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.

Common variable immunodeficiency disorder (CVID) is

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ABSTRACTical practice, but information concerning the prevalence and the clinical and immunological characteristics is not available. It is important to obtain insight into the frequency and severity of the clinical complications of IPH, to clarify whether IPH is a clinically relevant antibody deficiency, and to develop appropriate treatment strategies. In addition, analysis of immunological parameters will enable the comparison of pathophysiological aspects of IPH and CVID.Therefore, we aimed to determine the position of IPH in the spectrum of idiopathic antibody deficiencies through clinical and immunological comparison with CVID. First, we recorded the patients with the clinical phenotypes as established by Chapel et al. 2,7 In addition, we performed flow cytometric immunophenotyping in order to analyze T-cell dependent and independent memory B-cell subset counts 8 and blood B-cell patterns, which are associated with differences in pathophysiological background. 9 Methods PatientsPeripheral blood samples and clinical data were collected for 44 CVID patients and 21 IPH patients. IPH was diagnosed if patients had a reduction of IgG at least 2 SD below the mean for age, an onset of the immunodeficiency after two years of age, exclusion of defined causes of hypogammaglobulinemia and if they did not fulfill the CVID diagnostic criteria with respect to a reduction of two immunoglobulin isotypes and/or a reduced response to vaccination. The group of CVID patients includes the 37 patients who were reported in our original description of the B-cell patterns. 9 In addition, we collected blood from 130 healthy age-matched controls and 26 cord blood samples. This study was approved by the Medical Ethics Committee of the Erasmus MC. Clinical phenotypingClinical data were collected from all IPH and CVID patients to record their clinical phenotypes as previously described by Chapel et al. 2,5 These phenotypes are: 1) no disease-related complications (infections only); 2) autoimmune cytopenias; 3) polyclonal lymphoproliferation (granuloma/LIP/persistent unexplained lympadenopathy); and 4) unexplained persistent enteropathy. In addition, data were collected concerning the frequency and severity of infections and modes of treatment. Pneumococcal polysaccharide vaccination responses were interpreted according to Borgers et al. 10 as an adequate response to half of the measured pneumococcal serotypes. Flow cytometric analysis and assignment of B-cell patternsSix-color flow cytometric immunophenotyping of peripheral blood was performed on a CantoII (BD Biosiences) and data were analyzed using FACS Diva software (BD Biosiences). The following monoclonal antibodies were used: CD19-PerCP-Cy5.5, CD19-PE-Cy7, CD19-APC (all SJ25C1), CD5-APC ( L17F12 ), CD45-PerCP (2D1), CD19-APC (SJ25C1), CD38-PE, CD38-APC and CD38-PE-Cy7 ( HB7), CD27-APC (L128), CD3-PerCP-Cy5.5 (SK7) and CD8-APC-Cy7 (SK1) (all from BD Biosciences), polyclonal IgD-FITC, IgD-PE and IgM-PE (all from Southern Biotechnologies), polyclonal IgG-FITC (Kallestad), IgA-FITC a...