Francesca Rucci

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.

Mutations in OSTM1 (Grey Lethal) Define a Particularly Severe Form of Autosomal Recessive Osteopetrosis With Neural Involvement

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.

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.

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 Lig4R278H/R278H (Lig4R/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, Lig4R/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.

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.

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.

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.