Homozygous DNA ligase IV R278H mutation in mice leads to leaky SCID and represents a model for human LIG4 syndrome.

DNA ligase IV (LIG4) is an essential component of the nonhomologous end-joining (NHEJ) repair pathway and plays a key role in V(D)J recombination. Hypomorphic LIG4 mutations in humans are associated with increased cellular radiosensitivity, microcephaly, facial dysmorphisms, growth retardation, developmental delay, and a variable degree of immunodeficiency. We have generated a knock-in mouse model with a homozygous Lig4 R278H mutation that corresponds to the first LIG4 mutation reported in humans. The phenotype of homozygous mutant mice Lig4(R278H/R278H) (Lig4(R/R)) includes growth retardation, a decreased life span, a severe cellular sensitivity to ionizing radiation, and a very severe, but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID. Genomic instability is associated with a high rate of thymic tumor development. Finally, Lig4(R/R) mice spontaneously produce low-affinity antibodies that include autoreactive specificities, but are unable to mount high-affinity antibody responses. These findings highlight the importance of LIG4 in lymphocyte development and function, and in genomic stability maintenance, and provide a model for the complex phenotype of LIG4 syndrome in humans.

Induced pluripotent stem cells: a novel frontier in the study of human primary immunodeficiencies.

BACKGROUND: The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells (iPSCs) through the exogenous expression of transcription promises to revolutionize the study of human diseases. OBJECTIVE: Here we report on the generation of 25 iPSC lines from 6 patients with various forms of primary immunodeficiencies (PIDs) affecting adaptive immunity, innate immunity, or both. METHODS: Patients' dermal fibroblasts were reprogrammed by expression of 4 transcription factors, octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), Krueppel-like factor 4 (KLF4), and cellular myelomonocytosis proto-oncogene (cMYC), by using a single excisable polycistronic lentiviral vector. RESULTS: iPSCs derived from patients with PIDs show a stemness profile that is comparable with that observed in human embryonic stem cells. After in vitro differentiation into embryoid bodies, pluripotency of the patient-derived iPSC lines was demonstrated by expression of genes characteristic of each of the 3 embryonic layers. We have confirmed the patient-specific origin of the iPSC lines and ascertained maintenance of karyotypic integrity. CONCLUSION: By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of iPSC lines from patients with PIDs represents a unique resource to investigate the pathophysiology of hematopoietic and extrahematopoietic manifestations of these diseases and might assist in the development of novel therapeutic approaches based on gene correction.

Distinct pathogenic roles for resident and monocyte-derived macrophages in lupus nephritis.

Lupus nephritis is a serious complication of systemic lupus erythematosus, mediated by IgG immune complex (IC) deposition in kidneys, with limited treatment options. Kidney macrophages are critical tissue sentinels that express IgG-binding Fcγ receptors (FcγRs), with previous studies identifying prenatally seeded resident macrophages as major IC responders. Using single-cell transcriptomic and spatial analyses in murine and human lupus nephritis, we sought to understand macrophage heterogeneity and subset-specific contributions in disease. In lupus nephritis, the cell fate trajectories of tissue-resident (TrMac) and monocyte-derived (MoMac) kidney macrophages were perturbed, with disease-associated transcriptional states indicating distinct pathogenic roles for TrMac and MoMac subsets. Lupus nephritis-associated MoMac subsets showed marked induction of FcγR response genes, avidly internalized circulating ICs, and presented IC-opsonized antigen. In contrast, lupus nephritis-associated TrMac subsets demonstrated limited IC uptake, but expressed monocyte chemoattractants, and their depletion attenuated monocyte recruitment to the kidney. TrMacs also produced B cell tissue niche factors, suggesting a role in supporting autoantibody-producing lymphoid aggregates. Extensive similarities were observed with human kidney macrophages, revealing cross-species transcriptional disruption in lupus nephritis. Overall, our study suggests a division of labor in the kidney macrophage response in lupus nephritis, with treatment implications - TrMacs orchestrate leukocyte recruitment while MoMacs take up and present IC antigen.

Genetically determined lymphopenia and autoimmune manifestations.

Hypomorphic defects of V(D)J recombination in humans lead to residual T cell development. In these lymphopenic conditions, homeostatic lymphocyte proliferation occurs, and key mechanisms that normally maintain host tolerance are altered, allowing peripheral expansion of oligoclonal and autoreactive T cells. Recently described murine models support this notion. This review describes human and murine situations, in which genetically determined T and B cell lymphopenia is associated with autoimmune manifestations.

Human Germinal Center Reaction: Assessing T Follicular Helper-B Cells Interaction In Vitro.

Antibody responses deeply rely on the interaction of antigen-primed B cells and CD4 helper T cells in the context of germinal center reactions, through signals provided by costimulatory molecules and cytokines. B-cell proliferation and differentiation in antibody-secreting plasma cells are processes that critically depend on the helper function of a specific CD4 T-cell subset, known as follicular helper T cells (Tfh). Here, we describe a method that mimics in vitro the cross talk between Tfh and B cells occurring in the germinal center. The procedure is based on setting up a coculture system with B cells and Tfh isolated from blood of healthy donors, or tonsils removed upon surgical intervention, in order to recapitulate in vitro the Tfh-dependent mechanisms leading to B cells' activation, proliferation, and differentiation.

A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome.

Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.

Brain lipid composition in grey-lethal mutant mouse characterized by severe malignant osteopetrosis.

The grey-lethal mouse (gl/gl) mutant most closely resembles the severe human malignant autosomal recessive OSTM1-dependent form of osteopetrosis that it has been described to be associated with neurological abnormalities. For this reason, we have analyzed the brain lipid composition (gangliosides, neutral glycosphingolipids, phospholipids and cholesterol), from gl/gl mice at different ages of development and compared with wild type mice. Both cholesterol and glycerophospholipid content and pattern in the gl/gl and control mice were very similar. In contrast, significant differences were observed in the content of several sphingolipids. Higher amount of the monosialogangliosides GM2 and GM3, and lower content of sphingomyelin, sulfatide and galactosylceramide were observed in the gl/gl brain with respect to controls. The low content of sphingomyelin, sulfatide and galactosylceramide is consistent with the immunohistochemical results showing that in the grey-lethal brain significant depletion and disorganization of the myelinated fibres is present, thus supporting the hypothesis that loss of function of the OSTM1 causes neuronal impairment and myelin deficit.

Mutations in OSTM1 (grey lethal) define a particularly severe form of autosomal recessive osteopetrosis with neural involvement.

UNLABELLED: We report three novel osteopetrosis patients with OSTM1 mutations and review two that have been previously described. Our analysis suggests that OSTM1 defines a new subset of patients with severe central nervous system involvement. This defect is also present in the gl mouse, which could represent a good model to study the role of the gene in the pathogenesis of this disease. INTRODUCTION: Autosomal recessive osteopetrosis (ARO) is a severe hereditary bone disease whose cellular basis is in the osteoclast, but with heterogeneous molecular defects. In addition to the TCIRG1 and the ClCN7 genes, whose mutations account for approximately 55% and 10% of cases, respectively, the OSTM1 gene has been described thus far in only two ARO patients. materials and methods: We report here three novel ARO patients presenting with severe primary central nervous system involvement in addition to the classical stigmata of severe bone sclerosis, growth failure, anemia, thrombocytopenia, and visual impairment with optic atrophy. In addition we analyzed the brain morphology and histology of the grey lethal mutant mouse. RESULTS: The analysis of the OSTM1 gene in two patients, both from Kuwait, showed homozygous two nucleotide deletion in exon 2, leading to a frameshift and premature termination. The third (Lebanese) patient showed a single point mutation in exon 1, leading to a nonsense mutation. The clinical neurological evaluation of the two Kuwaiti patients by CT and MRI scans showed a defect in the white matter, with a specific diagnosis of severe cerebral atrophy. The gl brain showed a diffuse translucent appearance with loss of the normal demarcation between the white and the grey matter, features consistent with myelin loss or hypomyelination. Histological and myelin staining analysis evidenced an atrophy of the corpus callosum with loss of myelin fibers, and in cortical areas, loss of the normal lamination consistent with multiple foci of cortical dysplasia. CONCLUSIONS: These findings suggest that OSTM1-dependent ARO defines a new subset of patients with severe central nervous system involvement leading to a very poor prognosis. The fact that central nervous system involvement is also present in the gl mouse mutant suggests that this mouse is a good model to test possible therapies.

RAG-dependent primary immunodeficiencies.

Mutations in recombination activating genes 1 and 2 (RAG1 and RAG2) cause a spectrum of severe immunodeficiencies ranging from classical T cell-B cell-severe combined immunodeficiency (T(-)B(-)SCID) and Omenn syndrome (OS) to an increasing number of peculiar cases. While it is well established from biochemical data that the specific genetic defect in either of the RAG genes is the first determinant of the clinical presentation, there is also increasing evidence that environmental factors play an important role and can lead to a different phenotypic expression of a given genotype. However, a better understanding of the mechanisms by which the molecular defect impinges on the cellular phenotype of OS is still lacking. Ongoing studies in knock-in mice could better clarify this aspect.

Tissue-specific sensitivity to AID expression in transgenic mouse models.

Activation-induced cytidine deaminase (AID), an enzyme with homology to members of the APOBEC family, is involved in somatic hypermutation (SHM) of immunoglobulin (Ig) genes, either by direct deamination of DNA or by an indirect action through its putative RNA editing activity. AID is able to mutate both Ig-like reporter constructs and selected non-Ig genes in normal B cells and in other cells when ectopically overexpressed in mammalian cells and transgenic mice. However, in spite of the fact that in these transgenic animals AID activity was driven by an ubiquitous promoter, only T lymphomas and lung adenomas occurred. In the present work, we constructed three sets of transgenic mice in which AID was under the control of lck, HTLV-I and MMTV promoters, respectively. The lck/AID mice developed thymic lymphomas with variable but high efficiency, while no tumor was detected in HTLV-I/AID mice after two years of monitoring. Four MMTV/AID founder mice died with an atypical clinical picture, although no mammary tumor was found. These findings suggest that additional factors, present in thymocytes but not in other tissues or in lymphoid cells at different stages of differentiation, are needed for AID to fully manifest its tumorigenic potential in mouse. Alternatively, the display of full AID mutagenic and transforming activity could be related to the existence of physiologic DSBs which occur in both thymocytes and switching B cells.

Abnormalities of thymic stroma may contribute to immune dysregulation in murine models of leaky severe combined immunodeficiency.

Lymphostromal cross-talk in the thymus is essential to allow generation of a diversified repertoire of T lymphocytes and to prevent autoimmunity by self-reactive T cells. Hypomorphic mutations in genes that control T cell development have been associated with immunodeficiency and immune dysregulation both in humans and in mice. We have studied T cell development and thymic stroma architecture and maturation in two mouse models of leaky severe combined immune deficiency, carrying hypomorphic mutations in rag1 and lig4 genes. Defective T cell development was associated with abnormalities of thymic architecture that predominantly affect the thymic medulla, with reduction of the pool of mature medullary thymic epithelial cells (mTECs). While the ability of mTECs to express autoimmune regulator (Aire) is preserved in mutant mice, the frequency of mature mTECs expressing Aire and tissue-specific antigens is severely reduced. Similarly, the ability of CD4(+) T cells to differentiate into Foxp3(+) natural regulatory T cells is preserved in rag1 and lig4 mutant mice, but their number is greatly reduced. These data indicate that hypomorphic defects in T cell development may cause defective lymphostromal cross-talk and impinge on thymic stromal cells maturation, and thus favor immune dysregulation.

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency.

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.

Damaging-agent sensitivity of Artemis-deficient cell lines.

Defects in repairing double-strand breaks can lead to genome instability and tumorigenesis. In humans, most T(-)B(-) severe combined immunodeficiencies (SCID) have a defect in either the RAG1 or RAG2 gene, are not radiosensitive and do not show genome instability. On the contrary, a minority of T(-)B(-) SCID patients have abnormalities in the Artemis gene and are moderately radiosensitive. Artemis-deficient cells are unable to process hairpin ends after RAG cleavage, but hairpin opening activity alone does not explain the moderate X-ray sensitivity of Artemis-deficient cells. We report here that, at variance with what has been described in mice, cell lines from Artemis(-/-) patients are moderately sensitive to mitomycin C and show only a low to moderate increase in genomic instability, both spontaneously and after exposure to ionizing radiations. There is some heterogeneity in the levels of DNA damage sensitivity and genome instability, which could in part be due to different effects of the specific mutation involved or to genetic background, which may not always represent null alleles. This data supports the hypothesis that, in addition to playing a role in hairpin opening during the V(D)J recombination process, Artemis is involved in the repair of a subset of DNA damage whose exact nature is still undefined.

Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.

Autosomal recessive osteopetrosis (ARO) is a paradigm for genetic diseases that cause severe, often irreversible, defects before birth. In ARO, osteoclasts cannot remove mineralized cartilage, bone marrow is severely reduced, and bone cannot be remodeled for growth. More than 50% of the patients show defects in the osteoclastic vacuolar-proton-pump subunit, ATP6a3. We treated ATP6a3-deficient mice by in utero heterologous hematopoietic stem cell (HSC) transplant from outbred GFP transgenic mice. Dramatic phenotype rescue by GFP osteoclasts was obtained with engraftment, which was observed in most cases. Engraftment survived for variable periods. Recipients were not immunosuppressed, and graft-versus-host disease was not observed in all pups born after in utero treatment. Thus, differentiation of unmatched HSC transplanted in utero is sufficient to prevent fatal defects in ARO and may prevent complications of ARO unresponsive to conventional bone marrow transplantation. The presence of defective cells is not a barrier to the rescue of the phenotype by donor HSC.