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FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU‐induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss‐of‐function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic‐derived, Neuropilin‐1+ (NRP1+) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1− Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL‐2:anti‐IL‐2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11‐deficient Treg cells. Furthermore, these expanded Treg cells could mediate near‐normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss‐of‐function mutations that impair the expression of this critical immune‐regulatory protein. [ABSTRACT FROM AUTHOR]

PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development. • PR3-specific B cells may be identified from the blood of patients with PR3 autoantibodies, but at an extremely low frequency. • We report the immunoglobulin sequences from 19 PR3-specific B cells from a single patient. • PR3-specific B cells were enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length and IGHE gene expression. • PR3-antibody sequences showed low levels of somatic hypermutation & some germline-reverted sequences retained PR3 binding. • This suggests that PR3-self reactivity may occur early in B-cell development. [ABSTRACT FROM AUTHOR]

Objective: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane‐bound IgM‐RF in primary SS and identify unique heavy‐chain peptide signatures for RF clonotype tracking. Methods: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGHRNA from matched peripheral blood mononuclear cells (n = 7). RF‐specific heavy‐chain third complementarity‐determining region (CDR3) peptides were identified by searching RF heavy‐chain peptide sequences against the corresponding IGHRNA sequence libraries. Heavy‐chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. Results: Serum RFs were clonally restricted and composed of shared sets of IgM heavy‐chain variable region (Ig VH) 1–69, 3–15, 3–7, and 3–74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy‐chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH4–34 IgM‐RF decreased following immunosuppression and remission of MC. Conclusion: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones. [ABSTRACT FROM AUTHOR]

Cancer cells seem to exploit mechanisms that evolve as part of physiological tolerance, which is a complementary and often beneficial form of defense. The study of physiological systems of tolerance can therefore provide insights into the development of a state of host tolerance of cancer, and how to break it. Analysis of these models has the potential to improve our understanding of existing immunological therapeutic targets, and help to identify future targets and rational therapeutic combinations. The treatment of cancer with immune checkpoint inhibitors aims to reverse the progression to tolerance of cancer, and achieve an immunogenic, rather than tolerogenic, homeostasis. Broadening the efficacy and durability of checkpoint inhibitors focuses on reversing tolerance and stimulating immunogenicity in the cancer, host, and environment. Two examples of important physiological states of tolerance that may inform tolerance of cancer are microbial infection and placental reproduction. These states of tolerance result from bilateral shaping of host and non‐self, akin to immunoediting in cancer, and offer reliable models to study the immune tolerance paradigm. [ABSTRACT FROM AUTHOR]

Abstract: The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self‐antigens. Strong T‐cell receptor (TCR) engagement induces tolerance in self‐reactive thymocytes by stimulating apoptosis or selection into specialized T‐cell lineages, including intestinal TCRαβ+ CD8αα+ intraepithelial lymphocytes (IEL). TCR‐intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self‐reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T‐cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity‐determining region 3 (CDR3) of the TCRα or TCRβ chain. Compared to pre‐selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3+ regulatory T cells (T‐reg) and naïve T cells. These findings reveal a TCR‐intrinsic motif that distinguishes Type A IELp and IEL from T‐reg and naïve T cells. [ABSTRACT FROM AUTHOR]

Summary: Acquisition of T‐cell central tolerance involves distinct pathways of self‐antigen presentation to thymocytes. One pathway termed indirect presentation requires a self‐antigen transfer step from thymic epithelial cells (TECs) to bone marrow‐derived cells before the self‐antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context‐dependent, potentially due to variation in self‐antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane‐bound transgenic self‐antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow‐derived antigen‐presenting cells, insHEL‐mediated thymocyte deletion was abolished, whereas thyroHEL‐mediated deletion occurred at a later stage of thymocyte development and Foxp3+ regulatory T‐cell differentiation increased. Indirect presentation increased the strength of T‐cell receptor signalling that both self‐antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self‐reactive thymocytes. [ABSTRACT FROM AUTHOR]

Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses. [ABSTRACT FROM AUTHOR]

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation. [ABSTRACT FROM AUTHOR]

Inherited mutations in lipopolysaccharide-responsive beige-like anchor (LRBA) cause a recessive human immune dysregulation syndrome with memory B-cell and antibody deficiency (common variable immunodeficiency), inflammatory bowel disease, enlarged spleen and lymph nodes, accumulation of activated T cells and multiple autoimmune diseases. To understand the pathogenesis of the syndrome, C57BL/6 mice carrying a homozygous truncating mutation in Lrba were produced using CRISPR/Cas9-mediated gene targeting. These mice revealed that LRBA has a critical, cell-autonomous role in promoting cytotoxic T-lymphocyte antigen-4 (CTLA-4) accumulation within CD4 effector T cells and FOXP3+ T-regulatory cells (Tregs). In young mice, or in chimeric mice where only half of the T cells are LRBA deficient, low CTLA-4 was the only detectable abnormality in Tregs, whereas in old mice FOXP3 was also decreased. Low CTLA-4 did not translate into increased CD86 on B cells unless the LRBA-deficient mice were immunised, and neither immunisation nor chronic lymphocytic choriomeningitis virus infection precipitated immune dysregulation. LRBA deficiency did not alter antigen-specific B-cell activation, germinal centre (GC) formation, isotype switching or affinity maturation. Paradoxically, CD86 was decreased on GC B cells in LRBA-deficient mice, pointing to compensatory mechanisms for controlling CD86 in the face of low CTLA-4. These results add to the experimental rationale for treating LRBA deficiency with the CTLA4-Ig fusion protein, Abatacept, and pose questions about the limitations of laboratory experiments in mice to reproduce human disease in natura. [ABSTRACT FROM AUTHOR]

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adultmice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity. [ABSTRACT FROM AUTHOR]

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency. [ABSTRACT FROM AUTHOR]

Linkage analysis studies for autoimmune diabetes have revealed multiple non‐major histocompatibility complex (MHC) chromosomal regions linked to disease susceptibility. To date, more than 20 insulin‐dependent diabetes (Idd) loci linked to diabetes susceptibility have been identified in NOD mice and validated via congenic breeding. Importantly, evidence suggests that Idd loci may regulate at least two pathological steps during autoimmune diabetes development, namely the onset of insulitis and the transition from insulitis to overt diabetes. Here we assess the role of various non‐MHC Idd diabetes‐resistance loci, which have been validated in the non‐transgenic setting, on autoimmune diabetes progression in the transgenic setting. Specifically, we generated multiple Idd congenic strains in the 3A9‐TCR:insHEL NOD.H2k transgenic model and monitored their diabetes incidence. We show that 3A9‐TCR:insHEL NOD.H2k mice congenic for Idd3 or Idd5 display a reduction in diabetes development, whereas mice congenic for Idd9 or Idd13 exhibit an increase, in comparison with 3A9‐TCR:insHEL NOD.H2k mice. These results suggest that the presence of the 3A9‐TCR and hen egg lysosyme transgenes can offset the regulatory function of certain diabetes‐resistance genetic variants contained within the Idd loci, including Idd9 and Idd13. We propose the antigen‐specific 3A9‐TCR:insHEL transgenic model as a useful tool for the study of the genetics of autoimmune diabetes development. [ABSTRACT FROM AUTHOR]

Most humans harbor both CD177neg and CD177pos neutrophils but 1–10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation. [ABSTRACT FROM AUTHOR]

Thymocytes that bind strongly to self‐antigens are prevented from becoming naive T cells by several mechanisms. They undergo clonal deletion at two stages of development; wave 1 in immature thymocytes lacking the medulla‐homing chemokine receptor, CCR7, or wave 2 in more mature CCR7+ thymocytes. Alternatively, self‐reactive thymocytes upregulate Foxp3 to become T‐regulatory cells. Here, we describe the differential timing of the two waves of deletion and Foxp3 upregulation relative to the immature proliferating stage. Proliferating thymocytes were pulse‐labeled in normal C57BL/6 mice with 5‐ethynyl‐2'‐deoxyuridine (EdU). Thymocytes progressed into wave 1 (CCR7−) and wave 2 (CCR7+) of clonal deletion ~2 and 5 days after proliferation, respectively. Foxp3 upregulation occurred between 4 and 8 days after proliferation, predominantly in thymocytes with a Helios+ CCR7+ phenotype. These findings establish a timeline that suggests that wave 1 of clonal deletion occurs in the thymic cortex, whereas wave 2 and Foxp3 upregulation both occur in the thymic medulla. [ABSTRACT FROM AUTHOR]

Dynamic interactions between B and T cells underpin the development of adaptive humoral immune responses to infections and vaccines. Recent advances in the molecular and spatiotemporal control of these interactions during primary responses have contributed greatly to elucidating the molecular pathogenesis of numerous immunodeficiency and autoimmune diseases. The next challenge is to determine how and where memory B and T cells interact during secondary responses to facilitate the rapid and robust response that characterizes anamnestic immunity. [ABSTRACT FROM AUTHOR]

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. [ABSTRACT FROM AUTHOR]

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. Poly- Phen2, 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. [ABSTRACT FROM AUTHOR]

Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8+ T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies. [ABSTRACT FROM AUTHOR]

Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysrégulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysrégulation, a common disturbance of atypical clinical immune deficiencies. [ABSTRACT FROM AUTHOR]

Objective Systemic lupus erythematosus (SLE) is a chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole-exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. This study was undertaken to identify genetic causes of SLE using whole-exome sequencing. Methods We performed whole-exome sequencing in a 4-year-old girl with early-onset SLE and conducted biochemical analysis of the putative defect. Results Whole-exome sequencing in a 4-year-old girl with cerebral lupus identified a rare, homozygous mutation in the three prime repair exonuclease 1 gene ( TREX1) that was predicted to be highly deleterious. The TREX1 R97H mutant protein had a 20-fold reduction in exonuclease activity and was associated with an elevated interferon-α (IFNα) signature in the patient. The discovery and characterization of a pathogenic TREX1 variant in our proband has therapeutic implications. The patient is now a candidate for neutralizing anti-IFNα therapy. Conclusion Our study is the first to demonstrate that whole-exome sequencing can be used to identify rare or novel deleterious variants as genetic causes of SLE and, through a personalized approach, improve therapeutic options. [ABSTRACT FROM AUTHOR]

Most genetic defects that arrest B-cell development in the bone marrow present early in life with agammaglobulinemia, whereas incomplete antibody deficiency is usually associated with circulating B cells. We report 3 related individuals with a novel form of severe B-cell deficiency associated with partial persistence of serum immunoglobulin arising from a missense mutation in NFKB2. Significantly, this point mutation results in a D865G substitution and causes a failure of p100 phosphorylation that blocks processing to p52. Severe B-cell deficiency affects mature and transitional cells, mimicking the action of rituximab. This phenotype appears to be due to disruption of canonical and noncanonical nuclear factor κB pathways by the mutant p100 molecule. These findings could be informative for therapeutics as well as immunodeficiency. (Blood. 2014;124(19):2964-2972) [ABSTRACT FROM AUTHOR]

Quantitative reductions in T-cell receptor ( TCR) signalling are associated with severe immunodeficiency, yet in certain cases can lead to autoimmunity. Mutation of the tyrosine kinase ZAP-70 can cause either of these outcomes, yet the limits of its signal transducing capacity are not well defined. To investigate these limits we have made use of mrtless: a chemically induced mutation of Zap70 associated with T-cell deficiency. Unlike cells devoid of ZAP-70, mrtless thymocytes showed partial induction of CD5 and CD69, and were sensitive to TCR stimulation with a dose-response shifted approximately 10-fold. However, essentially no T cells were able to compensate for the mrtless mutation and mature beyond the CD4+ CD8+ stage. This outcome contrasts with a ZAP-70 Src Homology 2 domain mutant strain, where high-affinity self-reactive TCR are positively selected rather than deleted. We discuss these data with respect to current models of TCR signalling in thymocyte selection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility. [ABSTRACT FROM AUTHOR]

Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1+ NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease. [ABSTRACT FROM AUTHOR]

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing. [ABSTRACT FROM AUTHOR]

Summary: Accumulation of T follicular helper (Tfh) cells and proinflammatory cytokines drive autoantibody-mediated diseases. The RNA-binding protein Roquin-1 (Rc3h1) represses the inducible costimulator ICOS and interferon-γ (IFN-γ) in T cells to prevent Tfh cell accumulation. Unlike Rc3h1 san mice with a mutation in the ROQ domain of Roquin-1, mice lacking the protein, paradoxically do not display increased Tfh cells. Here we have analyzed mice with mutations that eliminate the RING domain from Roquin-1 or its paralog, Roquin-2 (Rc3h2). RING or ROQ mutations both disrupted Icos mRNA regulation by Roquin-1, but, unlike the ROQ mutant that still occupied mRNA-regulating stress granules, RING-deficient Roquin-1 failed to localize to stress granules and allowed Roquin-2 to compensate in the repression of ICOS and Tfh cells. These paralogs also targeted tumor necrosis factor (TNF) in nonlymphoid cells, ameliorating autoantibody-induced arthritis. The Roquin family emerges as a posttranscriptional brake in the adaptive and innate phases of antibody responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein--protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum. [ABSTRACT FROM AUTHOR]

Abstract: To understand the interactions between Notch1 and Ikaros in the evolution of T cell acute lymphoblastic leukemia (T-ALL), we traced the evolution of T-ALL in mice with an inherited Ikaros mutation, Ikzf1 Plstc which inactivates DNA binding. DNA-binding Ikaros repressed Notch1 response in transfected cell lines and in CD4+8+ (DP) thymocytes from young pre-leukemic Ikzf1 Plstc heterozygous mice. In DP thymocytes, a 50–1000 fold escalation in mRNA for Notch1 target genes Hes1 and Dtx1 preceded thymic lymphoma or leukemia and was closely correlated with the first detectable differentiation abnormalities, loss of heterozygosity (LOH) eliminating wild-type Ikzf1, and multiple missense and truncating Notch1 mutations. These findings illuminate the early stages of leukemogenesis by demonstrating progressive exaggeration of Notch1 responsiveness at the DP thymocyte stage brought about by multiple mutations acting in concert upon the Notch1 pathway. [Copyright &y& Elsevier]

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4+ or CD8+ T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells. [ABSTRACT FROM AUTHOR]

Foxp3+ regulatory T cells play a pivotal role in maintaining self-tolerance and immune homeostasis. In the absence of regulatory T cells, generalized immune activation and multiorgan T cell-driven pathology occurs. Although the phenomenon of immunologic control by Foxp3+ regulatory T cells is well recognized, the comparative effect over different arms of the immune system has not been thoroughly investigated. Here, we generated a cohort of mice with a continuum of regulatory T-cell frequencies ranging from physiologic levels to complete deficiency. This titration of regulatory T-cell depletion was used to determine how different effector subsets are controlled. We found that in vivo Foxp3+ regulatory T-cell frequency had a proportionate relationship with generalized T-cell activation and Th1 magnitude, but it had a surprising disproportionate relationship with Th2 magnitude. The asymmetric regulation was associated with efficient suppression of Th2 cells through additional regulations on the apoptosis rate in Th2 cells and not Th1 cells and could be replicated by CTLA4-Ig or anti-IL-2 Ab. These results indicate that the Th2 arm of the immune system is under tighter control by regulatory T cells than the Th1 arm, suggesting that Th2-driven diseases may be more responsive to regulatory T-cell manipulation. [ABSTRACT FROM AUTHOR]

Objective: To define cellular mechanisms by which B cells promote type 1 diabetes.Research Design and Methods: The study measured islet-specific CD4 T cell regulation in T-cell receptor transgenic mice with elevated frequencies of CD4 T cells recognizing hen egg lysozyme (HEL) autoantigen expressed in islet β-cells and thymic epithelium under control of the insulin-gene promoter. The effects of a mutation in Roquin that dysregulates T follicular helper (Tfh) cells to promote B-cell activation and anti-islet autoantibodies were studied, as were the effects of HEL antigen-presenting B cells and passively transferred or maternally transmitted anti-islet HEL antibodies.Results: Mouse anti-islet IgG antibodies-either formed as a consequence of excessive Tfh activity, maternally transmitted, or passively transferred-caused a breakdown of tolerance in islet-reactive CD4(+) cells and fast progression to diabetes. Progression to diabetes was ameliorated in the absence of B cells or when the B cells could not secrete islet-specific IgG. Anti-islet antibodies increased the survival of proliferating islet-reactive CD4(+) T cells. FcγR blockade delayed and reduced the incidence of autoimmune diabetes.Conclusions: B cells can promote type 1 diabetes by secreting anti-islet autoantibodies that act in an FcγR-mediated manner to enhance the expansion of islet-reactive CD4 T cells and cooperate with inherited defects in thymic and peripheral CD4 T-cell tolerance. Cooperation between inherited variants affecting CD4 T-cell tolerance and anti-islet autoantibodies should be examined in epidemiological studies and in studies examining the efficacy of B-cell depletion. [ABSTRACT FROM AUTHOR]

Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell-deficiency syndrome in mice caused by mutations in Atp11c, which encodes a member of the P4 ATPase family thought to serve as 'flippases' that concentrate aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11C resulted in a lower rate of phosphatidylserine translocation in pro-B cells and much lower pre-B cell and B cell numbers despite expression of pre-rearranged immunoglobulin transgenes or enforced expression of the prosurvival protein Bcl-2 to prevent apoptosis and abolished pre-B cell population expansion in response to a transgene encoding interleukin 7. The only other abnormalities we noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. Our results identify an intimate connection between phospholipid transport and B lymphocyte function. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4+ T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4+ T cells in prediabetic 3A9-TCR × insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4+ cells or the numbers of islet-specific CD4+ or CD4+Foxp3+ T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-β-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4+ cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4+ cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity. [ABSTRACT FROM AUTHOR]

Roquin is an E3 ubiquitin ligase with a poorly understood but essential role in preventing T-cell-mediated autoimmune disease and in microRNA-mediated repression of inducible costimulator ( Icos) mRNA. Roquin and its mammalian paralogue membrane-associated nucleic acid binding protein (MNAB) define a protein family distinguished by an ∼ 200 amino acid domain of unknown function, ROQ, that is highly conserved from mammals to invertebrates and is flanked by a RING-1 zinc finger and a CCCH zinc finger. Here we show that human, Drosophila and Caenorhabditis elegans Roquin and human MNAB localize to the cytoplasm and upon stress are concentrated in stress granules, where stalled mRNA translation complexes are stored. The ROQ domain is necessary and sufficient for localization to arsenite-induced stress granules and to induce these structures upon overexpression, and is required to trigger Icos mRNA decay. Gel-shift, SPR and footprinting studies show that an N-terminal fragment centred on the ROQ domain binds RNA from the Icos 3′-untranslated region comprising the minimal sequence for Roquin-mediated repression, adjacent to the miR-101 sequence complementarity. These findings identify Roquin as an RNA-binding protein and establish a specific function for the ROQ protein domain in mRNA homeostasis. Structured digital abstract • : TIA-1 (uniprotkb: ) and Roquin (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) • : RLE-1 (uniprotkb: ) and TIA-1 (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) • : DmRoquin (uniprotkb: ) and TIA-1 (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) • , : MNAB (uniprotkb: ) and TIA-1 (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) • : eIF3 (uniprotkb: ) and Roquin (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) • : DCP1A (uniprotkb: ) and TIA-1 (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) [ABSTRACT FROM AUTHOR]

We report two new mouse strains: Jasmine (C57BL/6J/Apb-Tap2jas/Apb), with a point mutation in the transporter associated with antigen processing (TAP)2 ; and Rose, (C57BL/6J/Apb-Tap1rose/Apb), with a point mutation in TAP1. These strains were detected as the result of ethyl nitroso urea (ENU) screens for recessive point mutations affecting the immune system. As expected in cases of defective TAP expression, the mice have very low major histocompatibility complex (MHC)-I cell-surface expression, and few CD8+ T cells. The Rose strain has an A to T substitution in exon 10 of TAP1, resulting in an asparagine to valine substitution at position 643. Jasmine has an A to C transversion in exon 5 of TAP2, resulting in a threonine to proline substitution at position 293 of the protein. The mutation does not affect mRNA levels, but results in a very severe reduction in TAP2 protein. TAP1 protein levels are also decreased in Jasmine mice, demonstrating a new role for mouse TAP2 in stabilizing TAP1 protein expression. Jasmine is the first strain available with defective TAP2. The two mouse strains provide additional animal models for the human condition Bare Lymphocyte syndrome type 1, and identify new residues important for TAP function. [ABSTRACT FROM AUTHOR]

The article focuses on a study which tested the hypothesis on the role of circulating T cells resembling follicular helper T (Tfh) cells in a subset of patients with systemic lupus erythematosus (SLE). The study examined the enumeration of circulating T cells resembling Tfh cells as a biomarker of this expansion in sanroque mice. Study authors concluded that Tfh effector molecules can be used as therapeutic targets in SLE patients.

The human β globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5′- HBE1, HBG2, HBG1, HBD, HBB-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the β-globin ACH, and HPFH. [ABSTRACT FROM AUTHOR]

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4α. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4α, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.Immunology and Cell Biology (2008) 86, 3–14; doi:10.1038/sj.icb.7100123; published online 16 October 2007 [ABSTRACT FROM AUTHOR]

The cause of common organ-specific autoimmune diseases is poorly understood because of genetic and cellular complexity in humans and animals. Recent advances in the understanding of the mechanisms of the defects underlying autoimmune disease in autoimmune polyendocrinopathy syndrome type 1 and non-obese diabetic mice suggest that failures in central tolerance play a key role in predisposition towards organ-specific autoimmunity. The lessons from such rare monogenic autoimmune disorders and well-characterized polygenic traits demonstrate how subtle quantitative trait loci can result in large changes in the susceptibility to autoimmunity. These data allow us to propose a model relating efficiency of thymic deletion to T-cell tolerance and susceptibility to autoimmunity. [ABSTRACT FROM AUTHOR]

The cause of common polygenic autoimmune diseases is not understood because of genetic and cellular complexity. Here, we pinpoint the action of a subset of autoimmune susceptibility loci in the NOD mouse strain linked to D1mit181, D2mit490, D7mit101, and D15mit229, which cause a generalized resistance to thymic deletion in vivo that applies equally to Aire-induced organ-specific gene products in the thymic medulla and to systemic antigens expressed at high levels throughout the thymus and affects CD4+, CD4+8+, and CD4+25+ thymocytes. Resistance to thymic deletion does not reflect a general deficit in TCR signaling to calcineurin- or ERK-induced genes, imbalance in constitutive regulators of apoptosis, nor excessive signaling to prosurvival genes but is distinguished by failure to induce the proapoptotic gene and protein, Bim, during in vivo encounter with high-avidity autoantigen. These findings establish defects in thymic deletion and Bim induction as a key mechanism in the pathogenesis of autoimmunity. [Copyright &y& Elsevier]

A central issue in understanding the hematolymphoid system is the generation of appropriate mutant alleles in mice to reveal the function of regulatory genes. Here we describe a mouse strain, Plastic, with a point mutation in a zinc finger of Ikaros that disrupts DNA binding but preserves efficient assembly of the full-length protein into higher order complexes. IkarosPlastic homozygosity is embryonically lethal with severe defects in terminal erythrocyte and granulocyte differentiation, excessive macrophage formation, and blocked lymphopoiesis, while heterozygotes display a partial block in lymphocyte differentiation. The contrast with more circumscribed effects of Ikaros alleles that ablate the full-length protein highlights the importance in mammals of generating recessive niche-filling alleles that inactivate function without creating a void in multimolecular assemblies. [Copyright &y& Elsevier]

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-κB 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. [Copyright &y& Elsevier]

Self-tolerance is achieved by deleting or regulating self-reactive lymphocytes at a series of cellular checkpoints placed at many points along the developmental pathways to plasma cells and effector T cells. At each checkpoint, what are the molecular pathways that determine whether a lymphocyte remains quiescent, begins dividing, differentiates or dies? In splenic B cells, the decision between quiescence, tolerance by anergy, and activation provides a tractable setting to explore these issues by global gene expression profiling on DNA microarrays. Here we discuss the application of microarrays to illuminate a set of cell fate decisions that appear to be determined by summation of numerous small changes in expression of stimulatory and inhibitory genes. Many genes with known or predicted inhibitory functions are highly expressed in naïve, quiescent B cells, notably the signal inhibitor SLAP and DNA-binding proteins of the Kr*uuml;ppel family (LKLF, BKLF, GKLF), Tsc-22, GILZ, Id-3, and GADD45. Activation of naïve B cells, triggered by acute binding of antigen to the B-cell receptor involves a rapid decrease in expression of these inhibitory genes. Promitotic genes are induced in parallel, including c-myc, LSIRF/IRF4, cyclin D2, Egr-1 and Egr-2, as are the anti-apoptotic gene A1 and genes for the T-cell-attracting chemokines MIP-1α and β. B-cell tolerance through the process of anergy, induced by chronic binding of sel antigen, maintains expression of the inhibitory genes found in quiescent B cells and induces in additional set of inhibitory genes. The latter include inhibitors of signallign--CD72, neurogranin, pcp4--and additional inhibitors of gene expression such as SATB1, MEF2C, and TGIF and Nab-2. The effects of tolerance, the immunosuppressive drug... [ABSTRACT FROM AUTHOR]