Proc. Natl. Acad. Sci. USA Vol. 95, pp. 13135–13140, October 1998 Immunology Defect in IgV gene somatic hypermutation in Common Variable Immuno-Deficiency syndrome YVES LEVY*†‡,NEETU GUPTA†,FRANC¸OISE LE DEIST§,CORINNE GARCIA†,ALAIN FISCHER§,JEAN-CLAUDE WEILL†, AND CLAUDE-AGNE`S REYNAUD† *Unite´d’immunologie clinique, Hoˆpital Henri Mondor, 51, avenue du Mare´chalde Lattre de Tassigny, 94010 Cre´teilCedex 10, France; †Institut National de la Sante´et de la Recherche Me´dicaleUnite´373, Faculte´deMe´decine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15, France; and §Institut National de la Sante´et de la Recherche Me´dicaleUnite´429, Hoˆpital Necker-Enfants Malades, 149, rue de Se`vres, 75730 Paris Cedex 15, France Edited by Max D. Cooper, University of Alabama, Birmingham, AL, and approved September 8, 1998 (received for review June 8, 1998) ABSTRACT Common Variable Immuno-Deficiency from eight patients (six CVID and 2 hypogammaglobulinemic (CVID) is the most common symptomatic primary antibody- patients with recurrent infections). In two CVID cases, 40 to 75% deficiency syndrome, but the basic immunologic defects under- of the pool of circulating IgG memory B cells were found totally lying this syndrome are not well defined. We report here that devoid of somatic mutation, suggesting that the process of Ig among eight patients studied (six CVID and two hypogamma- affinity maturation can be severely hampered in some CVID globulinemic patients with recurrent infections), there is in two patients. Finally, functional analysis of the T cell compartment, CVID patients a dramatic reduction in Ig V gene somatic including an in vitro assay of the capacity of peripheral T cells to hypermutation with 40–75% of IgG transcripts totally devoid of induce somatic hypermutation in a human B cell line, argues in mutations in the circulating memory B cell compartment. Func- favor of an intrinsic B cell defect for the two hypomutated cases. tional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these MATERIALS AND METHODS two cases. Cell Separation and Flow Cytometry. Patients and normal donors (ND) were studied after informed consent was obtained. Common Variable Immuno-Deficiency (CVID) is the most com- Peripheral blood mononuclear cells (PBMCs) from patients and mon symptomatic primary antibody deficiency syndrome. This NDs were isolated by Ficoll isopaque density centrifugation. heterogeneous syndrome is characterized by decreased levels of Enriched B cell populations were obtained after T cell elimina- serum Ig while the number of circulating B cells is low or normal. tion by E-rosetting using sheep erythrocytes (E cells). E cell The usual age at presentation is the second and third decade of preparations were stained with tricolor-conjugated anti-CD19- life. Most patients present recurrent pyogenic infections predom- mAb, fluorescein isothiocyanate-conjugated, anti-human IgD inantly of the upper and lower respiratory tracts and of the and phycoerythrin-conjugated anti-human IgM mAbs (Caltag, South San Francisco, CA) for 30 min at 4°C. Cells were separated gastrointestinal tract. Moreover, the incidence of malignancies, 1 1 1 1 2 2 such as gastric carcinoma and lymphoma, is significantly in- into CD19 IgM IgD and CD19 IgM IgD fractions with a creased in patients with CVID (1). The heterogeneity of clinical FACS Vantage (Becton Dickinson). and immunological presentations of CVID has hampered inves- Cloning and Sequencing of V3–23-Cg Transcripts. Total RNA tigations. Although the basic immunologic defects that cause was extracted from 0.5–1 3 106 PBMC by using the RNA-plus CVID are unknown, a number of in vitro immunologic abnor- extraction procedures (Quantum Bioprobe, Montreal, Canada). malities have been identified in patients with this syndrome (1, 2). Single strand cDNA synthesis was performed by using the Strat- Susceptibility genes for CVID within the major histocompatibility agene reverse transcription- (RT) PCR kit and a CgA(59- complex class II and III loci have been reported (3, 4). GTCCTTGACCAGGCAGCCCAG-39) primer. After an etha- No specific treatment for CVID is available and the patients nol precipitation step, the cDNA produced was resuspended in 20 usually require life-long injections of gammaglobulins. A clinical ml of water. PCR was performed with 0.5 unit of Pfu polymerase paradox characterizing some CVID patients is the absence of (Stratagene) on 1y20 of the cDNA. The following primers were absolute correlation between the serum IgG level and the inci- used for amplification: V3–23 leader exon (59-GGCTGAGCT- dence and recurrence of bacterial infections. Therefore, the GGCTTTTTCTTGTGG-39) and CgB(59-AAGACCGAT- decision to treat a patient with Ig replacement is not based on the GGGCCCTTGGTGG-39). CgA and CgB primers match equally absolute level of serum IgG but rather on the frequency and all g isotypes. The PCR conditions were 35 cycles (94°C, 45 sec; severity of infections. Based on this observation, we wanted to 65°C, 1.5 min; 72°C, 2 min). PCR products were cloned by using analyze whether qualitative abnormalities in the antibody matu- the TA cloning kit (Invitrogen). V3–23 positive colonies were ration process could be associated to the common quantitative sequenced with the dRhodamine dye terminator cycle sequenc- defect in Ig production in CVID patients. ing kit (Applied Biosystems) and analyzed with the Applied The affinity maturation of T cell-dependent antibody responses Biosystems prism 310 genetic analyzer. Cloning and Sequencing of the JH4-JH5 Intronic Regions. results from the accumulation of point mutations in the variable 1 2 2 (V) region of Ig genes followed by antigen-driven selection of the Total genomic DNA was extracted from 105 CD19 IgM IgD B lymphocytes expressing high affinity antibodies (5, 6). This B cells by proteinase K digestion. The JH4-JH5 intronic region process takes place in germinal centers where antigen-specific B was amplified with Pfu polymerase by using a consensus FR3 cells differentiate to memory andyor plasma cells after switching primer for all human VH (heavy chain variable region) sequences of the heavy chain isotypes (7–10). In this study, we examined the (59-ACTCTAGACACGGCYGTGTATTACTGTGC-39) and a frequency of mutation in Ig genes from peripheral B cells isolated This paper was submitted directly (Track II) to the Proceedings office. The publication costs of this article were defrayed in part by page charge Abbreviations: CVID, Common Variable Immuno-Deficiency; PHA, phytohemagglutinin; ND, normal donor; PBMC, peripheral blood payment. This article must therefore be hereby marked ‘‘advertisement’’ in mononuclear cell; BL, Burkitt lymphoma; VH, heavy chain variable accordance with 18 U.S.C. §1734 solely to indicate this fact. region. © 1998 by The National Academy of Sciences 0027-8424y98y9513135-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: yves.levy@ PNAS is available online at www.pnas.org. hmn.hop-ap-paris.fr. 13135 Downloaded by guest on October 3, 2021 13136 Immunology: Levy et al. Proc. Natl. Acad. Sci. USA 95 (1998) primer immediately 59 of the JH5 exon (59-ACGAATTCGAAC- Somatic mutation of Ig genes was studied by using two tech- CAGTTGTCACATTGTG-39). PCR conditions were 35 cycles nical procedures. In the first one, we studied the region flanking (94°C, 45 sec; 55°C, 1.5 min; 72°C, 2 min). Gel purified PCR the 39 side of the rearranged JH gene segment (12), which allows products were cloned after EcoRI–XbaI digestion in the the analysis of Ig gene mutation on unselected sequences without pGEM3Zf plasmid (Promega) and sequenced as described focusing on an individual V gene segment. In the second proce- above. dure, we focused on the pattern of somatic mutation of a single, In Vitro Induction of Somatic Hypermutation. The Burkitt well expressed member of the VH3 gene family, the V3–23 gene lymphoma cell line BL2 can be induced to mutate in vitro its (13). rearranged VH4 gene after anti-IgM stimulation and when Analysis of Mutation of JH4-JH5 Intron. CD191IgD2IgM2 cocultured with anti-CD3-activated, cloned T helper cells (11). blood memory B cells from two normal donors (ND1 and ND2) We adapted this culture to the use of phytohemagglutinin- and four patients (LE, MZ, DA, and MA) were purified by cell (PHA) activated PBMC as a source of T cell help. The BL2 cell sorting before DNA extraction. PCR amplification was per- line was cultured in DMEM F12 medium (GIBCOyBRL) sup- formed by using a 59-primer consensus for all human VH genes plemented with 10% horse serum and 1% ADCM (ABiO1, located at the end of the FR3 region and a 39-oligonucleotide Lyon, France). PBMC from ND and from patients LE and SO priming immediately upstream of the JH5 exon. Because the JH4 were activated in the presence of 2 mgyml PHA (Murex) and gene is used in '50% of rearranged B cells (14), this approach recombinant IL-2 (50 unitsyml) (Roche) for 4 days. For coculture enables the analysis of a large compartment of memory B cells experiments, PHA-activated PBMC irradiated with 2,500 rads undergoing somatic mutation on unselected sequences. Intronic were seeded at 105 cells per well in flat-bottomed, 48-well culture JH4-JH5 region of memory B cells from the two controls were plates coated with anti-CD3 antibodies. BL2 cells were incubated analyzed first. Compared with germline allele sequences (15), with a soluble polyclonal anti-human IgM antibody (The Jackson nearly all the sequences exhibited a significant number of single Laboratory) at 10 mgyml for 30 min at 4°C and seeded after nucleotide changes (17y19 and 10y12 sequences from ND1 and washing at 104 cells per well alone or over PHA-activated PBMC. ND2, respectively, have more than one mutation) (Table 2).