Silvia Giliani

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Partial V(D)J Recombination Activity Leads to Omenn Syndrome

Genomic rearrangement of the antigen receptor loci is initiated by the two lymphoid-specific proteins Rag-1 and Rag-2. Null mutations in either of the two proteins abrogate initiation of V(D)J recombination and cause severe combined immunodeficiency with complete absence of mature B and T lymphocytes. We report here that patients with Omenn syndrome, a severe immunodeficiency characterized by the presence of activated, anergic, oligoclonal T cells, hypereosinophilia, and high IgE levels, bear missense mutations in either the Rag-1 or Rag-2 genes that result in partial activity of the two proteins. Two of the amino acid substitutions map within the Rag-1 homeodomain and decrease DNA binding activity, while three others lower the efficiency of Rag-1/Rag-2 interaction. These findings provide evidence to indicate that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease the efficiency of V(D)J recombination.

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4+ T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDX1)

BACKGROUND X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Interleukin‐7 receptor α (IL‐7Rα) deficiency: cellular and molecular bases. Analysis of clinical, immunological, and molecular features in 16 novel patients

Summary:  Analysis of gene‐targeted mice and patients with severe combined immunodeficiency due to mutations of the α chain of the interleukin‐7 receptor (IL‐7Rα) has shown important differences between mice and humans in the role played by IL‐7 in lymphoid development. More recently, it has been shown that IL‐7Rα is also shared by the receptor for another cytokine, thymic stromal lymphopoietin (TSLP). In this review, we discuss recent advances in IL‐7‐ and TSLP‐mediated signaling. We also report on the clinical and immunological features of 16 novel patients with IL‐7Rα deficiency and discuss the results of hematopoietic stem cell transplantation.

Long-term outcome and lineage-specific chimerism in 194 patients with Wiskott-Aldrich syndrome treated by hematopoietic cell transplantation in the period 1980-2009: an international collaborative study.

In this retrospective collaborative study, we have analyzed long-term outcome and donor cell engraftment in 194 patients with Wiskott-Aldrich syndrome (WAS) who have been treated by hematopoietic cell transplantation (HCT) in the period 1980- 2009. Overall survival was 84.0% and was even higher (89.1% 5-year survival) for those who received HCT since the year 2000, reflecting recent improvement of outcome after transplantation from mismatched family donors and for patients who received HCT from an unrelated donor at older than 5 years. Patients who went to transplantation in better clinical conditions had a lower rate of post-HCT complications. Retrospective analysis of lineage-specific donor cell engraftment showed that stable full donor chimerism was attained by 72.3% of the patients who survived for at least 1 year after HCT. Mixed chimerism was associated with an increased risk of incomplete reconstitution of lymphocyte count and post-HCT autoimmunity, and myeloid donor cell chimerism < 50% was associated with persistent thrombocytopenia. These observations indicate continuous improvement of outcome after HCT for WAS and may have important implications for the development of novel protocols aiming to obtain full correction of the disease and reduce post-HCT complications.

Severe cutaneous papillomavirus disease after haemopoietic stem-cell transplantation in patients with severe combined immune deficiency caused by common γc cytokine receptor subunit or JAK-3 deficiency

Haemopoietic stem-cell transplantation is a life-saving treatment for severe combined immune deficiency. However, there has been little long-term follow-up of this treatment. There is evidence for the persistance of partial immunodeficiency associated with significant infections, including severe human papillomavirus (HPV) disease. We did a retrospective analysis of severe HPV disease in a group of 41 patients with severe combined immune deficiency from one centre who were alive 10 years or longer after haemopoietic stem-cell transplantation. Nine of the 41 patients had extensive chronic HPV disease limited to the skin, with a median onset at 8 years after transplantation. Four had lesions typical of epidermodysplasia verruciformis, a rare genodermatosis. Transplant characteristics, immune status, and chimerism of these nine patients did not differ significantly from those of the other patients. The nine patients with HPV disease had severe combined immune deficiency associated with either common gammac receptor cytokine subunit or Janus kinase-3 (JAK-3) deficiency. By contrast, patients with other forms of severe combined immune deficiency did not have any signs of HPV disease. That genetic causes are the only predisposing factor to be identified for severe combined immune deficiency, suggests that natural-killer cells or gammac/JAK-3-dependent signalling in keratinocytes could have a role in anti-HPV immunity.

Defective Expression of CD40 Ligand on T Cells Causes “X‐Linked Immunodeficiency with Hyper‐IgM (HIGM1)”

X-linked immunodeficiency with hyper-IgM (HIGM1) is a rare disorder, characterized by recurrent infections associated with very low or absent IgG and IgA, and normal to increased IgM serum levels. The disease has been earlier mapped to the q26-27 region of the X-chromosome. We have identified a novel molecule expressed on the surface of activated T cells, which was designated TRAP (Tumor necrosis factor Related Activation Protein), and could demonstrate that TRAP is a ligand for the CD40 receptor expressed on B cells. Our mapping of the TRAP gene to the Xq26.3-27.1 region suggested a causal relationship to HIGM1. Further work revealed that various mutations of the TRAP/CD40 ligand (CD40L) gene may lead to a defective expression of the TRAP/CD40L molecule on the T-cell surface in HIGM1 patients. A combination of structural and functional analyses finally demonstrated that the failure of TRAP/CD40L on T cells to interact with CD40 on B cells is responsible for the inefficient T-cell help for B cells observed in HIGM1. The observations made in HIGM1 allowed us to conclude that TRAP/CD40L is not required for IgM synthesis. In contrast, functional expression of TRAP is a prerequisite for effective immunoglobulin isotype switching and subsequent production of IgG, IgA and IgE by B cells in vivo. The interaction of TRAP/CD40L with CD40 thus provides a very critical link between the cellular and the humoral part of the immune system. The knowledge of TRAP/CD40L cDNA sequence, the availability of various reagents for the testing of expression and function of TRAP/CD40L, and our recent elucidation of the exon-intron structure of the TRAP/CD40L gene now provide all necessary tools for early diagnosis of affected patients and the detection of female carriers of HIGM1. The available information will also provide a basis for future attempts at gene therapy in this disease.

IL-21 is the primary common γ chain-binding cytokine required for human B-cell differentiation in vivo

SCID resulting from mutations in IL2RG or JAK3 is characterized by lack of T and natural killer cells; B cells are present in normal number, but antibody responses are defective. Hematopoietic cell transplantation (HCT) is curative for SCID. However, B-cell dysfunction persists in a substantial proportion of patients. We hypothesized that impaired B-cell responses after HCT in IL2RG/JAK3 deficiency results from poor donor B-cell engraftment and defective γc-dependent cytokine signaling in host B cells. To test this, and to identify which γc cytokine(s) is critical for humoral immunity, we studied 28 transplanted patients with IL2RG/JAK3 deficiency. Lack of donor B-cell engraftment associated with persistent humoral dysfunction and significantly reduced memory B cells. B-cell proliferation induced by CD40L alone or together with CpG, anti-Ig, IL-4, IL-10, or IL-13 was comparable in healthy controls and in post-HCT SCID patients, irrespective of their chimerism status. However, in vitro stimulation with CD40L/IL-21 induced B-cell proliferation, plasmablast differentiation, and antibody secretion in patients with donor B cells, but not in patients with autologous B cells. These data imply that IL-21-mediated signaling is critical for long-lived humoral immunity and to restore antibody responses in IL2RG/JAK3-deficient patients after HCT. Furthermore, in vitro stimulation with CD40L/IL-21 can predict in vivo B-cell immunity in IL2RG/JAK3 SCID after transplantation.

A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.

A homozygous mutation that gave rise to a stop codon in the WIPF1 gene resulted in WASP protein destabilization and in symptoms resembling those of Wiskott-Aldrich syndrome