Proc. Natl. Acad. Sci. USA Vol. 95, pp. 13135–13140, October 1998

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 - 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 , suggesting that the process of Ig among eight patients studied (six CVID and two hypogamma- can be severely hampered in some CVID globulinemic patients with recurrent infections), there is in two patients. Finally, functional analysis of the T 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 line, argues in in the circulating compartment. Func- favor of an intrinsic B cell defect for the two hypomutated cases. tional assays of the 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, ϩ ϩ ϩ ϩ Ϫ Ϫ 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-C␥ Transcripts. Total RNA tigations. Although the basic immunologic defects that cause was extracted from 0.5–1 ϫ 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 - (RT) PCR kit and a C␥A(5Ј- complex class II and III loci have been reported (3, 4). GTCCTTGACCAGGCAGCCCAG-3Ј) 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 ␮l of water. PCR was performed with 0.5 unit of Pfu polymerase paradox characterizing some CVID patients is the absence of (Stratagene) on 1͞20 of the cDNA. The following primers were absolute correlation between the serum IgG level and the inci- used for amplification: V3–23 leader exon (5Ј-GGCTGAGCT- dence and recurrence of bacterial infections. Therefore, the GGCTTTTTCTTGTGG-3Ј) and C␥B(5Ј-AAGACCGAT- decision to treat a patient with Ig replacement is not based on the GGGCCCTTGGTGG-3Ј). C␥A and C␥B primers match equally absolute level of serum IgG but rather on the frequency and all ␥ 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 ϩ Ϫ Ϫ (V) region of Ig genes followed by -driven selection of the Total genomic DNA was extracted from 105 CD19 IgM IgD B expressing high affinity (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 and͞or 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 (5Ј-ACTCTAGACACGGCYGTGTATTACTGTGC-3Ј) 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 0027-8424͞98͞9513135-6$2.00͞0 ‡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 5Ј of the JH5 exon (5Ј-ACGAATTCGAAC- of Ig genes was studied by using two tech- CAGTTGTCACATTGTG-3Ј). 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 3Ј 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. CD19ϩIgDϪIgMϪ 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 (GIBCO͞BRL) sup- formed by using a 5Ј-primer consensus for all human VH genes plemented with 10% horse serum and 1% ADCM (ABiOϩ, located at the end of the FR3 region and a 3Ј-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 ␮g͞ml PHA (Murex) and gene is used in Ϸ50% of rearranged B cells (14), this approach recombinant IL-2 (50 units͞ml) (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 ␮g͞ml for 30 min at 4°C and seeded after nucleotide changes (17͞19 and 10͞12 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). The A second anti-IgM activation was performed at day 7 of culture rate of mutation was comparable in both NDs with 3.8% and and cells were cultured with new irradiated PHA-activated 3.4%, respectively. Analysis of the pattern of mutations showed PBMC for 7 more days. At day 14, RNA was extracted from nine individual hotspots of mutation, six of them corresponding harvested cells, and cDNA was obtained by using random prim- in either DNA strand to known motifs of somatic mutation (16) ers. VH4-JH5 transcripts were amplified with primers on the (Fig. 1). Seven large insertions͞deletions (5–140 bp) and five leader V4-34 sequence (5Ј-TTCTTCCTCCTGCTGGTGGCG- insertions͞deletions in small mono- or di-nucleotide repeats were 3Ј) and the JH5 exon (5Ј-ACCTGAGGAGACGGTGACCAG- observed among the 31 sequences from controls, representing 3Ј). PCR conditions were 35 cycles (94°C, 45 sec; 64°C, 1.5 min; one event per 32 mutations (Fig. 2A for ND1). 72°C, 2 min). TA cloning and sequencing of PCR products were Strikingly, in one CVID patient (LE), nearly 65% of the performed as described above. sequences exhibited 0–1 mutation (Fig. 2B). The mutation level was decreased markedly in this patient (1.4%), whereas in the RESULTS three other cases, the rate of mutations was close to normal values Characteristics of Patients. Four adults (LE, SO, MA, and (2.3% to 3.5%) (Table 2). To confirm that this rate of mutation BE), two children (TH and FL) with CVID, and two hypogam- obtained on JH4-JH5 region concerned exclusively switched maglobulinemic children with recurrent infections (MZ and DA) memory B cells and not a fraction of nonmutated contaminating were studied. Three of them belong to the same family; FL and IgMϩ B cells (17), we used a second approach analyzing directly TH are siblings, and BE is the father. Clinical features of patients expressed IgG transcripts. are reported in Table 1. All patients had a history of recurrent Analysis of Mutation of V3–23-C␥ Transcripts. The V3–23 bacterial infection of the upper and lower respiratory tracts and gene is expressed in 4–10% of human B cells (13). V3–23-C␥ nodular lymphoid hyperplasia was diagnosed in three cases (LE, transcripts from two normal donors and six CVID patients were SO, and MA). The serum Ig level at diagnosis was variable studied. Two of them have been previously analyzed at the between these patients, from moderately (e.g., MZ and DA) to JH4-JH5 intron (LE and MA) and four were part of a new cohort severely (e.g., LE, MA, and BE) reduced (Table 1). The per- of patients (SO, BE, TH, and FL) in whom the small size of the centage of blood CD19ϩ B cells ranged from 3.5% to 23%. All blood sample prevented the sorting of CD19ϩIgMϪIgDϪ B cells. patients, but one (MZ), are treated with monthly i.v. Ig substi- A specific IgG constant region oligonucleotide was used to prime tution. first strand cDNA synthesis from RNA extracted from nonsepa-

Table 1. Characteristics of CVID and hypogammaglobulinemic patients

Serum Ig levels, mg͞ml* ϩ Age of CD19 cells, Patients diagnosis Age Symptoms IgG IgM IgA % LE 32 53 Sinopulmonary infections, 1.29 0.06 0.07 4 Inflammatory bowel disease, Insulinodependant diabetes, INLH‡ SO 12 36 Sinopulmonary infections, Lambliasis, 3.6 1 0.18 6 INLH‡ MA 21 47 Sinopulmonary infections, INLH‡ 2.64 0.12 0.04 3.5 BE† 39 43 Sinopulmonary infections 1.99 0.95 0.98 4.5 MZ§ 9 11 Pulmonary infections 6.11 0.69 0.3 14 TH† 4 9 Pulmonary infections 3.71 0.48 0.31 7 FL† 7 11 Pulmonary infections 4.2 0.99 0.62 13 DA§ 5 8 Pulmonary infections 5 1.4 0.5 23 *At the time of diagnosis. Range of normal values are: 4–5 years: IgG: 6.8–11.8; IgM: 0.5–1.1; IgA: 0.6–1.3; 7–9 years: IgG: 6.33–12.8; IgM: 0.7–1.3; IgA: 0.8–1.6; adults: IgG: 7–14; IgM: 0.6–3.3; IgA: 1–3.9. †Belong to the same family: BE is the father; TH and FL are siblings. ‡Ileal nodular lymphoid hyperplasia. §These patients are referred to as hypogammaglobulinemic with recurrent infections. Downloaded by guest on October 3, 2021 Immunology: Levy et al. Proc. Natl. Acad. Sci. USA 95 (1998) 13137

Table 2. Mutations in JH4-J5 introns flanking VDJH4 showed the major individual hotspots of mutation previously ϩ Ϫ Ϫ rearrangements from CD19 IgM IgD memory B cells reported for the V3–23 gene (18) and were broadly similar in Clones Mutations patients and NDs (not shown). As shown in Table 3, the ratio of replacement versus silent mutations (R͞S) argued against obvi- All 0 or 1 mutation Total per bp, % ous abnormalities in antigenic selection process in patients who Controls show a normal mutation frequency, as well as for patient SO. For ND1 19 2 242 3.8 patient LE, the low R͞S ratio in CDR might suggest a defective ND2 12 2 138 3.4 antigen-driven selection, although it is difficult to draw a defin- itive conclusion from the small number of mutations accumu- Patients lated. Interestingly, and as reported by others (19, 20), five LE 15 10 73 1.4 deletions͞insertions (two in NDs and three in patients) of one to MZ 16 3 127 2.3 three codons were identified among the 1,198 mutations seen in DA 10 0 94 2.7 97 V3–23-C␥ transcripts from controls and patients (Table 3), MA 7 0 84 3.5 representing one event for 240 mutations. In Vitro Induction of Somatic Hypermutation of IgVH Gene of rated PBMC. C␥ transcripts were amplified by using a 5Ј-V3–23 BL2 Cells. Dene´poux et al. (11) reported recently that the type I primer hybridizing to the V3–23 leader exon together with a 3Ј-C␥ Burkitt’s lymphoma cell line BL2 can be induced to mutate in primer. PCR products were cloned and sequenced. vitro its rearranged VH4 gene after anti-IgM stimulation and coculture with activated cloned helper T cells. We wanted to In the two controls, 100% of the individual sequences obtained adapt this system to the use of PBMC as source of T cell help for were heavily mutated with a mutation rate of 7.9% and 7.1% in the induction of mutations in BL2 cells as a new assay for T cell ND3 and ND4, respectively (Table 3). For patients LE and MA function (see Materials and Methods). The background level of previously analyzed, the mutation frequency of their rearranged mutations observed in BL2 bulk cultures, or after anti-IgM V3–23 gene correlated with the findings of the JH4-JH5 intron stimulation alone, was established after amplification of the VH4 analysis. Indeed, patient MA was close to NDs with 5.4% transcripts and was below 0.75 ϫ 10Ϫ4 mut͞bp (0 mutation in the mutation rate in the V3–23 gene. The low rate of mutation of the 37 sequences analyzed, i.e., 12,950 nucleotides). This background JH4-JH5 intron found in case LE was confirmed at the V3–23 value is lower than the one reported by Dene´poux et al. (11) due level with a 0.8% mutation rate (Table 3). Moreover, in this latter to the different PCR conditions used. In contrast, when anti-IgM case, 15 of 21 V3–23 sequences analyzed were totally germline stimulated BL2 cell line was cocultured with PHA-stimulated T (Fig. 3) confirming, here again, the results of the JH4-JH5 intron cells from ND, six mutations were introduced in 34 VH4 se- analysis. Interestingly, in the four remaining patients, another quences (11,900 nucleotides) representing 5 ϫ 10Ϫ4 mutation͞bp. case (SO) exhibited a reduced rate of somatic mutation in These results showed that signals necessary for the in vitro ␥ V3–23-C transcripts (1.6%), with 8 of the 19 V3–23 clones induction of somatic hypermutation in the BL2 cell line can be analyzed completely unmutated (Fig. 3). In the three last cases, provided by normal activated T cells. the mutation frequency was close to control values with 5.1% When PHA activated T cells from patient LE were used in (FL), 5.4% (BE), and 6.2% (TH) (Table 3). coculture with anti-IgM stimulated BL2 cells, four mutations Overall, the frequency of mutations of the V3–23 gene in were found in 37 sequences (12, 950 nucleotides) representing 3 ϫ patients varied from 0.8% to 6.2%. It appears that two CVID 10Ϫ4 mutation͞bp, indicating that T cells from this CVID patient cases may emerge among the eight patients studied with a severe are fully capable of inducing mutation in the BL2 cell line. defect in the Ig gene somatic mutation process because 40 to 75% However, in CVID patient SO, the level of mutations obtained of their circulating IgG B cells were unmutated. In addition, the was lower, with two mutations in 42 sequences, i.e., 1.4 ϫ 10Ϫ4 rate of nucleotide substitution on the remaining transcripts mutation͞bp, but above background value (Table 4). obtained in these patients was 2.8% for both cases, indicating that the frequency of mutation accumulated in these mutated B cells DISCUSSION remained still lower than the one found in NDs. In this study, we wanted to find out whether qualitative abnor- Analysis of the Mutation Pattern of V3–23 Transcripts. The malities of Ig affinity maturation could be associated in some pattern of mutations accumulated in the V3–23 gene in the NDs CVID patients to the common quantitative defect in Ig produc- tion. This study concerning a small cohort of patients (six CVID patients and two hypogammaglobulinemic patients with recur- rent infections) was based on the analysis of sequences from the intronic JH4-JH5 region flanking VH-JH4 rearrangements from purified circulating memory CD19ϩIgMϪIgDϪ B cells and on the analysis of V3–23-C␥ transcripts from total PBMC. The first approach allowed us to study the imprint of the hypermutation process in unselected genomic sequences representing one-half of the rearrangements without focusing on an individual VH gene; this approach also excludes any bias that can result from an analysis performed at the RNA level in which a few activated B cell clones can be over represented due to their high RNA content. For patients in whom there was not enough blood material available, sequences of selected V3–23-C␥ transcripts were obtained directly from a small blood sample by RT-PCR. These two approaches were concordant because in controls the great majority of intronic sequences from sorted memory cells and of V3–23-C␥ transcripts were heavily mutated. Moreover, we FIG. 1. Distribution of mutations along the JH4-JH5 intron in ͞ CD19ϩIgMϪIgDϪ memory B cells from normal donors. The number found a significant number of insertion deletion events in in- of mutations in 31 sequences obtained from controls is indicated. tronic sequences and, as reported by others for VH genes (19, 20), Mutation hotspots are indicated in a four-nucleotide context, with the also in V3–23 transcripts, indicating that these events are part of mutated position underlined. the hypermutation process: the insertion͞deletion per mutation Downloaded by guest on October 3, 2021 13138 Immunology: Levy et al. Proc. Natl. Acad. Sci. USA 95 (1998)

ϩ Ϫ Ϫ FIG. 2. Sequences of JH4-JH5 intron from CD19 IgM IgD memory B cells from normal donor and CVID patient LE. JH4-JH5 intronic sequences were compared with the sequence of the corresponding germline allele (15) (allelic variations are marked above the sequence). Nineteen sequences from ND1 (A) and 15 from patient LE (B) are shown. These sequences were checked by the criteria of their VDJH4 junction to be clonally unrelated. Asterisks represent deletions and insertions are indicated by vertical arrows. A slash indicates an incomplete sequence. 1͞19 sequences (13) from ND1 and 10͞15 sequences (1, 2, 4, 5, 8, 10–13, and 15) from patient LE displayed 0–1 mutation.

ratio corresponds to the type of sequence analyzed, being as high proportion of circulating IgG memory B cells harboring germline as one event per 32 mutations for unselected noncoding se- VH genes. To our knowledge, evidence of a large unmutated quences, and reduced to one event for 240 mutations for coding memory B cell compartment has never been reported in humans. sequences in which only a few in-phase insertions͞deletions can The process of affinity maturation of antibodies requires T be tolerated. helper cell-derived signals provided in soluble and membrane The analysis of the cohort of patients distinguishes clearly two forms (23, 24). Previous studies performed in CVID patients gave CVID cases who present 40 and 70% of unmutated switched B controversial results probably due to the heterogeneity of clinical cells in the peripheral blood. Most of their mutated IgG V and immunological features of patients. It has been reported that sequences carry less than 10 mutations, whereas in normal production of , such as IL-2, IL-4, IL-10, and Interfer- controls, V sequences harbor frequently 20–30 mutations. The on-␥, was impaired in CVID patients (25–27). More recently, it other six patients studied showed a level of mutation which varies was shown that the expression of CD40L is defective in some between 65 and 80% of the control level. It does not seem possible cases (28). Overall these data are consistent with an insufficient at this stage to assess whether these values remain in the normal in vivo B cell stimulation rather than with an intrinsic failure of variation range. The pattern of mutations, CDR clustering and B cells to differentiate. R͞S ratio appeared normal in all patients. However, for CVID patients LE and SO, examination of T cell In conclusion, while the majority of -switched B lym- phenotype, in vitro T cell proliferative responses to mitogens and phocytes derived from the peripheral blood display a high level , T cell production (IL-2, IL-4, Interferon-␥), of somatic mutation (refs. 21 and 22 and this report), we found and T-cell CD40L expression did not reveal obvious abnormal- two CVID cases among the eight patients studied with a large ities (not shown). In the LE case, we had the opportunity to Downloaded by guest on October 3, 2021 Immunology: Levy et al. Proc. Natl. Acad. Sci. USA 95 (1998) 13139

FIG.2 Continued.

review lymph node and ileal biopsies performed 19 years ago for cell help. In this assay, PBMC of patient LE appeared fully surgery for inflammatory colitis at a time when the CVID was capable of providing helper signals required for the induction of established already. New immunostaining analysis confirmed the Ig VH gene somatic hypermutation, thus eliminating the possi- presence of germinal centers (GC) in this patient, with a normal bility of major defects in T cell function in this case. PBMC from cellular organization: i.e., presence of B, T, and follicular den- patient SO behaved less efficiently, which might represent vari- dritic cells (not shown). Finally, we addressed directly the ques- ations inherent to such an in vitro assay. Altogether, taking into tion of the functionality of the T helper compartment in LE and SO cases by testing its capacity to induce mutation in a B cell line in vitro. The BL2 cell line can be induced to mutate its VH gene upon simultaneous IgM crosslinking and helper signals provided by a T cell (11). We show here that we can adapt this system of induction to the use of PHA-activated PBMC as a source of T

Table 3. Mutations in V3-23-C␥ transcript sequences from PBMC from controls and CVID patients Clones Mutations R͞S All Unmutated Total per bp, % CDRs FRs Controls ND3 18 0 410* 7.9 2.05 1.07 ND4 11 0 226* 7.1 4.68 1.2 Patients LE 21 15 50 0.8 1.75 1.5 SO 19 8 90 1.6 3.25 1.29 FL 8 0 118* 5.1 1.55 1.02 BE 8 0 125 5.4 3.54 1.08 ␥ MA 7 1 108* 5.4 3.6 1.36 FIG. 3. Frequency distribution of mutations within V3–23-C transcripts from normal donors and CVID patients LE and SO. Each TH 4 0 71* 6.2 4.16 2.54 histogram represents the percentage of V3–23 sequences displaying *Including one deletion or insertion. the number of mutations in a given range. Downloaded by guest on October 3, 2021 13140 Immunology: Levy et al. Proc. Natl. Acad. Sci. USA 95 (1998)

Table 4. In vitro induction of somatic mutation in BL2 cells in the We thank Michel Peuchmaur and Christine Copie for immuno- presence of activated T cells histological study of biopsy of patient LE, Serge Lebecque and Ste´phane De´nepouxfor advice with the BL2 assay, and Anne Durandy and Claire Mutation frequency, Hivroz for performing functional assays. This work was ϫ Ϫ4 ͞ BL2 T Sequences Events 10 mutation bp supported by grants from the Fondation Princesse Grace de Monaco, the ϩ —370 Ͻ0.75 Fondation de France, and the De´le´gation a`la Recherche Clinique de ϩ ND 34 6 5 l’Assistance Publique des Hoˆpitaux de Paris (CRC 97160). ϩ LE 37 4 3 ϩ SO 42 2 1.4 1. Sneller, M. C., Strober W., Eisenstein E., Jaffe, J. S. & Cunning- ham-Rundles, C. (1993) Ann. Intern. Med. 118, 720–730. 2. Spickett, G. P., Farrant, J., North, M. E., Zhang, J. G., Morgan, account the normal functional characteristics of the T cell com- L. & Webster, A. D. (1997) Immunol. Today 18, 325–328. partment, these results point toward a B cell defect in the two 3. Schaffer, F. M., Palermos, J., Zhu, Z. B., Barger, B. E., Cooper, hypomutated CVID analyzed in this study. M. D. & Volanakis, J. E. (1989) Proc. Natl. Acad. Sci. USA 86, What can we learn from these two cases on the complex 8015–8019. regulation leading to the generation of high affinity antibody 4. Olerup, O., Smith, C. I., Bjorkander, J. & Hammarstro¨m, L. molecules, and could there be any clinical consequences which (1992) Proc. Natl. Acad. Sci. USA 89, 10653–10657. can be drawn from this observation? In patient LE for example, 5. Weigert, M. G., Cesari, I. M., Yonkovich, S. J. & Cohn, M. (1970) 228, the normal histological aspect of GC, the physiological behavior Nature (London) 1045–1047. 6. Berek, C. & Milstein, C. (1988) Immunol. Rev. 105, 5–25. of T cell population, and the fact that some IgG sequences still 7. MacLennan, I. C. M. & Gray, D. (1986) Immunol. Rev. 91, 65–85. carry mutations imply that the different cellular steps that give 8. Jacob, J., Kelsoe, G., Rajewsky, K. & Weiss, U. (1991) Nature rise to mutated memory B cells can partially function. Different (London) 354, 389–392. scenarios could then explain the results observed: (i) targeting of 9. Berek, C., Berger, A. & Apel, M. (1991) Cell 67, 1121–1129. the hypermutation process to the Ig sequences could be ham- 10. Weiss, U. & Rajewsky, K. (1990) J. Exp. Med. 172, 1681–1689. pered. Such results have been obtained in mice with a Ig 11. Dene´poux, S., Razanajoana, D., Blanchard, D., Meffre, G., transgene carrying, for example, specific mutations in the Ig Capra, J. D., Banchereau, J. & Lebecque, S. (1997) 6, intronic enhancer (29), a sequence that was shown previously to 35–46. be indispensable for the hypermutation process to occur (30). In 12. Lebecque, S. G. & Gearhart, P. J. (1990) J. Exp. Med. 172, such a scheme, mutation can rarely occur properly, but if occur- 1717–1727. 13. Stewart, A. K., Huang, C., Stollar, B. D. & Schwartz, R. S. (1993) ring, sequences with a rather large number of mutations, as found J. Exp. Med. 177, 409–418. for LE and SO, can be observed, suggesting that once triggered 14. Yamada, M., Wasserman, R., Reichard, B. A., Shane, S., Caton, the process is able to continue within the same clone (29); (ii) the A. J. & Rovera, G. (1991) J. Exp. Med. 173, 395–407. precise molecular events allowing the introduction of mutation 15. Mattila, P. S., Schugk, J., Wu, H. & Ma¨kela¨,O. (1995) Eur. into the V sequences, or any step in the cascade of signal J. Immunol. 25, 2578–2582. transduction leading to the activation of the mutation process, 16. Rogozin, I. B. & Kolchanov, N. A. (1992) Biochim. Biophys. Acta could be altered but not completely abolished thereby explaining 1171, 11–18. the presence of germline and mutated sequences. A more precise 17. Klein, U., Ku¨ppers, R. & Rajewsky, K. (1993) Eur. J. Immunol. analysis of the patient’s B cell antigen receptor and intracellular 23, 3272–3277. signaling components should allow us to distinguish between 18. Wagner, S. D., Elvin, J. G., Norris, P., McGregor, J. M. & these different possibilities; (iii) the amount of B cells driven into Neuberger, M. S. (1996) Int. Immunol. 8, 701–705. 19. Wilson, P. C., de Bouteiller, O., Liu, Y. J., Potter, K., Banchereau, the GC could be largely reduced, this defect in the T-B cell J., Capra, J. D. & Pascual, V. (1998) J. Exp. Med. 187, 59–70. collaboration resulting in the abnormal expansion of a B cell 20. Goossens, T., Klein, U. & Ku¨ppers, R. (1998) Proc. Natl. Acad. population having switched before its entry into the GC (31). Sci. USA 95, 2463–2468. Along the same line, recycling of memory B cells into the GC 21. Klein, U., Ku¨ppers, R. & Rajewsky, K. (1994) J. Exp. 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