Barbara H. Barendregt

A new type of radiosensitive T–B–NK+ severe combined immunodeficiency caused by a LIG4 mutation

V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.

Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression

The role of specific transcription factors in the initiation and regulation of Ig gene rearrangements has been studied extensively in mouse models, but data on normal human precursor B cell differentiation are limited. We purified five human precursor B cell subsets, and assessed and quantified their IGH, IGK, and IGL gene rearrangement patterns and gene expression profiles. Pro-B cells already massively initiate DH-JH rearrangements, which are completed with VH-DJH rearrangements in pre-B-I cells. Large cycling pre-B-II cells are selected for in-frame IGH gene rearrangements. The first IGK/IGL gene rearrangements were initiated in pre-B-I cells, but their frequency increased enormously in small pre-B-II cells, and in-frame selection was found in immature B cells. Transcripts of the RAG1 and RAG2 genes and earlier defined transcription factors, such as E2A, early B cell factor, E2-2, PAX5, and IRF4, were specifically up-regulated at stages undergoing Ig gene rearrangements. Based on the combined Ig gene rearrangement status and gene expression profiles of consecutive precursor B cell subsets, we identified 16 candidate genes involved in initiation and/or regulation of Ig gene rearrangements. These analyses provide new insights into early human precursor B cell differentiation steps and represent an excellent template for studies on oncogenic transformation in precursor B acute lymphoblastic leukemia and B cell differentiation blocks in primary Ab deficiencies.

DNA microarrays for comparison of gene expression profiles between diagnosis and relapse in precursor-B acute lymphoblastic leukemia: Choice of technique and purification influence the identification of potential diagnostic markers

Microarrays for gene expression profiling are rapidly becoming important research tools for the identification of novel markers, for example, for novel classification of leukemias and lymphomas. Here, we review the considerations and infrastructure for microarray experiments. These considerations are illustrated via a microarray-based comparison of gene expression profiles of paired diagnosis–relapse samples from patients with precursor-B acute lymphoblastic leukemia (ALL), who relapsed during therapy or after completion of treatment. Initial experiments showed that several seemingly differentially expressed genes were actually derived from contaminating non-leukemic cells, particularly myeloid cells and T-lymphocytes. Therefore, we purified the ALL cells of the diagnosis and relapse samples if their frequency was lower than 95%. Furthermore, we observed in earlier studies that extra RNA amplification leads to skewing of particular gene transcripts. Sufficient (non-amplified) RNA of purified and paired diagnosis–relapse samples was obtained from only seven cases. The gene expression profiles were evaluated with Affymetrix U95A chips containing 12 600 human genes. These diagnosis–relapse comparisons revealed only a small number of genes (n=6) that differed significantly in expression: mostly signaling molecules and transcription factors involved in cell proliferation and cell survival were highly upregulated at relapse, but we did not observe any increase in drug-resistance markers. This finding fits with the observation that tumors with a high proliferation index have a poor prognosis. The genes that changed between diagnosis and relapse are currently not in use as diagnostic or disease progression markers, but represent potential new markers for such applications.

B-cell replication history and somatic hypermutation status identify distinct pathophysiologic backgrounds in common variable immunodeficiency.

Common variable immunodeficiency disorder (CVID) is the most prevalent form of primary idiopathic hypogammaglobulinemia. Identification of genetic defects in CVID is hampered by clinical and immunologic heterogeneity. By flow cytometric immunophenotyping and cell sorting of peripheral B-cell subsets of 37 CVID patients, we studied the B-cell compartment at the B-cell subset level using the κ-deleting recombination excision circle assay to determine the replication history and the Igκ-restriction enzyme hot-spot mutation assay to assess the somatic hypermutation status. Using this approach, 5 B-cell patterns were identified, which delineated groups with unique replication and somatic hypermutation characteristics. Each B-cell pattern reflected an immunologically homogenous patient group for which we proposed a different pathophysiology: (1) a B-cell production defect (n = 8, 18%), (2) an early peripheral B-cell maturation or survival defect (n = 4, 11%), (3) a B-cell activation and proliferation defect (n = 12, 32%), (4) a germinal center defect (n = 7, 19%), and (5) a postgerminal center defect (n = 6, 16%). The results of the present study provide for the first time insight into the underlying pathophysiologic background in 5 immunologically homogenous groups of CVID patients. Moreover, this study forms the basis for larger cohort studies with the defined homogenous patient groups and will facilitate the identification of underlying genetic defects in CVID.

Anti-TNF treatment blocks the induction of T cell-dependent humoral responses

Objective Experimental and human data suggest that tumour necrosis factor (TNF) blockade may affect B cell responses, in particular the induction of T cell-dependent (TD) humoral immunity. This study aimed to assess this hypothesis directly in patients with arthritis by analysing longitudinally the effect of TNF blockade on B cell activation and the maturation of humoral responses against TD and T cell-independent vaccines. Materials and methods Peripheral blood samples were obtained from 56 spondyloarthritis patients before and after treatment with either non-steroidal anti-inflammatory drug (NSAID) alone or TNF blockers and analysed for B cell activation, plasma cell differentiation, germinal centre versus extra-follicular B cell maturation, and somatic hypermutation. Vaccine responses to hepatitis B and Streptococcus pneumoniae were measured by ELISA. Results TNF blockade augmented B cell activation as reflected by the expression of early activation markers, CD40, and costimulatory molecules, without affecting differentiation towards plasmablasts. This was associated with a specific increase of the unswitched fraction of circulating memory B cells and a decreased level of somatic hypermutation in anti-TNF treated patients, indicating an impairment of the germinal centre-dependent B cell maturation. In agreement with these findings, TNF blockade profoundly suppressed the response to the TD vaccination against hepatitis B, whereas the T cell-independent response against pneumococcal polysaccharides was only modestly affected. Conclusions These data indicate that TNF blockade severely impedes the induction of primary TD humoral responses, probably by interfering with the germinal centre reaction.

Defective Artemis nuclease is characterized by coding joints with microhomology in long palindromic-nucleotide stretches

T–B–NK+ severe combined immunodeficiency (SCID) is caused by a defect in V(D)J recombination. A subset of these patients has a mutation in one of the non‐homologous end joining (NHEJ) genes, most frequently the Artemis gene. Artemis is involved in opening of hairpin‐sealed coding ends. The low levels of residual Dh‐Jh junctions that could be amplified from patients’ bone marrow precursor B cells showed high numbers of palindromic (P)‐nucleotides. In 25% of junctions, microhomology was observed in the P‐nucleotide regions, whereas this phenomenon was never observed in junctions amplified from bone marrow precursor B cells from healthy controls. We utilized this difference between Artemis‐deficient cells and normal controls to develop a V(D)J recombination assay to determine hairpin‐opening activity. Mutational analysis of the Artemis gene confirmed and extended the mapping of an N‐terminal nuclease active site, which contains several indispensable aspartate residues. C‐terminal deletion mutants did not show such severe defects in the V(D)J recombination assay using transient overexpression of (mutated) Artemis protein. However, a C‐terminal deletion mutation causes T–B–NK+ SCID, indicating that the Artemis C terminus is essential for V(D)J recombination at the normal Artemis expression level. The V(D)J recombination assays used in this study contribute to the diagnostic strategy for T–B–NK+ SCID patients.

Common variable immunodeficiency and idiopathic primary hypogammaglobulinemia: two different conditions within the same disease spectrum

Patients with hypogammaglobulinemia who do not fulfill all the classical diagnostic criteria for common variable immunodeficiency (reduction of two immunoglobulin isotypes and a reduced response to vaccination) constitute a diagnostic and therapeutic dilemma, because information concerning the clinical and immunological characteristics of these patients with idiopathic primary hypogammaglobulinemia is not available. In 44 common variable immunodeficiency and 21 idiopathic primary hypogammaglobulinemia patients we determined the clinical phenotypes and performed flow cytometric immunophenotyping to assess the pathophysiological B-cell patterns and memory B-cell subset counts. Age-matched B-cell subset reference values of 130 healthy donors were generated. Severe pneumonia and bronchiectasis occurred at similar frequencies in idiopathic primary hypogammaglobulinemia and common variable immunodeficiency. Although IgG levels were only moderately reduced compared to common variable immunodeficiency, 12 of 21 idiopathic primary hypogammaglobulinemia patients required immunoglobulin replacement. Non-infectious disease-related clinical phenotypes (autoimmune cytopenia, polyclonal lymphocytic proliferation and persistent unexplained enteropathy) were exclusively observed in common variable immunodeficiency and were associated with early peripheral B-cell maturation defects or B-cell survival defects. T-cell dependent memory B-cell formation was more severely affected in common variable immunodeficiency. Furthermore, 14 of 21 idiopathic primary hypogammaglobulinemia patients showed normal peripheral B-cell subset counts, suggestive for a plasma cell defect. In conclusion, idiopathic primary hypogammaglobulinemia patients who do not fulfill all diagnostic criteria of common variable immunodeficiency have moderately decreased immunoglobulin levels and often a normal peripheral B-cell subset distribution, but still suffer from serious infectious complications.

A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells

Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B+T−NK− X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined immunodeficiencies with a variety of clinical presentations. We analyzed peripheral blood mononuclear cells of a 6-year-old boy with normal lymphocyte counts, who suffered from recurrent pneumonia and disseminated mollusca contagiosa. Since proliferative responses of T cells and NK cells to γc -cytokines were severely impaired, we performed IL2RG gene analysis, showing a heterozygous mutation in the presence of a single X-chromosome. Interestingly, an IL2RG reversion to normal predominated in both naïve and antigen-primed CD8+ T cells and increased over time. Only the revertant CD8+ T cells showed normal expression of CD132 and the various CD8+ T cell populations had a different T-cell receptor repertoire. Finally, a fraction of γδ+ T cells and differentiated CD4+CD27− effector-memory T cells carried the reversion, whereas NK or B cells were repeatedly negative. In conclusion, in a patient with a novel IL2RG mutation, gene-reverted CD8+ T cells accumulated over time. Our data indicate that selective outgrowth of particular T-cell subsets may occur following reversion at the level of committed T progenitor cells.

Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Oz- polymorphism in the human Ig lambda locus

Two polymorphisms of the human Igλ (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the Cλ2-Cλ3 region. The second polymorphism is the Mcg−Ke+Oz− isotype, which has only been defined via serological analyses in Bence-Jones proteins of multiple myeloma patients and was assumed to be encoded by a polymorphic Cλ2 segment because of its high homology with the Mcg−Ke−Oz− Cλ2 isotype. It has been speculated that the Mcg−Ke+Oz− isotype might be encoded by a Cλ gene segment of the amplified Cλ2-Cλ3 region. We now unraveled both IGL gene polymorphisms. The amplification polymorphism appeared to result from a duplication, triplication, or quadruplication of a functional J-Cλ2 region and is likely to have originated from unequal crossing over of the J-Cλ2 and J-Cλ3 region via a 2.2-kb homologous repeat. The amplification polymorphism was found to result in the presence of one to five extra functional J-Cλ2 per genome regions, leading to decreased Igκ:Igλ ratios on normal peripheral blood B cells. Via sequence analysis, we demonstrated that the Mcg−Ke+Oz− isotype is encoded by a polymorphic Cλ2 segment that differs from the normal Cλ2 gene segment at a single nucleotide position. This polymorphism was identified in only 1.5% (2 of 134) of individuals without J-Cλ2 amplification polymorphism and was not found in the J-Cλ2 amplification polymorphism of 44 individuals, indicating that the two IGL gene polymorphisms are not linked.

Increased PI3K/Akt activity and deregulated humoral immune response in human PTEN deficiency.

_To the Editor:_ Autosomal-dominant germline mutations in _PTEN_ are associated with phosphate and tensin homologue deleted on chromosome 10 (PTEN) hamartoma tumor syndromes (PHTS), including Cowden syndrome, characterized by hamartomas, malignant tumors, macrocephaly, and neurodevelopmental delay. Immunodeficiency has recently been reported in PHTS, but the mechanism of disease is not clear. Therefore, we performed a detailed study of the peripheral B-cell development of 9 patients with PHTS, to explore the role of phosphatidyl inositol 3-kinase (PI3K)/Akt signaling in the humoral immune response in these patients (details are described in this article’s Methods section in the Online Repository at _www.jacionline.org_).