Francesco Frugoni

B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.

First reported case of Omenn syndrome in a patient with reticular dysgenesis

To the Editor: Reticular dysgenesis (RD) is a rare form of autosomalrecessive severe combined immunodeficiency characterized by lack of circulating T lymphocytes, severe congenital neutropenia, and sensorineural deafness.1,2 The disease is caused by mutations in the gene encoding adenylate kinase 2 (AK2), a mitochondrial protein important for regulating intracellular levels of adenosine diphosphate and maintaining mitochondrial membrane potential. A similar function is also mediated by the cytoplasmic enzyme AK1. While most tissues express both AK1 and AK2 enzymes, neutrophils, T lymphocytes, and cells of the stria vascularis in the inner ear uniquely express AK2, thus explaining the RD phenotype.3,4 Omenn syndrome (OS) has been described in association with several genetic defects responsible for severe combined immunodeficiency and results from residual development of oligoclonal T lymphocytes that undergo peripheral expansion and infiltrate various tissues including the skin, gut, liver, and lymphoid organs.5 We report the first case of OS in a patient with RD. A male infant of Kuwaiti origin was born to consanguineous parents who had a previously affected daughter with RD due to a homozygous missense AK2 mutation (c.524 G>A, p.R175Q). The sister’s first hematopoietic stem cell transplant from cord blood with reduced intensity conditioning failed, but she later had a successful matched related transplant with conditioning consisting of busulfan and cyclophosphamide.6 At birth, the infant was vigorous with an otherwise unremarkable physical examination; however, laboratory studies demonstrated leukopenia, absence of neutrophils, and undetectable IgA and IgM levels (Table I), and a chest radiograph lacked a thymic shadow. Immunologic evaluation at our institution at 6 weeks of age revealed undetectable T-cell receptor excision circles with detectable RNAse P, near-absence of natural killer cells, and B-cell lymphopenia. The T-cell count was slightly reduced, composed entirely of CD45RA-cells. T-lymphocyte proliferation to mitogens was decreased (Table I). Auditory brain stem response-evoked potential testing documented profound hearing loss. A diagnosis of RD was presumed, and mutation analysis by using genomic DNA derived from fibroblasts confirmed homozygosity for the same AK2 mutation as the infant’s sister. The patient was started on prophylactic antibiotics and antifungal medications along with intravenous gammaglobulin. At 8 weeks, the infant developed desquamative erythroderma, pachydermia (see Fig E1 in this article’s Online Repository at www.jacionline.org), diarrhea, and generalized lymphadenopathy. The CD3+ T-cell count increased to 10,032 cells/μL (Table I). Treatment with methylprednisolone 1 mg/kg given twice daily and cyclosporine was started; improvement was noted clinically, with laboratory studies documenting decreased circulating T lymphocytes (Table I). The infant underwent a 9/10 HLA-A mismatched unrelated donor bone marrow transplant at 3 months of age, with conditioning consisting of 4 days of busulfan given every 6 hours with area under the curve dosing targeted to 800 to 1200 μmol*min/L. cyclophosphamide 50 mg/kg daily for 4 days, and equine antithymocyte globulin 30 mg/kg daily for 3 days. Cyclosporine and 1 mg/kg per day of methylprednisolone were continued for graft versus host prophylaxis. There was no evidence for liver involvement secondary to the OS as the pretransplant liver function test results were normal. Vitamin E and ursodiol were administered for veno-occlusive disease prophylaxis. He achieved neutrophil engraftment on day +16, with 100% donor chimerism in the peripheral blood documented on day +21. The child unfortunately developed severe veno-occlusive disease with portal venous thrombosis and was treated with defibrotide. He subsequently developed progressive multiorgan dysfunction with fevers, respiratory insufficiency, hemodynamic instability, renal insufficiency, and coagulopathy. Similar to published cases of infants transplanted for immunodeficiency treated with corticosteroids,10 he developed cardiomyopathy with new severe concentric left ventricular hypertrophy. Cardiac biopsy on day +25 showed a mild T lymphocyte and histiocyte infiltrate and lack of myocyte hypertrophy. Rectal biopsy on day +28 demonstrated severe colitis consistent with acute graft versus host disease. Methylprednisolone was increased to 3 mg/kg per day, but despite this therapy, the patient died on day +31 after developing asystole. Postmortem examination was not performed. TABLE I Hematologic and immunologic characteristics of the patient We sought to confirm the diagnosis of RD by assessing AK2 protein expression and function. We also examined the etiology of this infant’s rash, diarrhea, and lymphadenopathy in the setting of the expanded CD3+ T-lymphocyte population, considering maternal engraftment or OS as possible causes of the presentation. AK2 protein was detected at reduced levels in the patient’s CD3+ T cells and fibroblasts by Western blot (Fig 1, A). To determine whether this AK2 protein was derived from autologous or maternally engrafted T cells, we performed fluorescence in situ hybridization of peripheral blood cells (99% carried a Y chromosome), short tandem repeat analysis of purified CD3+ cells (0% maternal), and flow cytometry analysis (0.5% bearing the noninherited maternal HLA-A2 allele), thus ruling out maternal T-cell engraftment as the source of expanded T cells (Fig 1, B). Somatic reversion in T cells was ruled out by detecting the same AK2 mutation in purified CD3+ T lymphocytes (see Fig E2 in this article’s Online Repository at www.jacionline.org). OS is characterized by the production and expansion of oligoclonal T cells, and indeed, the infant’s CD3+, CD4+, and CD8+ T-cell receptor repertoire was highly oligoclonal, with only 5 of 24, 2 of 24, and 3 of 24 TCRs variable region families falling in the normal range, respectively (Fig 1, C). Purified CD3+ T lymphocytes showed susceptibility to apoptosis, evidenced by increased depolarization of the mitochondrial membrane potential at 3, 6, and 9 hours of incubation with staurosporin, indicating abnormal function of the mutant AK2 (Fig 1, D) (see additional information in this article’s Methods section in the Online Repository at www.jacionline.org). FIG 1 Characteristics of the infant’s presentation. A, Expression of AK2 protein detected in patient’s PBMC and fibroblasts (Pt) compared with normal control (Ctrl). Expression of β-actin is shown as a loading control. B, Maternal engraftment ... This infant’s presentation of desquamative erythroderma, diarrhea, and generalized lymphadenopathy in the setting of an expanded and oligoclonal autologous CD3+ T-cell population is consistent with OS. This case indicates that at least some missense AK2 mutations may result in residual T-lymphocyte development, oligoclonal expansion, and symptoms of OS. Thus, AK2 should be added to the list of severe combined immunodeficiency–causing genes that may manifest as OS.

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency

BACKGROUND The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation

To the Editor: We would like to share a relevant and interesting immunodeficiency case that would be of interest to the readers of the Journal of Allergy and Clinical Immunology. This report describes an adult-onset idiopathic T-cell lymphopenia due to recombinase activating gene 1 (RAG1) deficiency in an HIV-negative male patient with no recurrent or opportunistic infections presenting at the age of 38 years with chronic dermatitis, pruritus, and hyperkeratosis. Clinical and immunologic examination revealed eosinophilia with modestly elevated IgE (747 kU/L), normal IgG, IgA, and IgM, profound pan–T-cell lymphopenia, high normal total B cells, and slightly reduced natural killer cells. Genetic analysis revealed a heterozygous frameshift mutation in the RAG1 gene, resulting in a truncated RAG1 protein. This is a late clinical presentation of an idiopathic T-cell lymphopenia secondary to a heterozygous hypomorphic RAG1 mutation and has important implications for the considerable phenotypic variability related to molecular defects in genes typically associated with severe combined immunodeficiency or Omenn syndrome (OS). A 38-year-old man presented with significant pruritic skin rash on his legs and eosinophilia that had been present for 2 years with poor resolution using over-the-counter topical treatments. Medical history revealed a healthy childhood and adulthood until 2 years earlier when the skin rash first appeared on the lower extremities. Gross examination of the skin revealed a generalized dermatitis with hyperkeratosis without histologic evidence of granulomas (Fig 1, A). There was also keratoderma of the feet and onychodystrophy. Skin biopsy of the lesions on the leg revealed a chronic dermatitis with eosinophilia, which was negative for IgG, IgM, or IgA with weak, discontinuous granular deposition of C3 along the basement membrane zone. Complete blood cell count with differential revealed significant lymphopenia, which on detailed lymphocyte subset evaluation by flow cytometry revealed profound pan–T-cell lymphopenia (Table I). There was a relative increase in the frequency of activated T cells (CD4+CD25+ and HLA-DR+ CD4 and CD8 T cells). Thymic function was significantly impaired for age, with almost absent CD4 recent thymic emigrants and TREC. Genetic analysis revealed the following heterozygous frameshift mutation (c.256_257delAA, K86VfsX33) in exon 2 of the gene encoding RAG1 that has been previously reported to be associated with SCID and OS, when present as a homozygous or compound heterozygous mutation.1 No other pathogenic genetic variations were found in either the known coding or promoter regions of the RAG1 gene or in any of the following SCID-associated genes: RAG2, JAK3, IL7R, ADA, CD3D, CD3E, DCLRE1C, and IL2RG. Genetic analysis for the above RAG1 mutation was performed not only on DNA extracted from whole blood but also from PBMCs and buccal brushing. In all 3 sample types, the same heterozygous mutation was identified, indicating that this was not a somatic mutation but a germline variation in the RAG1 gene. Genetic testing for this specific RAG1 mutation was performed on PBMCs isolated from whole blood of both parents of this patient, and the father was shown to have the same heterozygous mutation as the patient; however, the father was clinically, phenotypically, and immunologically normal with normal T, B, and natural killer cell counts. A second mutation was not identified in the patient or the mother. The patient has a single older male sibling who was not evaluated but was negative for relevant clinical history. FIG 1 A, Photograph showing skin rash. Diffuse skin rash was present on the extremities and, to a lesser extent, on the trunk of this patient with a heterozygous RAG1 mutation. B, Determination of recombinase activity level of wild-type and mutant RAG1. Stable ... TABLE I Lymphocyte subset quantitation by flow cytometry Further immunologic analysis revealed robust antibody responses to vaccine antigens such as tetanus and diphtheria toxoids, but significantly abnormal lymphocyte proliferation to mitogens (PHA, PWM) and antigens (Candida and tetanus toxoid) and stimulation with soluble anti-CD3. RAG1 function was determined by recombinase activity (Fig 1, B), and the 256–257delAA mutant had only 2.67% ± 0.58% activity as compared with wild-type RAG1. Although overexpression experiments have shown that this mutation can induce the production of an N-terminal–truncated protein by using a second internal ATG1 (which might explain the residual recombination activity), we failed to detect the expression of the mutant RAG1 on retrovirus-mediated transduction of Rag1−/− pro–B cells. Detailed B-cell subset analysis revealed low total memory B cells with low class-switched memory and marginal zone B cells with a significant increase in naive B cells (CD27−) and transitional B cells (CD19+CD24++CD38++). Functional B-cell analysis revealed that the patient’s B cells were capable of plasmablast differentiation on stimulation with CD40L and IL-21 or CpG, though it was decreased than that in a healthy control (data not shown). B-cell proliferation in response to CD40L + IL-21 was lower than in control B cells, but almost comparable to that in control with CpG. Also, the distribution of immunoglobulin light chains (kappa/lambda) was normal, arguing against significant defects of receptor editing of B cells. Therefore, it would appear that patient’s B cells are capable of responding to helper T-cell signals, and the reduced frequency of memory B cells is likely related to the low numbers of such T cells. There was evidence of autoantibody production with high titer of ANA, double-stranded DNA and SS-A antigens in addition to antibodies to 14 other autoantigens. Although there was no clinical evidence of specific autoimmune disease or granulomatous disease and renal function was preserved,2,3 the diffuse skin rash, which was responsive to steroids, suggests that a clear inflammatory process was present (prior to treatment). Molecular T-cell receptor repertoire analysis (T-cell receptor Vb spectratyping) revealed an overall polyclonal T-cell repertoire for most T-cell receptor Vb families with oligoclonality of a few (family 2 was absent and families 3–1, 6–4, 13, and 16 were oligoclonal with limited diversity). There was a significant increase in peripheral levels of IL-7, which is related to the presence of T-cell lymphopenia. Clinically, the patient responded well to wet dressings with ammonium lactate topical (AmLactin), a keratolytic, and treatment with triamcinolone. He was treated with prophylactic antibiotics with no evidence of infection. In a mouse model of a hypomorphic Rag1 variant, progressive T-cell lymphopenia with age was demonstrated,4 suggesting that heterozygosity for RAG defects may lead to premature immune senescence and T-cell dysfunction. This case is unique and exemplifies the magnitude of phenotypic variations that can be seen in molecular defects typically associated with SCID, or hypomorphic mutations in genes that are associated with OS,2 but in this case represents an idiopathic T-cell lymphopenia, adding to the spectrum of previously reported mutations.5 What is remarkable is the apparent adult-onset presentation of eosinophilia, skin rash, and low T-cell counts in this patient with only a single copy of a frameshift variation in the 5′ open reading frame of the RAG1 gene that usually causes an autosomal-recessive disorder. There is evidence that frameshift mutations in the 5′ end of RAG1 can maintain V(D)J recombination activity,1 and this may partially explain the late onset of the phenotype and normal B-cell numbers in the context of T-cell lymphopenia. As seen in the mouse model of hypomorphic Rag1 mutation,4 it is possible that the T-cell lymphopenia is due to an age-related decline in immunocompensatory mechanisms that permit the phenotype to be revealed later than typical. There are several differences between OS and idiopathic T-cell lymphopenia, with the former typically being clinically severe and characterized by elevated eosinophils and IgE along with erythrodermia, hepatosplenomegaly, lymphadenopathy, T-cell counts that are variable but generally oligoclonal in diversity, absent B cells, and normal natural killer cells.6 Patients with OS usually present early in life with recurrent and opportunistic infections, and the clinical course is fulminant and typically managed with hematopoietic cell transplantation. Adult-onset patients with idiopathic T-cell lymphopenia may have an overall milder clinical phenotype and are unlikely to require hematopoietic cell transplantation; nonetheless, these patients require careful monitoring to control significant infections as well as the development of autoimmunity and subsequent end-organ damage. Adult-onset ADA deficiency is the only other SCID-associated defect that has been reported with delayed manifestations in the context of heterozygous mutations in the ADA gene,7 resulting in partial ADA deficiency and a milder phenotype. In the partial ADA deficiency, immune function has been shown to decline over time, resulting in an immunologic and clinical phenotype. This phenomenon appears to be reproduced in this patient with the heterozygous RAG1 mutation. The presence of severe T-cell lymphopenia with dysregulated B-cell responses raises intriguing questions about the impact of hypomorphic mutations on immune function, and further the lack of an immunologic or clinical phenotype in the parent who is a carrier for this mutation suggests that there is an underlying additional genetic, epigenetic, or environmental modulation of RAG1 function in this patient.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

Next-Generation Sequencing Reveals Restriction and Clonotypic Expansion of Treg Cells in Juvenile Idiopathic Arthritis.

Treg cell–mediated suppression of Teff cells is impaired in juvenile idiopathic arthritis (JIA); however, the basis for this dysfunction is incompletely understood. Animal models of autoimmunity and immunodeficiency demonstrate that a diverse Treg cell repertoire is essential to maintain Treg cell function. The present study was undertaken to investigate the Treg and Teff cell repertoires in JIA.

Intronic SH2D1A mutation with impaired SAP expression and agammaglobulinemia.

X-linked lymphoproliferative (XLP) disease is a primary immunodeficiency syndrome associated with the inability to control Epstein-Barr virus (EBV), lymphoma, and hypogammaglobulinemia. XLP is caused by mutations in the SH2D1A gene, which encodes the SLAM-associated protein (SAP), or in the BIRC4 gene, which encodes the X-linked inhibitor of apoptosis protein (XIAP). Here we report a patient with recurrent respiratory tract infections and early onset agammaglobulinemia who carried a unique disease-causing intronic loss-of-function mutation in SH2D1A. The intronic mutation affected SH2D1A gene transcription but not mRNA splicing, and led to markedly reduced level of SAP protein. Despite undetectable serum immunoglobulins, the patients B cells replicated and differentiated into antibody producing cells normally in vitro.

RAG1 Reversion Mosaicism in a Patient with Omenn Syndrome

PurposeTo identify mechanisms of disease in a child born to consanguineous parents, who presented with Omenn syndrome (OS) and was found to carry a heterozygous RAG1 mutation in peripheral blood DNA.MethodsMutation analysis was performed on whole blood and buccal swab DNA. Recombination activity of the mutant RAG1 protein and diversity of T cell repertoire were tested.ResultsApparent heterozygosity for a novel, functionally null RAG1 mutation in peripheral blood DNA from a patient with OS was shown to be secondary to true somatic reversion. Analysis of T cell repertoire demonstrated expression of various TCRBV families, but an overall restricted pattern.ConclusionsThis is the first case of true somatic reversion of a RAG1 mutation in a patient with OS. The reversion event likely occurred at a stage where only a limited pool of T cell progenitors capable of performing V(D)J recombination could be generated. This work emphasizes the importance of performing functional studies to investigate the significance of novel genetic variants, and to consider somatic reversion as a possible disease modifier in SCID.

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype

Background: Mutations in recombination‐activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. Objective: Using a flow cytometry–based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. Methods: Abelson virus–transformed Rag2−/− pro‐B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild‐type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP‐expressing cells was evaluated by using flow cytometry, and high‐throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. Results: The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild‐type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. Conclusions: Our data support genotype‐phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease‐causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype. Graphical abstract Figure. No caption available.

A119: Deep Sequencing Analysis of the T Regulatory and T Effector Repertoire in Juvenile Idiopathic Arthritis

T regulatory (Treg) function has been shown to be impaired in juvenile idiopathic arthritis (JIA) as synovial fluid (SF) Tregs are unable to suppress T effector cells (Teffs) found in the joint. The cause of this breach in immunologic tolerance is not fully understood, meriting the further study of Tregs and Teffs in JIA. The T cell receptor (TCR) endows T lymphocytes with antigen specificity and mediates interactions between T cells and their environment. Yet, little is known about the comparative specificities, clonality, and diversity of Tregs and Teffs in JIA. Further, the Treg repertoire has not been studied in adult or pediatric inflammatory arthritis. Therefore, we endeavored to characterize these TCR repertoires in JIA through deep sequencing technology.