The P4-type ATPase ATP11C is essential for B lymphopoiesis in adult bone marrow.

B lymphopoiesis begins in the fetal liver, switching after birth to the bone marrow, where it persists for life. The unique developmental outcomes of each phase are well documented, yet their molecular requirements are not. Here we describe two allelic X-linked mutations in mice that caused cell-intrinsic arrest of adult B lymphopoiesis. Mutant fetal liver progenitors generated B cells in situ but not in irradiated adult bone marrow, which emphasizes a necessity for the affected pathway only in the context of adult bone marrow. The causative mutations were ascribed to Atp11c, which encodes a P4-type ATPase with no previously described function. Our data establish an essential, cell-autonomous and context-sensitive function for ATP11C, a putative aminophospholipid flippase, in B cell development.

A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity.

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.

Increased susceptibility to DNA virus infection in mice with a GCN2 mutation.

The downregulation of translation through eIF2α phosphorylation is a cellular response to diverse stresses, including viral infection, and is mediated by the GCN2 kinase, protein kinase R (PKR), protein kinase-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI). Although PKR plays a major role in defense against viruses, other eIF2α kinases also may respond to viral infection and contribute to the shutdown of protein synthesis. Here we describe the recessive, loss-of-function mutation atchoum (atc) in Eif2ak4, encoding GCN2, which increased susceptibility to infection by the double-stranded DNA viruses mouse cytomegalovirus (MCMV) and human adenovirus. This mutation was identified by screening macrophages isolated from mice carrying N-ethyl-N-nitrosourea (ENU)-induced mutations. Cells from Eif2ak4(atc/atc) mice failed to phosphorylate eIF2α in response to MCMV. Importantly, homozygous Eif2ak4(atc) mice showed a modest increase in susceptibility to MCMV infection, demonstrating that translational arrest dependent on GCN2 contributes to the antiviral response in vivo.

Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells.

Despite their low frequency, plasmacytoid dendritic cells (pDCs) produce most of the type I IFN that is detectable in the blood following viral infection. The endosomal Toll-like receptors (TLRs) TLR7 and TLR9 are required for pDCs, as well as other cell types, to sense viral nucleic acids, but the mechanism by which signaling through these shared receptors results in the prodigious production of type I IFN by pDCs is not understood. We designed a genetic screen to identify proteins required for the development and specialized function of pDCs. One phenovariant, which we named feeble, showed abrogation of both TLR-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving TLR responses intact in other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1) and has not previously been implicated in pDC function. The identification of the feeble mutation led to our subsequent observations that AP-3, as well as the BLOC-1 and BLOC-2 Hermansky-Pudlak syndrome proteins are essential for pDC signaling through TLR7 and TLR9. These proteins are not necessary for TLR7 or TLR9 signaling in conventional DCs and thus comprise a membrane trafficking pathway uniquely required for endosomal TLR signaling in pDCs.

Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells.

A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L --> Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised.

A mutation of Ikbkg causes immune deficiency without impairing degradation of IkappaB alpha.

Null alleles of the gene encoding NEMO (NF-kappaB essential modulator) are lethal in hemizygous mice and men, whereas hypomorphic alleles typically cause a syndrome of immune deficiency and ectodermal dysplasia. Here we describe an allele of Ikbkg in mice that impaired Toll-like receptor signaling, lymph node formation, development of memory and regulatory T cells, and Ig production, but did not cause ectodermal dysplasia. Degradation of IkappaB alpha, which is considered a primary requirement for NEMO-mediated immune signaling, occurred normally in response to Toll-like receptor stimulation, yet ERK phosphorylation and NF-kappaB p65 nuclear translocation were severely impaired. This selective loss of function highlights the immunological importance of NEMO-regulated pathways beyond IkappaB alpha degradation, and offers a biochemical explanation for rare immune deficiencies in man.

Commitment to the regulatory T cell lineage requires CARMA1 in the thymus but not in the periphery.

Regulatory T (T(reg)) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4(+) T cells in a cytokine dependent manner. T(reg) cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4(+)Foxp3(+) T(reg) cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T(reg) cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2). In vivo, a small peripheral T(reg) pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T(reg) cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.

Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5-deficient mice.

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus.

Using the Unc93b1 3d mutation that selectively abolishes nucleic acid-binding Toll-like receptor (TLR) (TLR3, -7, -9) signaling, we show these endosomal TLRs are required for optimal production of IgG autoAbs, IgM rheumatoid factor, and other clinical parameters of disease in 2 lupus strains, B6-Fas(lpr) and BXSB. Strikingly, treatment with lipid A, an autoAb-inducing TLR4 agonist, could not overcome this requirement. The 3d mutation slightly reduced complete Freund's adjuvant (CFA)-mediated antigen presentation, but did not affect T-independent type 1 or alum-mediated T-dependent humoral responses or TLR-independent IFN production induced by cytoplasmic nucleic acids. These findings suggest that nucleic acid-sensing TLRs might act as an Achilles' heel in susceptible individuals by providing a critical pathway by which relative tolerance for nucleic acid-containing antigens is breached and systemic autoimmunity ensues. Importantly, this helps provide an explanation for the high frequency of anti-nucleic acid Abs in lupus-like systemic autoimmunity.

ENU-induced phenovariance in mice: inferences from 587 mutations.

BACKGROUND: We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. FINDINGS: In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. CONCLUSIONS: Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Rapid identification of a disease allele in mouse through whole genome sequencing and bulk segregation analysis.

In a pedigree of C57BL/6J mice homozygous for germline mutations induced by the mutagen N-ethyl-N-nitrosourea (ENU), numerous animals died under specific pathogen-free (SPF) conditions between 6 and 7 months of age. Death was caused by nephritic syndrome, which progressed to renal failure associated with focal segmental glomerulosclerosis. To identify the mutation responsible for renal disease, we sequenced genomic DNA from an affected animal using the Applied Biosystems SOLiD sequencing platform. Approximately 74% of the nucleotides comprising coding sequences and splice junctions in the mouse genome were covered at least three times. Within this portion of the genome, 64 discrepancies were flagged as potential homozygous mutations and 82 were flagged as potential heterozygous mutations. A total of 10 of these calls, all homozygous, were validated by capillary sequencing. One of the validated mutations disrupted splicing of the Col4a4 transcript. Genetic mapping by bulk segregation analysis excluded all mutations but this one as the cause of renal disease in Aoba mice. Col4a4 has not been targeted in the mouse, and this strain, named Aoba, represents the first functionally null allele in this species. Our study demonstrates the speed and utility of whole genome sequencing coupled with low resolution meiotic mapping as a means of identifying causative mutations induced by ENU.

The germinal center response is impaired in the absence of T cell-expressed CXCR5.

Germinal centers support the differentiation of memory B cells and long-lived antibody-secreting cells during infection or upon vaccination. Here, we constructed mice with T cells that selectively lack the chemokine receptor CXCR5 to determine if expression of this receptor by T cells is mandatory for germinal center formation and function. In these animals, germinal centers that are properly localized in B cell follicles and contain T cells do form after immunization with a thymus-dependent antigen. However, fewer and smaller germinal centers form, resulting in a significant reduction in the frequency of germinal center B cells. The defect in germinal center formation is paralleled by decreased frequencies of isotype-switched antibody-secreting cells in the spleen and bone marrow and reduced serum concentrations of total and high-affinity hapten-specific IgG1. The results demonstrate that although CXCR5-dependent T cell positioning is important for maximal induction and expansion of germinal centers, stimulation of isotype class switching, and development of antibody-secreting cells that seed the spleen and bone marrow, it is not absolutely required for the formation and function of follicular germinal centers.

Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues.

Lymphocytes travel throughout the body to carry out immune surveillance and participate in inflammatory reactions. Their path takes them from blood through tissues into lymph and back to blood. Molecules that control lymphocyte recruitment into extralymphoid tissues are well characterized, but exit is assumed to be random. Here, we showed that lymphocyte emigration from the skin was regulated and was sensitive to pertussis toxin. CD4(+) lymphocytes emigrated more efficiently than CD8(+) or B lymphocytes. T lymphocytes in the afferent lymph expressed functional chemokine receptor CCR7, and CCR7 was required for T lymphocyte exit from the skin. The regulated expression of CCR7 by tissue T lymphocytes may control their exit, acting with recruitment mechanisms to regulate lymphocyte transit and accumulation during immune surveillance and inflammation.

Antigen-specific lymphocyte sequestration in lymphoid organs: lack of essential roles for alphaL and alpha4 integrin-dependent adhesion or Galphai protein-coupled receptor signaling.

Selective lymphocyte sequestration was described over 30 years ago as the transient withdrawal of Ag-specific lymphocytes from the circulation as a result of their activation in secondary lymphoid organs. We used a TCR-transgenic adoptive transfer system to further characterize the Ag and adjuvant dependence of this process in mice. In addition, we examined the contribution of the alpha(L) and alpha(4) integrin chains as well as Galpha(i) protein-coupled receptor signaling to the retention of Ag-specific T cells in peripheral lymph nodes. Our results demonstrate that selective lymphocyte sequestration is T cell autonomous and adjuvant independent, and that the duration of sequestration is not controlled by the continued presence of Ag in secondary lymphoid organs. This process is not critically dependent on the alpha(L) and alpha(4) integrin chains or Galpha(i) protein-coupled receptor signaling. Selective lymphocyte sequestration may be mediated by redundant mechanisms and/or controlled by novel or nonclassical adhesion or trafficking molecules.