CD40 Ligand Deficiency.

CD40 ligand deficiency (CD40L), currently classified as an inborn error of immunity affecting cellular and humoral immunity, prevalently emerges in boys within the first two years of life. It manifests itself as a decrease in serum IgG, IgA and IgE, with normal or high IgM, defects in T cell proliferation, and decrease in soluble CD40L. These accompany sinopulmonary and/or gastrointestinal infections, and there may be infections caused by pyogenic bacteria, opportunistic infections, autoimmune diseases, and neoplasms. Mild and moderate cases of this deficiency may respond well to prophylactic antibiotic therapy or to human immunoglobulin replacement therapy, in addition to the early treatment of infections. Severe cases can be treated with hematopoietic stem cell transplantation, which allows the healing of such patients, rather than sequelae and a poor progression. Thus, its differential diagnosis with other inborn errors of immunity is essential, especially CD40 deficiency and variable common immunodeficiency; the reason why we have proposed the present literature review.

CD40 ligand deficiency: treatment strategies and novel therapeutic perspectives.

INTRODUCTION: CD40 ligand (CD40L) deficiency or X-linked Hyper-IgM syndrome is a severe primary immunodeficiency caused by mutations in the CD40L gene. Despite currently available treatments, CD40L-deficient patients remain susceptible to life-threatening infections and have poor long term survival. Areas covered: Here, we discuss clinical and immunological characteristics of CD40L deficiency as well as current therapeutic strategies used for patient management. This review highlights that beyond B cell defects, patients' susceptibility to opportunistic pathogens might be due to impaired T cell and innate immune responses. In this context, we discuss how better knowledge of CD40L function and regulation may result in the development of new treatments. Expert opinion: Despite the introduction of hematopoietic stem-cell transplantation, immunoglobulin replacement, granulocyte colony-stimulating factor (G-CSF) administration, and prophylactic antibiotic therapies, life-threatening infections still cause high morbidity and mortality among CD40L-deficient patients. The reasons for this inadequate response to current therapies remains poorly understood, but recent reports suggest the involvement of CD40L-CD40 interaction in early stages of the innate immune system ontogeny. The development of novel gene therapeutic approaches and the use of redirected immunotherapies represent alternative treatment methods that could offer reduced morbidity and mortality rates for patients with CD40L deficiency.

CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-γ.

BACKGROUND: Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. OBJECTIVES: We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. METHODS: We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. RESULTS: Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. CONCLUSION: Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

Successful Sequential Liver and Hematopoietic Stem Cell Transplantation in a Child With CD40 Ligand Deficiency and Cryptosporidium-Induced Liver Cirrhosis.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is curative in patients with primary immunodeficiencies. However, pre-HSCT conditioning entails unacceptably high risks if the liver is compromised. The presence of a recurrent opportunistic infection affecting the biliary tree and determining liver cirrhosis with portal hypertension posed particular decisional difficulties in a 7-year-old child with X-linked CD40-ligand deficiency. We aim at adding to the scanty experience available on such rare cases, as successful management with sequential liver transplantation (LT) and HSCT has been reported in detail only in 1 young adult to date. METHODS: A closely sequential strategy, with a surgical complication-free LT, followed by reduced-intensity conditioning, allowed HSCT to be performed only one month after LT, preventing Cryptosporidium parvum recolonization of the liver graft. RESULTS: Combined sequential LT and HSCT resolved the cirrhotic evolution and corrected the immunodeficiency so that the infection responsible for the progressive sclerosing cholangitis did not recur. CONCLUSIONS: Hopefully, this report of the successful resolution of a potentially fatal combination of immunodeficiency and chronic opportunistic infection with end-stage organ damage in a child will encourage others to adapt a sequential transplant approach to this highly complex pathology. However, caution is to be exercised to carefully balance the risks intrinsic to transplant surgery and immunosuppression in primary immunodeficiencies.

X-linked Hyper IgM Syndrome Presenting as Pulmonary Alveolar Proteinosis.

PURPOSE: X-linked hyper IgM syndrome (XHIGM) is a combined immunodeficiency caused by mutations in the CD40 ligand (CD40L) gene that typically results in decreased or absent CD40L expression on activated T cells, leading to defective class switching and somatic hypermutation. We describe an infant who presented with respiratory failure due to pulmonary alveolar proteinosis (PAP) with a novel damaging missense mutation in the CD40L gene. METHODS: Whole exome sequencing (WES) was used to identify a mutation in the CD40L gene. CD40L expression and function were determined by flow cytometry. RESULTS: A 5-month-old previously-healthy male presented with respiratory failure and diffuse pulmonary ground glass opacities on CT scan of the chest. Laboratory evaluation revealed an undetectable IgG, normal IgA, and elevated IgM. A bronchoalveolar lavage demonstrated pulmonary alveolar proteinosis. WES demonstrated a c.608G > C mutation in the CD40L gene resulting in p.R203T. Flow cytometry demonstrated normal CD40L expression on activated T cells but absent binding of CD40-Ig to CD40L on activated patient T cells. CONCLUSIONS: The clinical manifestations of XHIGM in our patient had several unique features, including the presentation with PAP, normal serum IgA, and expression of non-functional CD40L on activated T cells. To our knowledge, this is the first published case of PAP in a patient with XHIGM.

Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome.

Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.

Hematopoietic Stem Cell Transplantation for CD40 Ligand Deficiency: Single Institution Experience.

BACKGROUND: X-linked hyper-IgM syndrome (X-HIGM) due to mutations in the gene encoding CD40 ligand results in failure of Ig class switching and an increased propensity for recurrent sinopulmonary and other infections, and thus decreased life expectancy. Allogeneic hematopoietic stem cell transplantation (HSCT) is curative, but long-term follow-up data are limited. PROCEDURES: We conducted a retrospective analysis of seven patients who have undergone allogeneic HSCT for HIGM syndrome at Duke University Medical Center. RESULTS: Median age at transplant was 5.2 years (range 0.7-19.3). None of the patients had active hepatic or pulmonary disease immediately prior to transplant, but all had a history of serious infections. Five patients received myeloablative conditioning, and two patients received reduced intensity conditioning. Graft sources included bone marrow, peripheral blood, and unrelated umbilical cord blood. Post-transplantation complications included veno-occlusive disease, hemorrhagic cystitis, adenoviremia, and cryptosporidium recurrence in one patient each. Two patients developed acute GVHD grades II-IV that resolved promptly with treatment and none developed extensive chronic GVHD. All patients are intravenous IgG-independent and 6/7 have normal antibody titers. Immunoglobulin (Ig) A levels normalized in all but one patient and T and B cell numbers and function are otherwise normal in all. All patients are alive at a median follow-up of 9.7 (range 9.7-16.1) years post-transplantation with predominantly donor chimerism and no recurrent infections. CONCLUSIONS: Allogeneic HSCT results in excellent survival and sustained immune reconstitution in patients with CD40 ligand deficiency using both myeloablative and reduced intensity conditioning approaches and various graft sources, including bone marrow, peripheral blood, and umbilical cord blood.

CD40 Ligand Deficient C57BL/6 Mouse Is a Potential Surrogate Model of Human X-Linked Hyper IgM (X-HIGM) Syndrome for Characterizing Immune Responses against Pathogens.

Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT) and C57-CD40L deficient (C57-CD40L(-/-)) mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L(-/-) mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L(-/-) animals, orally inoculated with 2 × 10(9) CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L(-/-) mice infected with 1 × 10(7) CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1 × 10(7) CFU), collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L(-/-) animals had lower IgG and IgG2b titres than WT mice, C57-CD40L(-/-) mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L(-/-) mice are capable of producing antibodies that are protective. C57-CD40L(-/-) mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens.

A novel mutation in the CD40 ligand gene in a Chinese boy with X-linked hyper-IgM syndrome.

X-linked hyper-IgM Syndrome (XHIGM) is caused by a mutation of CD40 ligand (CD40L), which is normally expressed on activated CD4+ T cells and is responsible for immunoglobulin class switching. A 7-year-old boy with recurrent sino-pulmonary infections since the age of 3 months had normal CD3+, CD4+, CD8+T lymphocytes, and CD19+B lymphocytes and NK cells, but significantly elevated IgM and extremely decreased IgG and IgA. Sequencing of genomic DNA revealed that the patient had a 34 base deletion in intron 3 and exon 4 of CD40L(g.8172_8205del34bp), which lead to the entire deletion of exon 4 in cDNA (c.347_409del63bp, i.e.,exon 4 skipping) and an in-frame deletion of 21 amino acids in CD40L protein. Moreover, the patient had negligible CD40L expression on activated CD3+CD8-T lymphocytes. His mother and sister were carriers of the CD40L mutation. Our studies demonstrated a novel mutation in CD40L, which, to our knowledge, has not been reported previously.

Immunoglobulin class switch recombination deficiency type 1 or CD40 ligand deficiency: from bedside to bench and back again.

The immunoglobulin class switch recombination deficiency or hyper-IgM syndrome is characterized by normal or elevated serum IgM and low serum levels of other immunoglobulins. Since the first reported patient with hyper-IgM, more than 200 patients with this phenotype resulted from CD40 ligand deficiency have been reported. However, in addition to this common finding, they presented with different manifestations like opportunistic infections, autoimmunity and malignancies each of them are worth a detailed look. In this review, we will focus on different underlying mechanisms of these presentations to review what we have learned from our patients. In the end, we will discuss different treatment options available for these patients using this knowledge.

The polymorphism p.G219R of CD40L does not cause immunological alterations in vivo: conclusions from a X-linked hyper IgM syndrome kindred.

Hyper-IgM syndromes (HIGM) are characterized by low levels of IgG, IgA and IgE and normal to high levels of IgM. Patients with these syndromes present recurrent infections due to an impaired immunoglobulin maturation. The most prevalent form of HIGM, X-linked hyper IgM syndrome (XHIM), is caused by mutations in the gene encoding the CD40 ligand (CD40LG). We present two siblings with XHIM caused by a large CD40LG deletion affecting more than half of the gene, and extended from the end of intron 3 to far upstream of the promoter regions. Genetic analysis in the maternal family discovered the CD40L(G219R) polymorphism in several members. Segregation of this polymorphism in the kindred indicated that the deletion of CD40LG was a de novo mutation in the mother. Although half of her CD4+ T cells did not express CD40L and the other half expressed the CD40L(G219R) variant, the mother was healthy. This suggests that this polymorphism is not pathogenic by itself although it has been recently related to X-linked lymphoproliferative syndrome.

CD40 agonist antibody mediated improvement of chronic Cryptosporidium infection in patients with X-linked hyper IgM syndrome.

X-linked hyper-IgM syndrome (XHM) is a combined immune deficiency disorder caused by mutations in CD40 ligand. We tested CP-870,893, a human CD40 agonist monoclonal antibody, in the treatment of two XHM patients with biliary Cryptosporidiosis. CP-870,893 activated B cells and APCs in vitro, restoring class switch recombination in XHM B cells and inducing cytokine secretion by monocytes. CP-870,893 infusions were well tolerated and showed significant activity in vivo, decreasing leukocyte concentration in peripheral blood. Although specific antibody responses were lacking, frequent dosing in one subject primed T cells to secrete IFN-g and suppressed oocyst shedding in the stool. Nevertheless, relapse occurred after discontinuation of therapy. The CD40 receptor was rapidly internalized following binding with CP-870,893, potentially explaining the limited capacity of CP-870,893 to mediate immune reconstitution. This study demonstrates that CP-870,893 suppressed oocysts shedding in XHM patients with biliary cryptosporidiosis. The continued study of CD40 agonists in XHM is warranted.

Molecular Diagnostic Challenges and Complex Management of Consecutive Twin Pregnancies in a Family with CD40 Ligand Deficiency.

X-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency disorder (PID) caused by mutation in the gene encoding the CD40 ligand (CD40L) expressed on activated T cells. Prenatal genotyping in carriers with twin pregnancies is more challenging than in women with singleton pregnancies. In addition, women with twin pregnancies may decide on selective termination for which the risk of loss of the healthy foetus may exceed 7%. We report here on a family affected by XHIGM. Diagnosis of the disease was made in a male patient as late as 33 years of age. After family screening, the sister of the proband conceived male twins in two consecutive pregnancies. In the first pregnancy, one of the male foetuses was hemizygous for the c.521A>G (Q174R) mutation in the CD40L gene. In the second pregnancy, ultrasound scan showed one foetus to have exencephaly and karyotyping revealed this foetus to have trisomy 18. Several options were discussed, but the parents decided on selective termination in both pregnancies. The interventions were successful in both cases, and the mother now has two healthy sons. This report demonstrates the way in which advanced technologies in molecular medicine and obstetric interventions may assist families with decisions about possible selective termination in case of life-threatening molecular or chromosomal disorders. Diagnosis of CD40L deficiency at the age of 33 years in the proband was striking and indicated that PIDs are still neglected as disease entities in the evaluation of patients with recurrent severe infectious diseases.

Dendritic cells from X-linked hyper-IgM patients present impaired responses to Candida albicans and Paracoccidioides brasiliensis.

BACKGROUND: Patients with X-linked hyper-IgM syndrome (X-HIGM) due to CD40 ligand (CD40L) mutations are susceptible to fungal pathogens; however, the underlying susceptibility mechanisms remain poorly understood. OBJECTIVE: To determine whether monocyte-derived dendritic cells (DCs) from patients with X-HIGM exhibit normal responses to fungal pathogens. METHODS: DCs from patients and controls were evaluated for the expression of costimulatory (CD80 and CD86) and MHC class II molecules and for their ability to produce IL-12 and IL-10 in response to Candida albicans and Paracoccidioides brasiliensis. We also evaluated the ability of C albicans- and P brasiliensis-pulsed mature DCs to induce autologous T-cell proliferation, generation of T helper (T(H)) 17 cells, and production of IFN-γ, TGF-ß, IL-4, IL-5, and IL-17. RESULTS: Immature DCs from patients with X-HIGM showed reduced expression of CD80, CD86, and HLA-DR, which could be reversed by exogenous trimeric soluble CD40L. Most important, mature DCs from patients with X-HIGM differentiated by coculturing DCs with fungi secreted minimal amounts of IL-12 but substantial amounts of IL-10 compared with mature DCs from normal individuals. Coculture of mature DCs from X-HIGM patients with autologous T cells led to low IFN-γ production, whereas IL-4 and IL-5 production was increased. T-cell proliferation and IL-17 secretion were normal. Finally, in vitro incubation with soluble CD40L reversed the decreased IL-12 production and the skewed T(H)2 pattern response. CONCLUSION: Absence of CD40L during monocyte/DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with X-HIGM.

CD40 ligand deficiency with grade III liver fibrosis, transplanted by a treosulphan-based conditioning regimen.

X-linked Hyper IgM is characterized by an absence of the CD40 ligand on activated T lymphocytes resulting in defects of both cellular and humoral immunity. Patients usually present with recurrent bacterial and opportunistic infections. Chronic liver disease is seen in about 75% of patients as a complication. Here, we report a 3.5-year-old boy with X-linked Hyper IgM referred to our clinic for bone marrow transplant. He was transplanted from an HLA-identical sibling donor using a new conditioning agent, treosulphan, together with cyclophosphamide. Since 6 months of age, he has had recurrent respiratory infections, and his XHIGM was diagnosed when he was 1.5 years old. The diagnosis was confirmed by sequence analysis of the CD40L gene. On physical examination, growth failure, bilateral fine crackles in both lungs, and hepatosplenomegaly were detected. The results of his liver function tests were abnormal, and a liver biopsy showed grade III fibrosis and compensated cirrhosis. After conditioning with treosulphan (12 g/m(2)/d x 3 d) and cyclophosphamide (50 mg/kg/d x 4 d), bone marrow from his HLA-identical sister was infused. CD40L expression on activated lymphocytes of the patient was 84% on day +21. His posttransplant period was uneventful. He is now at posttransplant 2 years, with full donor chimerism, and mild, chronic, graft-versus-host disease on his tongue. In conclusion, treosulphan is a new agent for conditioning regimen with less toxicity in patients with severe liver disease.

Partial immune reconstitution of X-linked hyper IgM syndrome with recombinant CD40 ligand.

X-linked hyper IgM syndrome (XHM) is a combined immune deficiency disorder caused by genetic alterations in CD40 ligand. The purpose of this study was to investigate the safety and efficacy of recombinant CD40 ligand (rCD40L) in the treatment of the disease. Three children were administered rCD40L subcutaneously 3 times per week at 0.03 mg/kg for 22 weeks, and after a 12-week drug-free interval, the dose was increased to 0.05 mg/kg for an additional 22 weeks of treatment. Although specific antibody responses to T cell-dependent antigens was lacking, administration of rCD40 resulted in acquisition of the capacity to mount cutaneous delayed type hypersensitivity reactions that disappeared during the drug-free interval as well as the postbiologic follow-up period. With rCD40L treatment, patient T cells developed a new capacity to respond to T-cell mitogens with synthesis of IFN-γ and TNF-α. Intracellular cytokine staining studies showed that both CD4(+) and CD8(+) T cells participated in this response. Finally, CD40L therapy was associated with changes in lymph node size and architecture based on comparison of biopsies taken before and after therapy. This clinical study showed that rCD40L is capable of improving T cell-immune function in patients with XHM.

Flow cytometry assays in primary immunodeficiency diseases.

The primary immunodeficiency diseases (PIDs) encompass an extremely large and diverse number of clinical disorders caused by mutations in genes that affect virtually every measurable component of our immune systems. Many of the genetic mutations lead to abnormalities that can be detected in circulating peripheral blood cells of suspected patients by flow cytometry and the appropriate combinations of reagents and in vitro manipulations. The flow cytometry procedures that have been developed to detect abnormalities in peripheral blood cells of primary immunodeficiency patients can barely be covered in an entire book, let alone one chapter. Instead of attempting to cover each disease with a specific assay or test, we review three procedures each covering a global aspect of the observed immune abnormality, i.e., detection of lymphocyte subset abnormalities, lymphocyte "marker" abnormalities, and leukocyte function abnormalities.