X-linked hyper-IgM Syndrome Patients Research Articles

316. Successful Editing of the CD40LG Locus in Human Hematopoietic Stem Cells

X-Linked Hyper-IgM Syndrome (X-HIGM) is a genetic disorder caused by mutations in CD40LG that result in the loss of functional CD40L protein on the T cell surface. CD40L is required for T cells to provide “help” to B cells during an immune challenge; thus X-HIGM patients have impaired immunoglobulin class-switching and somatic hypermutation, and suffer recurrent infections. Murine gene transfer and X-HIGM patient studies suggest that safe and effective gene therapies for this disorder need to replicate cell surface expression patterns of WT endogenous CD40L, as well as knock-out expression of the mutant CD40L; requirements that are unlikely to be achievable using viral gene replacement. We recently reported a gene editing approach combining mRNA-delivered TALEN (targeting just upstream of the coding sequence of CD40LG) with an rAAV donor template for homology directed repair, targeting a promoter-less CD40L cDNA to the ATG start codon of the endogenous allele. This approach restored regulated cell surface expression of CD40L to X-HIGM T cells, disrupted expression of the mutant protein, and resulted in T cells that induced B cell class-switching in vitro, thus demonstrating it's potential as a T cell therapy for X-HIGM.Although the pathologies of X-HIGM are attributed mostly to the T cell defects, CD40L is expressed in most hematopoietic lineages. Besides providing a stable X-HIGM treatment, editing of autologous hematopoietic stem cells (HSC) would likely rescue regulated CD40L expression in all lineages. A selective advantage for gene corrected cells is not anticipated; however, patients with mixed donor chimerism post-transplantation have substantial improvements when as few as 10% of HSC have the WT allele. Here we report gene editing of CD40LG in adult human CD34+ PBSC at rates that are anticipated to provide such clinical benefit. Initial experiments using mRNA delivery of TALEN pairs targeting exon 1 of the CD40LG locus in human CD34+ cells demonstrated indel frequencies of >50%. To investigate the potential for HDR at the CD40LG locus in adult CD34+ cells, we combined delivery of the TALEN with an AAV6 donor template containing an MND promoter-GFP expression cassette flanked by 1 kb CD40LG homology arms. This donor template allowed us to track editing rates within the CD40LG locus (normally silent in HSCs) by flow cytometry. Using this co-delivery strategy, we consistently achieved editing rates of ~30% across multiple control human stem cell donors. Edited cells demonstrated minimal loss of viability, and expansion rates in culture equivalent to controls. Edited cells have been transplanted into immune deficient, NSG mice in order to track engraftment and differentiation potential. In parallel, we are currently evaluating editing rates using a more clinically relevant, promoter-less donor template encoding the CD40L cDNA. In summary, we have achieved clinically relevant rates of gene editing within the endogenous CD40LG locus in human HSC, setting the stage for additional work required to translate this approach into clinical application.

Targeted Gene Editing Restores Regulated CD40L Expression and Function in X-HIGM T cells.

Abstract Loss of CD40L expression or function results in X-Linked Hyper-IgM 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 TALEN-induced double-strand break and a donor template delivered by recombinant Adeno-Associated Virus (rAAV). HDR mediated insertion of a coding sequence (GFP 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 (3′-UTR) in the transgene preserved post-transcriptional 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-muIg binding, and rescued IgG class switching of naïve 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.

482. Editing of the CD40L Gene Restores Regulated CD40L Expression in X-HIGM Patient T Cells

X-linked hyper-IgM syndrome (X-HIGM) is a primary immunodeficiency syndrome characterized by insufficient CD40 Ligand (CD40L) expression, resulting in defective T-cell help, impaired immunoglobulin class-switching and recurrent opportunistic infections. Previously, constitutive CD40L expression in murine bone marrow cells and thymocytes was achieved by retroviral gene transfer and successfully corrected immune function. However, treated animals developed severe lymphoproliferative disease, likely due to constitutive CD40L surface expression. This demonstrated the importance of preserving endogenous gene regulation in this and other gene therapy endeavors. Here, we report efficient, on-target Homology Directed Repair (HDR) editing of the CD40L locus in primary human T cells using a combination of TALEN gene targeting and a donor template delivered transiently by recombinant Adeno-Associated Virus (rAAV). Our TALEN and donor template reagents were designed to insert a coding sequence (GFP or CD40L) upstream of the endogenous translation start site within Exon 1, thus allowing transgene expression to be regulated by the endogenous CD40L promoter, and included the CD40L 3’UTR to preserve known post-transcriptional regulation features. The kinetics of GFP and edited CD40L surface expression after PMA-Ionomycin stimulation paralleled that of endogenous CD40L in unedited T-cells. Additionally, activated T-cells expressing edited CD40L were able to bind CD40-Ig similar to unedited cells. When T-cells from X-HIGM patients were edited in this fashion, CD40L expression and CD40-Ig binding was restored, and repaired activated X-HIGM T-cells induced the expression of early activation markers and class switching of naive B-cells in vitro. Finally, we found no differences in TCR repertoire or engraftment and stability in NSG mice between T cells that underwent gene editing vs. unedited cells. These results demonstrate the feasibility of site-directed gene repair to restore normally regulated CD40L protein expression and functional T-cell help.

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.

Expanding the Clinical and Genetic Spectrum of Human CD40L Deficiency: The Occurrence of Paracoccidioidomycosis and Other Unusual Infections in Brazilian Patients

CD40 ligand (CD40L) deficiency or X-linked hyper-IgM syndrome (X-HIGM) is a well-described primary immunodeficiency in which Pneumocystis jiroveci pneumonia is a common clinical feature. We have identified an unusual high incidence of fungal infections and other not yet described infections in a cohort of 11 X-HIGM patients from nine unrelated Brazilian families. Among these, we describe the first case of paracoccidioidomycosis (PCM) in X-HIGM. The molecular genetic analysis of CD40L was performed by gene sequencing and evaluation of CD40L protein expression. Nine of these 11 patients (82%) had fungal infections. These included fungal species common to CD40L deficiency (P. jiroveci and Candida albicans) as well as Paracoccidioides brasiliensis. One patient presented with PCM at age 11 years and is now doing well at 18 years of age. Additionally, one patient presented with a simultaneous infection with Klebsiella and Acinetobacter, and one with condyloma caused by human papilloma virus. Molecular analysis revealed four previously described CD40L mutations, two novel missense mutations (c.433 T > G and c.476 G > C) resulting in the absence of CD40L protein expression by activated CD4(+) cells and one novel insertion (c.484_485insAA) within the TNFH domain leading to a frame shift and premature stop codon. These observations demonstrated that the susceptibility to fungal infections in X-HIGM extends beyond those typically associated with X-HIGM (P. jiroveci and C. albicans) and that these patients need to be monitored for those pathogens.

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

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. To determine whether monocyte-derived dendritic cells (DCs) from patients with X-HIGM exhibit normal responses to fungal pathogens. 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-β, IL-4, IL-5, and IL-17. 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, invitro incubation with soluble CD40L reversed the decreased IL-12 production and the skewed T(H)2 pattern response. 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.

Pitfalls of “hyper”-IgM syndrome: a new CD40 ligand mutation in the presence of low IgM levels. A case report and a critical review of the literature

Here, we report on a male infant with low serum IgG, IgA and IgM levels who suffered from Pneumocystis jirovecii and cytomegalovirus (CMV) pneumonia. The patient was tested to be HIV-negative. Absolute and relative numbers of lymphocyte subsets were normal, excluding the diagnosis of an X-linked agammaglobulinaemia (Bruton's disease). Despite the decreased serum IgM level, an X-linked hyper-IgM syndrome (X-HIGM) was considered. X-HIGM is a rare immunodeficiency usually characterised by recurrent severe opportunistic infections, low serum IgG and IgA, but normal or increased serum IgM. The syndrome is caused by mutations of the CD40 ligand (CD40L) gene. In our patient, CD40L mutation analysis proved a novel mutation at codon 257 associated with non-detectable expression of CD40L on the surface of activated T cells. A literature search revealed that approximately 6.4% of X-HIGM patients had been found to have low serum IgM levels. Our statistical analysis of the IgM levels as reported by different studies arouses suspicion that many patients with low IgM levels may not have undergone diagnostic procedures for X-HIGM. In summary, in this report and critical review of the literature, we described a new mutation of CD40L and highlighted the pitfalls of the diagnosis of X-HIGM.

Allogeneic hematopoietic stem cell transplantation for seven children with X-linked hyper-IgM syndrome: a single center experience.

X-linked hyper-IgM syndrome (XHIM), or hyper-IgM syndrome type 1 (HIGM1), is a rare primary immunodeficiency disorder susceptible to recurrent bacterial infection and opportunistic infection such as Pneumocystis carinii and Cryptosporidium parvum. The long-term outcome is quite poor, and allogeneic hematopoietic stem cell transplantation (HSCT) offers the only cure. Seven patients with XHIM, from age 3 to 19 years (mean 11.3 years), underwent allogeneic HSCT in our institution. Details of pre- and post-transplantation data and transplantation procedure were analyzed retrospectively. The donors were HLA-identical siblings for three patients and HLA-identical unrelated donors for four patients. All but one received conventional conditioning regimen consisting of busulfan and cyclophosphamide and prophylaxis for graft-versus-host disease (GVHD) consisting of cyclosporine and methotrexate. Five out of seven patients are alive and well with normal CD40L expression, and four of these five are free of intravenous immunoglobulin supplementation. The two patients who died had prolonged episodes of severe and recurrent infections and organ damage. We conclude that conventional allogeneic HSCT from HLA matched related or unrelated donors is curative and feasible for XHIM patients, if performed before significant infections and organ damage occur. For the high-risk patients, an alternative approach including nonmyeloablative HSCT may be more feasible.

Asymptomatic cryptosporidiosis with atypical cholangitis and eosinophilic hepatitis in X-linked hyper-IgM syndrome (XHIM)

Rationale XHIM is a rare form of immunodeficiency due to abnormally low expression of CD40 Ligand on CD4+ T-cells. XHIM patients present with variably diminished IgG, IgA, IgE and normal to high IgM levels. XHIM patients are predisposed to infections with fungi, encapsulated organisms and opportunistic organisms such as Cryptosporidium (Cry.). Cry. infection is usually associated with gastrointestinal symptoms but may also cause cholangitis in immune deficient patients. Methods 8 year old asymptomatic Hispanic male with XHIM and elevated liver enzymes was diagnosed with Cry. infection on fecal exam for oocytes and antigen. After failing metronidazole therapy, Cry. cleared from his stools after fourteen months of paromomycin therapy. Persistently elevated liver enzymes prompted an abdominal ultrasound (AUS), endoscopic retrograde cholangiopancreatography (ERCP) and liver biopsies. Results AUS showed mild dilatation throughout the biliary tract. ERCP revealed marked beading of the intrahepatic biliary tree suggesting cholangitis. Liver biopsy specimens obtained at three months and twelve months from the onset of elevated liver enzymes revealed eosinophilic inflammation with little fibrosis and no demonstrable organisms. Conclusions XHIM patients with Cry. infection and asymptomatic elevation of hepatic enzymes are at an increased risk for developing cholangitis and hepatic inflammation despite effective clearing of infection from the gastrointestinal tract. XHIM patients should be monitored closely for these complications.

Evidence for the requirement of T cell costimulation in the pathogenesis of natural Pneumocystis carinii pulmonary infection

Pneumocystis carinii pneumonia (PCP) is a frequent and serious opportunistic infection in immunocompromized patients. Although the pathogenesis of PCP-mediated lung injury is poorly understood, a central involvement of host inflammatory responses has been implicated. We have found that while the loss of specific T cell costimulatory signals increases susceptibility to the spontaneous pneumocystis infection, PCP-induced pulmonary injury (and subsequent morbidity and mortality) involves other intact costimulatory pathways. Mice that are genetically deficient for the costimulatory receptor CD154 (CD154 knockout (ko) mice) spontaneously developed PCP, consistent with the increased susceptibility of X-linked hyper IgM syndrome patients (caused by CD154 gene mutations) to P. carinii infection. In these mice PCP was manifested by progressive weight loss, dyspnea and death. In contrast, CD154 ko mice also genetically lacking ICAM1 (CD154 ko×ICAM1 ko) or CD28 (CD154 ko×CD28 ko) costimulatory receptors had later onset of weight loss and significantly prolonged survival. Although onset of infection and age-matched P. carinii organism burden were equivalent, the CD154 single knockout mice had evidence of greater pulmonary inflammation vs. the double ko's. These findings suggest that costimulation-dependent T cell-mediated inflammation plays an important role in both susceptibility to and pathogenesis of PCP, and may identify potential molecular targets for novel immunomodulatory treatment approaches.

The Role of CD40–CD40 Ligand (CD154) Interactions in Immunoglobulin Light Chain Repertoire Generation and Somatic Mutation

To determine whether CD40 ligation influences the molecular and selective mechanisms that govern the development of the human Ig light chain repertoire, analysis of the Vκ and Vλ repertoires of CD19+ B cells obtained from a patient with X-linked hyper IgM syndrome (XHIM) and a nonfunctional CD154 was carried out. The nonproductive Vκ and Vλ repertoires were largely comparable to that of the normals with respect to V gene and J segment distribution as well as CDR3 length and VLJL joint complexity. Comparison of the nonproductive and productive repertoires indicated that a limited number of VL genes were positively and negatively selected in the XHIM patient. Although mutations were observed in the XHIM VL repertoires, the frequency of mutations was significantly lower than in normals. Typical targeting of these mutations into RGYW/WRCY motifs was significantly reduced and subsequent selection of RGYW/WRCY mutations, which is normally observed, was not found. These results indicate that CD40 ligation is not required for generation of the light chain repertoire, positive selection of some Vk rearrangements, negative selection of specific VL genes, and some degree of somatic mutation. Importantly, however, targeting of mutations to RGYW/WRCY motifs and subsequent selection of these mutated motifs does not occur in the absence of CD40 ligation.

Coexpression of Normal and Mutated CD40 Ligand with Deletion of a Putative RNA Lariat Branchpoint Sequence in X-Linked Hyper-IgM Syndrome

We describe a novel CD40 ligand (CD40L) splicing mutation in a patient with X-linked hyper-IgM syndrome (X-HIM) associated with alternate splicing of exon 3, resulting in the expression of both full-length and exon-3-skipped CD40L mRNA. The mutation is an 8-bp deletion 25 bp upstream of the intron 2/exon 3 junction which overlaps a putative RNA branchpoint, suggesting that it may impair RNA lariat formation. The exon-3-skipped CD40L transcript encodes a truncated protein (CD40LDeltaE3) encompassing the cytoplasmic, transmembrane, and extracellular stalk domains, but lacking the CD40L receptor binding domain. CD40LDeltaE3 protein expression was readily detectable in transfected Cos cells by immunofluorescence. In cells cotransfected with CD40LDeltaE3 and wild-type CD40L, expression of CD40LDeltaE3 did not inhibit the expression of wild-type CD40L monomers, but strongly inhibited staining by the conformationally sensitive anti-CD40L mAb 5c8, suggesting that CD40LDeltaE3 acts in a dominant negative manner to inhibit the assembly of functional CD40L trimers. This mechanism may contribute to the pathophysiology of CD40L deficiency in X-HIM patients with leaky splice site mutations.

The influence of CD40-CD154 interactions on the expressed human V(H) repertoire: analysis of V(H) genes expressed by individual B cells of a patient with X-linked hyper-IgM syndrome.

Analysis of the V(H)DJ(H) repertoire of peripheral blood IgM(+) B cells from a patient with X-linked hyper-IgM syndrome (X-HIgM) was undertaken to determine whether the distribution of V(H) families in the productive repertoire might be regulated by in vivo CD40-CD154 interactions. The distribution of V(H) genes in the non-productive repertoire of IgM(+) B cells was comparable in X-HIgM and normals. Unlike the normal productive V(H) repertoire, however, in the X-HIgM patient the V(H)4 family was found at almost the same frequency as the V(H)3 family. This reflected a diminution in the positive selection of the V(H)3 family observed in normals and the imposition of positive selection of the V(H)4 family in the X-HIgM patient. Unique among the V(H)3 genes, V(H)3-23/DP-47 was positively selected in both normals and the X-HIgM patient. No major differences in the usage of J(H) or D segments or the complementarity-determining region (CDR) 3 were noted, although the foreshortening of the CDR3 noted in the mutated V(H) rearrangements of normals was absent in the X-HIgM patient. Finally, a minor degree of somatic hypermutation was noted in the X-HIgM patient. These results have suggested that specific influences on the composition of the V(H) repertoire in normals require CD40-CD154 interactions.

T cells can induce somatic mutation in B cell receptor-engaged BL2 Burkitt's lymphoma cells independently of CD40-CD40 ligand interactions.

The B cell surface trigger(s) and the molecular mechanism(s) of somatic hypermutation remain unknown, partly because of the lack of amendable in vitro models. Recently, however, we reported that upon B cell receptor cross-linking and coculture with activated T cells, the Burkitt's lymphoma cell line BL2 introduces mutations in its IgVH gene in vitro. We now confirm the relevance of our culture model by establishing that the entire spectrum of somatic mutations observed in vivo, including insertions and deletions, could be found in the DNA of BL2 cells. Additionally, we show that among four human B cell lines, only two with a centroblast-like phenotype can be induced to mutate. Triggering of somatic mutations in BL2 cells requires intimate T-B cell contacts and is independent of CD40-CD40-ligand (CD40L) interactions as shown by 1) the lack of effect of anti-CD40 and/or anti-CD40L blocking Abs on somatic mutation and 2) the ability of a CD40L-deficient T cell clone (isolated from an X-linked hyper-IgM syndrome patient) to induce somatic mutation in B cell receptor-engaged BL2 cells. Thus, our in vitro model reveals that T-B cell membrane interactions through surface molecules different from CD40-CD40L can trigger somatic hypermutation.

CD40 Ligand Mutants Responsible for X-linked Hyper-IgM Syndrome Associate with Wild Type CD40 Ligand

CD40 ligand (CD40L) is a 33-kDa type II membrane glycoprotein mainly expressed on activated CD4(+) T cells in trimeric form. When it is mutated, the clinical consequences are X-linked hyper-IgM syndrome (XHIM), a primary immunodeficiency disorder characterized by low levels of IgG, IgA, and elevated or normal levels of IgM. Mutated CD40L can no longer bind CD40 nor provide signals for B cells to proliferate and to switch from IgM to other immunoglobulin isotypes. When considering gene therapy for XHIM, it is important to address the possibility that the mutated CD40L associates with transduced wild type CD40L, and as a consequence, immune reconstitution is not attained. In this study, we demonstrate that the various mutated CD40L species we have identified in patients with XHIM, including both full-length and truncated mutants, associate with wild type CD40L on the cell surface of co-transfected COS cells. The association between wild type and mutated CD40L was also observed in CD4(+) T cell lines established from XHIM patients with leaky splice site mutations. The clinical phenotype of these patients suggests that this association between wild type and mutated CD40L species may result in less efficient cross-linking of CD40.

Mutations of the CD40 Ligand Gene and Its Effect on CD40 Ligand Expression in Patients With X-Linked Hyper IgM Syndrome

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus.