiased spectrum of random mutations arising de novo. Here we perform such a test comparing the predicted and actual effects of de novo mutations in 23 genes with essential functions for normal immunity and all possible mutations in the TP53 tumor suppressor gene. These results highlight an important gap in our ability to relate genotype to phenotype in clinical genome sequencing: the inability to differentiate immediately clinically relevant mutations from nearly neutral mutations. Each person’s genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea–treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation’s functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.

Analysis of an Ethylnitrosourea-generated Mouse Mutation Defines a Cell Intrinsic Role of Nuclear Factor κB2 in Regulating Circulating B Cell Numbers

The number of circulating follicular B lymphocytes is normally kept within a precise range despite their dispersion through the body and daily overproduction of precursors in the bone marrow. By establishing a genome wide recessive mutation screen in C57BL/6 mice to identify critical components of immune system regulation, we identified a mutant strain with selective deficiency in recirculating B cells but not immature or peritoneal B1 cells. Analysis of mixed bone marrow chimeras established that the mutation affects a cell autonomous process within B cells that is required for their accumulation after emigrating to peripheral lymphoid organs. The defect is caused by a point mutation in the gene encoding transcription factor nuclear factor (NF)-κB2, terminating the encoded protein within the DNA-binding domain. These findings establish the feasibility of analyzing immune regulation by genome wide mutant screens and demonstrates an intrinsic requirement for NF-κB2 in regulating circulating follicular B cell numbers.

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8− dendritic cells require the intramembrane endopeptidase SPPL2A

Mice lacking activity of the intramembrane protease SPPL2A exhibit humoral immunodeficiency and lack mature B cell subsets.

Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.

Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids

LFA-1 expression allows liver-resident CD8+ T cells to patrol hepatic sinusoids yet remain in the liver. LFA-1 helps T cells feel at home in the liver Liver-resident T cells are critical to defense against infections such as malaria and hepatitis B virus. To protect the hepatic sinusoids, these cells must enter circulation yet still remain in the liver. McNamara et al. now report that liver-resident CD8+ T cells up-regulate LFA-1 and that LFA-1–ICAM-1 (intercellular adhesion molecule–1) interactions are critical for T cell patrolling of the hepatic sinusoids. Moreover, in the absence of LFA-1, CD8 T cells do not form liver-resident memory populations, even after infection with either Plasmodium or lymphocytic choriomeningitis virus. Thus, LFA-1 expression acts as a sort of invisible fence, allowing the liver-resident memory T cells free reign within the boundaries of the liver. Liver-resident CD8+ T cells are highly motile cells that patrol the vasculature and provide protection against liver pathogens. A key question is: How can these liver CD8+ T cells be simultaneously present in the circulation and tissue-resident? Because liver-resident T cells do not express CD103—a key integrin for T cell residence in epithelial tissues—we investigated other candidate adhesion molecules. Using intravital imaging, we found that CD8+ T cell patrolling in the hepatic sinusoids is dependent on LFA-1–ICAM-1 (intercellular adhesion molecule–1) interactions. Liver-resident CD8+ T cells up-regulate LFA-1 compared with effector memory cells, presumably to facilitate this behavior. Last, we found that LFA-1–deficient CD8+ T cells failed to form substantial liver-resident memory populations after Plasmodium immunization or lymphocytic choriomeningitis virus infection. Collectively, our results demonstrate that it is adhesion through LFA-1 that allows liver-resident memory CD8+ T cells to patrol and remain in the hepatic sinusoids.

Genes, pathways and checkpoints in lymphocyte development and homeostasis

A plethora of genes involved in murine B and T cell development have been identified, and developmental pathways within the primary lymphoid tissues have been well delineated. The generation of a functional, but non‐self reacting lymphocyte repertoire results from the completion of several checkpoints during lymphocyte development and competition for survival factors in the periphery. Improved knowledge of these developmental checkpoints and homeostatic mechanisms is critical for understanding human immunodeficiency, leukaemia/lymphoma and autoimmunity, which are conditions where checkpoints and homeostasis are likely to be deregulated.

Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes

Significance Mammalian B lymphocytes make antibodies of five different heavy chain isotypes, IgM, IgD, IgG, IgE, and IgA. The different isotypes are produced at discrete stages in B-cell development from a single immunoglobulin heavy chain (Igh) gene, either by irreversible rearrangement of the gene to make IgG, IgE, or IgA or by alternative splicing of the RNA transcribed from the Igh gene to coexpress IgM and IgD. Developmentally regulated trans-acting factors have been hypothesized to control IgM and IgD expression from large Igh RNAs, but these factors have remained elusive for several decades. Here, using a genome-wide mutation screen in mice, we identify an obscure gene, Zfp318, as encoding a specific and essential factor promoting IgD expression in mature B cells. IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.