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Local Somatic Hypermutation and Class Switch Recombination in the Nasal Mucosa of Allergic Rhinitis Patients

This information is current as Heather A. Coker, Stephen R. Durham and Hannah J. Gould of September 23, 2021. J Immunol 2003; 171:5602-5610; ; doi: 10.4049/jimmunol.171.10.5602 http://www.jimmunol.org/content/171/10/5602 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Local Somatic Hypermutation and Class Switch Recombination in the Nasal Mucosa of Allergic Rhinitis Patients

Heather A. Coker,1* Stephen R. Durham,† and Hannah J. Gould2*

Immunoglobulin E is produced by nasal B cells in response to . We have analyzed IgE VH region sequences expressed in ؉ the nasal mucosa of patients suffering from allergic rhinitis. VH region sequences were amplified by RT-PCR from IgE B cells from nasal biopsies. In two of six patients, sequence analysis clearly demonstrated the presence of closely related IgE؉ B clones: cells displaying identical signature regions across CDR3/FWR4, indicating a common clonal ancestry, but a mixture of ؉ shared and diverse somatic across the VH region. Furthermore, in one of the two patients exhibiting related IgE ,clones, five IgA؉ B cell clones, related to the IgE؉ B cell family, were also isolated from the patient’s nasal mucosa. This evidence Downloaded from combined with the local expression of mRNA transcripts encoding activation-induced , suggests that local somatic hypermutation, clonal expansion, and class switch recombination occur within the nasal mucosa of allergic rhinitics. The presence of related B cells in the nasal mucosa does not appear to result from the random migration of IgE؉ cells from the systemic ⑀ pool, as analysis of a nonatopic subject with highly elevated serum IgE did not exhibit any detectable VH-C transcripts in the nasal mucosa. We have provided evidence that suggests for the first time that the nasal mucosa of allergic rhinitics is an active site

for local somatic hypermutation, clonal expansion, and class switch recombination, making it of major significance for the http://www.jimmunol.org/ targeting of future therapies. The Journal of Immunology, 2003, 171: 5602–5610.

llergic reactions occur mainly in the mucosal tissues of rheumatoid arthritis (3, 4). In the local mucosal environment, sig- the respiratory tract, gut, and skin. Susceptibility at these nals to initiate SHM and CSR are available (5, 6), as in vitro A sites is due to the presence of mast cells bearing the high studies have demonstrated that, in the presence of Ag, T cells have affinity IgE receptor, Fc⑀RI, sensitized by Abs of the IgE class. the ability to induce SHM and CSR by the production of Cross-linking of the receptors by allergen binding to IgE Abs trig- (such as IL-4) and also their interaction via CD40:CD40- gers immediate . Mast cells in the nasal mucosa of and CD80:CD28 with B cells (7). Evidence suggesting local CSR patients with allergic rhinitis are sensitized by IgE Abs that are has been presented for IgA in the murine gut mucosa (8), for IgE by guest on September 23, 2021 produced locally by resident plasma cells (1). In an allergic indi- in the human nasal mucosa of allergic rhinitics (9Ð11), and in the vidual, local IgE production persists for long periods in the ab- human lung mucosa in allergic asthma (12, 13). Evidence for local sence of allergen, enabling an immediate response upon re-expo- SHM has been presented for IgE in the lung mucosa of an allergic sure to allergen. However, little is known about the history of the asthmatic patient (13). We have examined the evidence for local IgE-producing B cells, in particular when and where the precursor SHM and CSR in six patients with allergic rhinitis. cells underwent class switch recombination (CSR)3 to IgE (involv- SHM results from the stepwise accumulation of predominantly ing rearrangement of the C region genes encoding the various Ab single nucleotide substitutions into the V region DNA. This step- classes, e.g., C␮ to C⑀) and affinity maturation by somatic hyper- wise accumulation of mutations enables the genealogy of a B cell (SHM). to be traced, relying on the unique complementarity-determining Both SHM and CSR are stimulated by Ag in the germinal cen- region 3/ 4 (CDR3/FWR4) clonal signature gen- ters of lymphoid tissue (2), but it is becoming increasingly appar- erated by the VDJ recombination of the progenitor cell. It is esti- ent that these processes may also occur locally at sites of chronic mated that mutations are introduced at a rate of 10Ϫ4-10Ϫ3 per or recurrent Ag stimulation, as has been clearly demonstrated for per generation (14). A single mutation may bring about as much as a 10-fold increase in Ab affinity (14, 15). Mutations are introduced at a high level across the CDRs, but additionally the *The Randall Centre, King’s College London, London, United Kingdom; and †Upper RGYW motif (in which R ϭ AorG,Yϭ CorT,Wϭ AorT, Respiratory Medicine, Imperial College School of Medicine at National Heart and and G is mutated) or the reverse complement WRCY is a frequent Lung Institute, London, United Kingdom target of mutation, particularly at the serine codons AGC and Received for publication April 21, 2003. Accepted for publication September 4, 2003. AGT (16). The molecular mechanism of SHM has not been The costs of publication of this article were defrayed in part by the payment of page fully elucidated, although errors are thought to be introduced by charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. an activation-induced cytidine deaminase (AID) and error- 1 H.A.C. is supported by a Biotechnology and Biological Sciences Research Council prone polymerase-dependent process (17Ð19). Several DNA PhD studentship. polymerases have been suggested as candidates, including DNA 2 Address correspondence and reprint requests to Dr. H. J. Gould, The Randall Centre, polymerase ␩, ␬, ␫, and ␨ (20Ð23). AID has also been shown to King’s College London, Guy’s Campus, London, SE1 1UL, United Kingdom. E-mail be required for CSR (17, 18). address: [email protected] We present evidence generated by RT-PCR and DNA sequenc- 3 Abbreviations used in this paper: CSR, class switch recombination; AID, activation- ⑀ induced cytidine deaminase; CDR, complementarity-determining region; FWR, ing of VH-C sequences from the nasal mucosa of allergic rhinitis framework region; RAST, radioallergosorbent test; SHM, somatic hypermutation. patients. Our work is the first to demonstrate in two allergic rhinitis

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 5603

ϩ patients the clear presence of related IgE B cell clones in the resuspended in 20 ␮l RNase-free water. The concentration of RNA was nasal mucosa, B cells that exhibit shared ancestry (judged by iden- determined from the absorbance at 260 nm. tical CDR3/FWR4 motifs), and both shared and diverse somatic ⑀ mutations. In addition, detailed investigation enabled the detection RT-PCR of VH-C transcripts ϩ of sequences from IgA B cell clones from the nasal biopsy of one RNA (5 ␮g) was included in a 40 ␮l cDNA reaction with Moloney murine patient. These sequences exhibited shared ancestry and both shared leukemia virus reverse transcriptase (Invitrogen) using an oligo(dT) primer and diverse somatic mutations with the related IgEϩ B cell clones (Invitrogen). A total of 5 ␮l of cDNA was added to a 50 ␮l PCR, which included each VH leader region class-specific primer (VH1L-VH6L) at 0.5 isolated from the same nasal biopsy sample. IgE VH-CH region ␮M and the C⑀1-specific primer (C⑀1) at 0.5 ␮M, dNTPs at 0.25 mM, and amplification from the nasal mucosa of a healthy nonatopic subject contained 1.26 U PFU DNA polymerase (Promega). The reaction was ini- with highly elevated serum IgE was negative, implying that the tially denatured at 95¡C for 2 min, then subjected to 30 cycles of denatur- families of related clones seen in allergic patients were unlikely to ation at 94¡C for 1 min, annealing at 56¡C for 2 min, and extension at 72¡C ␮ have resulted from the random migration of IgEϩ B cells from a for 2 min before a final extension at 72¡C for 10 min. A total of 5 lofthe first PCR was then transferred into a second nested PCR, which differed systemic pool. only in that it included VH class-specific primers homologous to FWR1 ⑀ ⑀ Furthermore, RT-PCR analysis demonstrated the presence of (VH1F-VH6F) and an internal C 1 primer (C 2). This reaction was incu- mRNA-encoding AID in the nasal mucosa in five of seven allergic bated with an initial denaturation at 95¡C for 2 min, then 30 cycles of rhinitis patients, the first reported instance of local AID expression denaturation at 94¡C for 1 min, annealing at 65¡C for 2 min, extension at 72¡C for 2 min, then a final extension at 72¡C for 10 min. in humans. We propose that local somatic hypermutation, clonal ⑀ The PCR for amplification of the separate VH classes of the VH-C expansion, and class switch recombination take place in the nasal transcripts in the PBMC of the nonatopic subject GJ29 used the same Downloaded from mucosa of allergic rhinitis patients. We suggest that this local ac- conditions as above, using the appropriate VH primer in each reaction. Ј tivity is fundamental to the pathogenesis of allergic disease. The primers used were as follows: VH1L, 5 -CCATGGACTGGACCT Ј Ј Ј Ј GGA-3 ;VH2L, 5 -CAGATGGACATACTTTGTTCCAC-3 ;VH3L, 5 - Ј Ј CCATGGAGTTTGGGCTGAGC-3 ;VH4L, 5 -CGATGAAACACCTGT Materials and Methods Ј Ј Ј Ј GGTTCTT-3 ;VH5L, 5 -ATGGGGTCAACCGCCATCCT-3 ;VH6L, 5 - Samples from allergic rhinitis patients Ј Ј GATGTCTGTCTCCTTCCTCAT-3 ;VH1F,5 -CAGGTGCAGCTGGTGC Ј Ј Male and female donors between the ages of 18 and 55 were recruited from AGTCTG-3 ;VH2F, 5 -GTCTTGTCCCAGGTCAACTTAAGGGAGTC TT-3Ј;V3F, 5Ј-GAGGTGCAGCTGGTGGAGTCTG-3Ј;V4F, 5Ј- H H http://www.jimmunol.org/ the Royal Brompton Hospital Clinic (London, U.K.) or by adver- Ј Ј CAGGTGCAGCTGCAGGAGTCGG-3 ;VH5F, 5 -GAGGTGCAGCTGC tisement in the local press. None had received immunotherapy, and any Ј Ј TGCAGTCTG-3 ;VH6F, 5 -CTGTCACAGGTACAGCTGCAGCAGTC medication was discontinued at least 2 wk before nasal biopsy. Biopsies Ј ⑀ were performed at the Royal Brompton Hospital with the approval of the AG-3 (based on VH region primers published previously (25, 26)); C 1, 5Ј-TGTCCCGTTGAGGGAGCCTGT-3Ј;C⑀2,5Ј-GGGTCGACAGTCACGG local Ethics Committee and the patients’ written informed consent. The Ј ⑀ allergic status of the donors was assessed by medical history, skin-prick AGGTGGCATT-3 (based on C primers published previously (27)). tests, and serum allergen-specific IgE (radioallergosorbent test (RAST)). ␮ ␣ ␥ Six patients who exhibited a total IgE of over 200 IU/ml were selected for RT-PCR of VH-C ,VH-C , and VH-C transcripts ⑀ the study of VH-C transcripts. Biopsies were taken according to a previous ϩ ϩ ϩ detailed protocol (24) in which a 2.5-mm3 biopsy was randomly taken from PCR amplification of sequences from IgM , IgG ,orIgA B cells was the undersurface of the inferior turbinate, behind the anterior insertion into conducted as separate reactions based on a multiple step PCR published the lateral wall of the nose and 8Ð10 cm distal to the nearest lymphoid previously (28, 29). by guest on September 23, 2021 tissue, Waldeyer’s ring in the pharynx. The random nature of the biopsies prevented the sampling of any defined cell populations from within the PCR 1 inferior turbinate. Biopsies were placed in 1.5 ml HBSS (Invitrogen, Pais- ⑀ ley, U.K.) and washed to remove blood before being transferred to a cryo- The initial nested PCR was conducted as for the amplification of VH-C , Ϫ o except that only the VH region primer specific to the VH class of interest tube and snap frozen in liquid N2 before being stored at 70 C. was used, e.g., reaction 1 used VH5L in conjunction with each of the A total of 25 ml blood was taken from each patient, and PBMC was ␮ ␣ ␥ isolated by Ficoll-Paque density-gradient centrifugation. The PBMC pellet individual outer CH primers (C 1, C 1, or C 1). All other conditions was snap frozen and stored at Ϫ70oC. The samples were treated to extract remained the same as those specified above for IgE and the reaction de- RNA and manufacture cDNA as for the tissue samples, with the exception natured at 95¡C for 2 min, followed by 30 cycles of denaturation at 94¡C that 1 ml RNAWIZ buffer was added to the PBMC pellet (ϳ4 ϫ 107 cells) for 1 min, annealing at 56¡C for 2 min, extension at 72¡C for 2 min, and and the cells were resuspended by pipetting before dividing into four a final extension of 72¡C for 15 min. 250-␮l aliquots, one of which was used for future molecular analysis. PCR 2 Samples from the nonatopic subject Reaction 2 of the nested PCR for amplification of sequences from IgMϩ, A healthy 55-year-old male (G.J.) was recruited with no previous medical ϩ ϩ IgG ,orIgA cells used an inner VH5 primer, VH5Fm, with each of the history of any allergic symptoms and a negative skin-prick test and RAST ␮ ␣ ␥ inner CH primers (C 2, C 2, or C 2) to further amplify the VH5-CH se- for all common (a slightly raised RAST of 0.71 IU/ml (normal quences. All other conditions remained the same, except initial denatur- Ͻ 0.35 IU/ml) was detected for Aspergillus fumigatus). No other abnormal ation was conducted at 95¡C for 2 min, followed by 30 cycles of denatur- medical conditions were reported, although the subject’s total serum IgE ation at 94¡C for 1 min, annealing at 54¡C for 2 min, extension at 72¡C for was 1834 IU/ml. A nasal biopsy and PBMC sample were taken according 2 min, then a final extension at 72¡C for 15 min. to the protocol above. The PCR products from each second PCR were subjected to electro- Amplification of GAPDH from the samples used the following PCR phoresis on a 1% agarose gel, and the DNA was excised. A total of 5 ␮l ␮ ␮ method: 5 l of cDNA was added to a 50 l PCR, which included both a of the gel-extracted PCR product was carried forward into a third, semi- ␮ GAPDH forward and reverse primer each at 1 M (GAPDH forward, nested PCR for each . 5Ј-ATTTGGTCGTATTGGGCGCCTGGTC-3Ј; GAPDH reverse, 5Ј- TCATACTTCTCATTGTTCACACCCATG-3Ј), dNTPs at 0.25 mM, and included 1.26 U PFU DNA polymerase (Promega, Madison, WI). The PCR 3 reaction was initially denatured at 95¡C for 2 min, then subjected to 30 Reaction 3 was seminested, using the gel-extracted PCR 2 products with cycles of denaturation at 94¡C for 1 min, annealing at 59¡C for 2 min, and the inner of each set of C primers and a CDR3/FWR4 primer specific for extension at 72¡C for 2 min before a final extension at 72¡C for 10 min. H each clonal family (e.g., B16V5 specific for the SO16 VH5 family) to Ј RNA extraction amplify from CDR3 to the 5 of CH. The conditions for PCR 3 were as detailed above, except the initial denaturation 95¡C was for 2 min, then 20 Biopsies were transferred to an RNase-free tube and homogenized in 250 cycles of denaturation at 94¡C for 1 min, annealing at 53¡C for 2 min, ␮l RNAWIZ buffer (Ambion, Austin, TX) with 300 vigorous pulses using extension at 72¡C for 2 min, and a final extension at 72¡C for 15 min. PCR an Eppendorf homogenizer (Anachem, Luton, U.K.). Total RNA was ex- products were cloned and sequenced as below before proceeding to the tracted from each sample, according to the manufacturer’s protocol, and following stage. 5604 LOCAL SOMATIC HYPERMUTATION AND CLASS SWITCH RECOMBINATION

PCR 4 94¡C for 1 min, annealing at 60¡C for 1 min, extension at 72¡C for 2 min, then a final extension at 72¡C for 5 min. ␮ To obtain the VH region sequences from specific IgA clones, 5 lofthe Details of the primers are as follows: AID1P, 5Ј-GAGGCAAGAAGA gel-extracted PCR 2 products was used in the seminested reaction 4 with CACTCTGG-3Ј; AID2P, 5Ј-GTGACATTCCTGGAAGTTGC-3Ј (based conditions as for the previous reactions, except for the use of the VH5Fm on AID primers published previously (17)); AID3P, 5Ј-TAGACCCTGGC primer and SO16A4 (specific for the D-J junction of the family) with initial CGCTGCTACC-3Ј; AID4P, 5Ј-CAAAAGGATGCGCCGAAGCTGTCT denaturation at 95¡C for 2 min, then 20 cycles of denaturation at 94¡C for GGAG-3Ј (based on AID primers published previously (32)). 1 min, annealing at 54¡C for 2 min, extension at 72¡C for 2 min, and a final extension at 72¡C for 15 min. Cloning and sequencing of PCR products PCR 5 All PCR mixes were subjected to electrophoresis on 1% agarose gels, except for the C␮,C␣, and C␥ PCR products from reaction 3 and the AID An additional experiment was conducted to reamplify the complete se- PCR products that were subjected to electrophoresis on 2 or 1.5% agarose quence of the VH5-IgA 1 from CDR1 in the VH region through the gels, respectively. Bands of the appropriate size were gel purified (Qiagen, ␣ D and JH region to C (previously only analyzed by PCR 3 and 4 gener- Valencia, CA) and cloned using the Zero Blunt TOPO PCR Cloning Kit ating different fragments of the full sequence). The seminested PCR 5 (Invitrogen). EcoRI digests confirmed the presence of the cloned insert, included: 5 ␮l of the gel-extracted PCR 2 products with conditions as and subsequent minipreps were sequenced using M13F or M13R primers above, except that the specific CDR1 primer (B16V5C1) was used in con- ( Unit, King’s College London, and ABC Sequencing junction with the inner C␣ primer. The reaction was then incubated for an Service, Imperial College). initial denaturation at 95¡C for 2 min, then 20 cycles of denaturation at 94¡C for 1 min, annealing at 53¡C for 2 min, extension at 72¡C for 2 min, Identification of sequences and a final extension at 72¡C for 15 min. Assignment of V , D, and J genes and their somatic mutations was con- The primers used were as follows: C␮1, 5Ј-GCTCGTATCCGACGGG H H Ј ␮ Ј Ј ␣ Ј ducted according to their homology with the germline sequences detailed Downloaded from GAAT-3 ;C2, 5 -CGAGGGGGAAAAGGGT-3 ;C1, 5 -GGGAC on the VBase database (www.mrc-cpe.cam.ac.uk). The identity of a D gene CACGTTCCCATCT-3Ј;C␣2, 5Ј-CTCAGCGGGAAGACC-3Ј;C␥1, 5Ј- Ј ␥ Ј Ј given a score of less than 50 by VBase was regarded as undefined (in which CGGTTCGGGGAAGTAGTCCTT-3 ;C 2, 5 -CAGGGGGAAGACCGAT-3 ϩ5 is awarded for a nucleotide match and Ϫ4 for a mismatch). Where the (C␮2 and C␣2 based on that published previously (30, 31)); V 5Fm, H alignment of the V and D gene or D and J was seen to overlap, prece- 5Ј-TGCAGCTGCTGCAGTCTG-3Ј; B16V5, 5Ј-AGACATAAGAGTG H H dence was given to V and J so that a truncated D gene sequence was GCTCG-3Ј; SO16A4, 5Ј-TGGCCCCAGTAGTCAGC-3Ј; b16V5C1, H H Ј Ј presented. The CDR3/FWR4 region of each sequence was used to deter- 5 -TATAAGTTTGCCACCTATGCC-3 . mine clonality. Genealogical trees were constructed based on the premise http://www.jimmunol.org/ RT-PCR of AID transcripts that shared mutations were acquired early, and individual mutations were acquired later in the evolution of the clone. Ab isotype and AID sequence To amplify AID mRNA transcripts by RT-PCR, cDNA was manufactured homology were established using the National Center for Biotechnology from nasal biopsy samples taken from seven allergic rhinitis patients, as Information BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). above. A total of 5 ␮l cDNA was added to each 25 ␮l PCR, which included the forward primer AID1P at 0.2 ␮M and the reverse primer AID2P at 0.2 Results ␮M, dNTPs at 0.2 mM, MgCl at 1 mM, and included 1.25 U Platinum ϩ 2 VH sequence analysis and IgE B cell clonality TaqDNA polymerase (Invitrogen, San Diego, CA). The reaction was ini- ⑀ tially denatured at 94¡C for 5 min, then subjected to 30 cycles of denatur- We have analyzed VH-C sequences from the nasal biopsy sam- ation at 94¡C for 1 min, annealing at 56¡C for 1 min, and extension at 72¡C ples of six patients (five multiallergics and one grass pollen mono- ␮ for 2 min before a final extension at 72¡C for 5 min. A total of 5 lofthe allergic), characterized by skin-prick test, RAST, and a medical by guest on September 23, 2021 first PCR was then transferred into a second PCR, which differed only in that it included an inner forward primer AID3P at 0.2 ␮M and an inner history of allergic rhinitis for at least 2 years (Table I). The se- reverse primer AID4P at 0.2 ␮M. This reaction was incubated with an lected patients exhibited serum IgE levels Ͼ200 IU/ml to ensure initial denaturation at 94¡C for 5 min, then 30 cycles of denaturation at success in RT-PCR amplification of VH regions (27, 33). Fifteen

Table I. Patients’ clinical and experimental data

No. of B Cell Families (No. of Status Total Members Isolated From at Serum Specific Each Clonal Family) in No. of V Region Patient Biopsya IgE (IU/ml) IgE (IU/ml)b Allergiesc Nasal Biopsy Sequences Analyzed

JB7 O/P 236 G ϭ 9.25 HDM, G, C, D, CL 15 C ϭ 11.1

CM10 O/P 382 HDM ϭ 1.09 HDM, G, M, T, B, 15 C ϭ 3.36 C, D, H, CL G ϭ 101

HD14 I/P 787 HDM ϭ 2.68 HDM, G 15 G ϭ 101

SO16 I/P 514 G ϭ 101 G, T, B, D, 2 (2,3) 15

HD17 I 2745 G ϭ 101 G 15

AP19 I/P 414 HDM ϭ 1.23 HDM, G, C, D, H, 1 (3) 15 C ϭ 32.10 CL G ϭ 60.20

GJ29 Nonatopic 1834 AP ϭ 0.71

a In season (I), out of season (O), perennial (P). b Determined by RAST for the allergens; house dust mite (HDM), grass (G), cat (C), dog (D), tree (T), A. fumigatus (AF). c House dust mite (HDM), grass pollen (G), mugwort (M), three trees (T), silver birch (B), cat (C), dog (D), horse (H), A. fumigatus (AF), C. herbarum (CL), A. alternata (A), determined by skin-prick test. The Journal of Immunology 5605

⑀ sequences were analyzed from the nasal biopsy samples of each VH-C sequences containing mutations from the germline that patient. followed the expected trends of SHM were isolated from all nasal All samples were PCR amplified using PFU DNA polymerase, biopsy samples (Table II). Multiple copies of identical sequences which exhibits a proofreading ability resulting in a very low ex- were isolated from all samples. All sequences contained in-frame perimental error rate compared with other DNA polymerases. The rearrangements, implying successful translation of mRNA into IgE experimental error rate generated in our system of nested RT-PCR protein. ϳ amplification was determined to be 1 in 4720 bp, the equivalent Detailed analysis of VH region sequences sharing identical of 1 mutation introduced experimentally in approximately every CDR3/FWR4 regions was conducted in each sample, allowing the ϩ 17 VH region sequences amplified in this manner. Experimental construction of families of clonally related IgE B cells (nucleo- error rate was determined by analysis of the number of mutations tide sequences submitted to EMBL, accession numbers AJ491904- ⑀ ⑀ introduced into the C DNA amplified by the VH-C PCR (data not AJ491915). Due to the experimental error rate in our system, two shown). families (each comprising only two B cell clones) were discarded ⑀ Initial analysis of VH-C sequences generated by RT-PCR dem- on the basis that the single mutations linking each of the related onstrated that we were able to detect all VH gene families with the clones may have been experimental artifacts. When PBMC from exception of VH2, one of the most infrequently expressed VH gene each of the patients was analyzed (and three small families, again, families (34). VH, D, and J genes were assigned using the VBase each comprising two clones linked by a single nucleotide differ- database (www.mrc-cpe.cam.ac.uk), allowing mutations from the ence, were discarded), only two distantly related B cell clones germline to be identified. were detected (from patient HD17, who exhibited the highest total Downloaded from serum IgE of all the patients). These differed by 19 VH mutations,

4D,and1JH mutation, while still exhibiting clear homology across the signature region (data not shown). ϩ ϩ Table II. VH region sequences isolated from IgE B cells in the nasal Clear evidence of clonally related IgE B cells, closely linked mucosa of allergic rhinitis patients by multiple differences in their VH mutations, was however evi- dent in two of the six nasal biopsy samples analyzed (patients Germline VH Gene Percentage of SO16 and AP19). Sequences suggesting two families of related http://www.jimmunol.org/ with Greatest Mutation from No. of Times ϩ Patient Sequence Homologya Germline Isolated IgE B cell clones were isolated from SO16. One family of B cell

clones expressed a VH3 gene and comprised three B cell clonal JB7 B7C14 1Ð46 22.7 1 members (of which one was a hypothetical intermediate, not iso- B7C9 3Ð23 5.5 1 B7C17 3Ð74 4.8 8 lated experimentally). The other family of B cell clones isolated B7C22 4Ð04 7.3 5 from SO16 expressed a VH5 gene and comprised four B cell CM10 B10C7 3Ð30/3Ð30.5 2.9 1 clones, of which one was a hypothetical intermediate. The family B10C13 3Ð30/3Ð30.5 4.4 1 of B cells from AP19 also expressed V 5. The alignment of these B10C1 3Ð66 10.9 1 H B10C2 3Ð66 10.7 12 related clones (Fig. 1) enabled the formation of genealogical trees, by guest on September 23, 2021 HD14 B14C16 1Ð02 5.9 1 demonstrating the clonal relationships between the B cells (Fig. 2). B14C9 3Ð15 9.9 1 In only one instance did sequence data suggest the presence of an B14C4 3Ð30 11.0 1 IgEϩ B cell clone in the PBMC that was distantly related to B cell B14C2 3Ð30 7.0 8 clones detected in the nasal mucosa (one clone from the PBMC of B14C11 4Ð59 6.4 1 B14C19 5Ða 4.5 1 SO16 shared five mutations and the same VH-D-JH rearrangement ϩ B14C7 5Ð51 7.4 1 (data not shown) with the family of VH3 IgE B cell clones de- B14C12 5Ð51 4.8 1 picted in Fig. 2A). SO16 B16C12 3Ð20 3.3 2 B16C18 3Ð23 1.8 1 # B16C2 3Ð30 4.1 1 Identification of related IgE and IgA B cell clones in the nasal B16C3# 3Ð30 5.2 2 B16C10 3Ð33 5.6 2 mucosa B16C1 3Ð65 ND 1 Further investigation of patients SO16 and AP19 was undertaken B16C13 4Ð39 3.7 1 to determine whether sequences from cells related to the families B16C5 5Ð51 2.9 1 ϩ B16C8* 5Ð51 8.5 1 of IgE B cell clones, but which expressed isotypes other than IgE, B16C11* 5Ð51 7.7 1 could be detected in the nasal mucosa. A three-stage PCR ap- B16C14* 5Ð51 7.7 2 proach based on that documented previously (28, 29) was used to HD17 B17C13 1Ð69 4.8 1 achieve this, using a nested reaction (PCR 1 and 2) to amplify VH3 B17C1 3Ð30 9.6 1 ϩ ϩ ϩ B17C16 4ÐND 12.1 1 or VH5 sequences from either IgM , IgG ,orIgA B cells in B17C9 4Ð30.1/4Ð31 8.4 1 each cDNA sample. The third PCR in each case used a seminested B17C37 4Ð30.1/4Ð31 8.8 1 approach utilizing a forward primer specific for the clonal signa- B17C15 4Ð30.2 7.7 5 ture region (VH-D junction) of interest, combined with the inner B17C31 4Ð30.2 8.5 3 set of primers for IgM, IgG, or IgA. B17C32 4Ð59 5.6 1 B17C7 7Ð04.1 3.7 1 No sequences suggesting the presence of B cell clones of any ⑀ AP19 B19C16 1Ð18 2.2 1 other isotype related to the VH3-C family from SO16, or the ⑀ B19C6 4Ð04 5.2 1 VH5-C family from AP19, were detected. In contrast, successful B19C3 4Ð30.1/4Ð31 2.5 2 $ amplification of a signature region from an IgA2 B cell related to B19C2 5Ð51 7.1 8 ϩ B19C4$ 5Ð51 6.4 1 the VH5 family of IgE B cell clones was isolated from the nasal B19C8 5Ð51 ND 1 mucosa of patient SO16 (Fig. 3A) (nucleotide sequence submitted B19C14$ 5Ð51 7.5 1 to EMBL, accession number AJ491916), indicating the presence ϩ a of at least one IgA2 B cell sharing a common ancestry with the VH gene locus detailed such that 5Ð51 denotes a VH5 gene. ϩ *,# and $ denote members of each IgEϩ B cell clonal family. previously isolated family of IgE B cells. 5606 LOCAL SOMATIC HYPERMUTATION AND CLASS SWITCH RECOMBINATION Downloaded from

FIGURE 1. Alignment of VH-D-J sequences am- plified by RT-PCR from the nasal mucosa of allergic rhinitis patients SO16 and AP19 indicated the pres- ence of clonally related IgEϩ B cells. Sequences are http://www.jimmunol.org/ detailed such that, e.g., sequences isolated from AP19 have closest homology with the germline se- quence of a VH gene from the VH5 family, locus 5-51 (VH5-51), the D gene D1-26, and the JH gene JH3b. (Although the D1-26 germline sequence in reading frame 2 used in AP19 encodes a stop codon, denoted with an asterisk, the nontemplated insertion between the VH and D gene is such that rearranged sequences demonstrate an open reading frame.) CDR

regions are indicated and PCR primer regions are by guest on September 23, 2021 underlined. Mutations in the primer regions were not included.

Another, separate, seminested PCR (PCR 4) was used to am- that exhibited a complex pattern of shared and unique somatic ϩ plify the full VH region sequence from the related IgA B cell(s) mutations throughout the full length of the VH region sequence from which the signature region had been amplified in PCR 3. (nucleotide sequences submitted to EMBL, accession numbers Sequences were detected from five IgAϩ B cell clones that were AJ536081Ð5). These sequences were aligned, and homology with related to each other and to the IgEϩ family of B cell clones and the sequences from the related IgEϩ B cell clones, also isolated The Journal of Immunology 5607 Downloaded from http://www.jimmunol.org/

FIGURE 2. Sequences isolated from related IgEϩ B cell clones amplified from the nasal mucosa enabled the construction of genealogical trees for allergic rhinitis patients SO16 (A) and AP19 (B). The closest germline gene homology is depicted at the top of each tree in a box. Sequences isolated from clones are depicted in an oval. Hypothetical intermediates are denoted with X. Mutations are detailed such that 27(3) represents a single nucleotide substitution occurring at codon 27, nucleotide 3. Each family is detailed such that, e.g., sequences isolated from AP19 have closest homology with the by guest on September 23, 2021 germline sequence of a VH gene from the VH5 family, locus 5-51 (VH5-51), the D gene D1-26, and the JH gene JH3b.

from the nasal mucosa of patient SO16, could clearly be seen shown). In addition, a strong IgE signal was obtained from PCR

(Fig. 3B). amplification of IgE VH regions from the subject’s PBMC (Fig. ⑀ To eliminate the possibility that the IgA clones were PCR arti- 4A). When VH-C transcripts from the subject’s PBMC were am- facts (unlikely as both the shared and diverse mutations were dis- plified using primers to amplify the different VH class in separate persed throughout the VH region), we successfully reamplified in reactions, the subject’s IgE was shown to be comprised of B cells ␣ Ј PCR 5 the complete VH-D-JH-C 2 sequence 3 of CDR1 from the expressing VH1, VH3, VH4, VH5, and VH6 (Fig. 4B), thereby dem- IgA clone C1 that had previously been partially amplified by PCR onstrating that the repertoire of IgEϩ B cells in the PBMC was 4 (nucleotide sequence submitted to EMBL, accession number diverse and that a mono/oligoclonal lymphoproliferative disorder AJ536086) (data not shown). Both the signature region and the did not account for the subject’s raised total serum IgE. somatic mutations were identical with that amplified previously, confirming that the sequences presented in this study were unlikely to be PCR artifacts. Local AID mRNA transcripts in the nasal mucosa of allergic rhinitis patients ⑀ VH-C sequences were not isolated from the nasal mucosa of a To investigate the presence of local AID mRNA transcripts in nonatopic subject with highly elevated serum IgE allergic rhinitis, a nested PCR technique was applied to nasal bi- To investigate whether random migration of IgEϩ B cells from the opsy samples from seven patients. Two of these samples (CM10 ⑀ circulation to the nasal mucosa occurs in subjects exhibiting high and AP19) had also been used in the VH-C transcript analysis systemic IgE levels (a possible explanation for the presence of (restricted sample size unfortunately prevented the analysis of the ⑀ related B cell clones seen in the nasal mucosa of allergic rhinitis other five nasal biopsies on which VH-C analysis had been con- patients), a nasal biopsy was taken from nonatopic healthy subject ducted). All of the patients in which AID analysis was studied GJ29, who exhibited extremely elevated serum IgE (total serum were either biopsied within the grass pollen season or suffered ϭ ⑀ IgE 1834 IU/ml). No VH-C sequences could be isolated from from perennial allergic rhinitis. When the PCR products were sub- the nasal biopsy sample (Fig. 4A), although the nasal biopsy ex- jected to agarose gel electrophoresis, AID mRNA was shown to be ␮ ␣ hibited a strong GAPDH signal (Fig. 4C) and VH-C ,VH-C , and present in the nasal mucosa of five of the seven patients (Fig. 5). ␥ ϩ VH-C PCR products, corresponding to IgM, IgA, and IgG B The PCR products were confirmed by Southern blot analysis using cells, respectively, could be amplified from the sample (data not a probe that spanned exons 3 and 4 (data not shown). 5608 LOCAL SOMATIC HYPERMUTATION AND CLASS SWITCH RECOMBINATION Downloaded from http://www.jimmunol.org/

ϩ ϩ FIGURE 3. Alignment of the signature region and VH-D-JH sequence RT-PCR amplified from related IgE and IgA B cell clones from the nasal Ј mucosa of allergic rhinitis patient SO16. The signature region sequence (which was joined to the 5 end of the IgA2 CH region) generated by PCR 3 and also the corresponding VH-D-JH sequences from the related IgA clones (amplified by PCR 4) are both detailed, as the join to IgA was only explicit in the signature region sequence. A, An identical signature region sequence was amplified by PCR 3 from both IgE and IgA2 B cell clones. Sequences had greatest homology with the germline VH5 gene V5-51, D4-17, and JH4b (positions indicated). Primers used in PCR 3 are marked in bold, and regions of homology between the sequences are denoted with vertical lines. B, The VH region sequences of the related IgA clones amplified by PCR 4 were aligned with the related IgE clones and exhibited both shared and unique VH region mutations. The sequences had greatest homology with the germline VH5VH gene V5-51 and D4-17 (indicated). The JH gene JH4b was only partially amplified and was completely encompassed by the primer region. Primer regions are underlined.

Mutations in the primer regions were not included. by guest on September 23, 2021

Discussion subsequent clonal expansion are a likely explanation for the pres- ϩ Allergic rhinitis is increasingly prevalent in the Western world. ence of these families of closely related IgE B cell clones in the Studies at the molecular level have enabled the investigation of mucosa, rather than the migration of all members of each family of IgE-mediated immediate hypersensitivity at specific locations. Pre- related B cell clones from lymphoid tissue to the same location in vious work has demonstrated the occurrence of local IgE protein the nasal mucosa. production in the nasal mucosa of patients suffering from allergic Additional evidence supporting the theory of local events in the rhinitis (1), but no PCR-based investigation has been undertaken nasal mucosa was provided by the further investigation of patient ϩ until now to determine whether DNA class switch recombination SO16. Sequences were identified from five different IgA B cells to generate B cells committed to IgE synthesis and concurrent that exhibited identical CDR3/FWR4 clonal signature regions to ϩ somatic hypermutation also occur locally in the nasal mucosa of those from one of the IgE families of related B cell clones also allergic rhinitis patients (as has been shown for the asthmatic lung isolated from the same nasal biopsy sample. These IgA sequences mucosa (13)). These processes are potential therapeutic targets. exhibited a range of mutations shared with and also different from ϩ To investigate whether local CSR and SHM occur locally in the the IgE sequences. Again, it is unlikely that these IgA cells were nasal mucosa of allergic rhinitis patients, we have amplified IgEϩ derived from CSR in lymphoid tissue and that they then homed to 3 ϩ VH region sequences by RT-PCR from the nasal mucosa of six the same 2.5 mm of nasal mucosa as the related IgE B cells. ϩ ϩ allergic rhinitis patients. These sequences have provided clear ev- Detection of related IgE and IgA B cell clones instead implies idence of families of closely related IgEϩ B cell clones in the nasal that it is more likely that the different Ab isotypes were generated mucosa of two of the six patients. This was in stark contrast both by local CSR, as has been previously suggested, in the lung mu- to the one instance in which two very distantly related IgEϩ B cell cosa of allergic asthmatics (13). clones were isolated from the PBMC of one patient and the one Further studies were conducted to investigate the origin of the instance in which an IgEϩ clone from the PBMC was found to be related B cell clones observed in the nasal mucosa of the allergic distantly related to a family of IgEϩ clones from the nasal mucosa rhinitis patients. Analysis of a healthy nonatopic subject with (possibly resulting from the diffusion of related cells from lym- highly elevated IgE indicated that whereas (as expected) a strong phoid tissue before SHM and clonal expansion in the nose, or from RT-PCR product resulted from the amplification of IgE VH regions the nasal mucosa into the circulation). from the subject’s PBMC, no IgE VH region sequences could be Each family of related B cell clones originates from a common isolated from the subject’s nasal mucosa sample even though a precursor B cell that proliferated and was subjected to differing positive GAPDH signal could be amplified and VH region se- extents of SHM. We propose that the occurrence of local SHM and quences from other isotypes were detected. This suggests that a The Journal of Immunology 5609

FIGURE 5. Amplification of AID mRNA transcripts by RT-PCR from the nasal mucosa of five of seven allergic rhinitis patients. AID RT-PCR products from the nasal mucosa of seven allergic rhinitis patients were subjected to electrophoresis on a 1.5% agarose gel. Lane 1, 100-bp DNA ladder (500 bp indicated); lane 2, patient CM10; lane 3, patient AP19; lane 4, patient DR20; lane 5, patient JH23; lane 6, patient SJ24; lane 7, patient TL25; lane 8, patient ZC27; lane 9, negative control (no DNA). The correct 335-bp AID RT-PCR product was observed in five of the seven samples.

of allergic rhinitis patients, and to provide clear molecular evi- dence for the occurrence of localized AID mRNA expression and Downloaded from indirectly, evidence of local SHM, CSR, and clonal expansion in an area of the nasal mucosa distant from lymphoid tissue. In ad- dition, we suggest that while the presence of related B cell clones in two of six nasal biopsy samples and of AID in five of seven samples is of importance in itself, it is likely that the nasal biopsy samples that appeared negative for related B cell clonal families http://www.jimmunol.org/ ⑀ may have done so as a result of sample variation. It is likely that FIGURE 4. VH-D-J-C regions were RT-PCR amplified from the PBMC, but not the nasal mucosa of a nonatopic subject with highly ele- related B cell clones in the nasal mucosa of allergic rhinitis pa- ⑀ vated levels of serum IgE. A,VH-D-J-C RT-PCR products were subjected tients are located in clusters, as observed by immunohistochemical to electrophoresis on a 1% agarose gel. Lane 1, 100-bp DNA ladder (500 staining of CD19ϩ B cells from nasal biopsy sections. Our pre- bp indicated); lane 2, nonatopic subject GJ29 nasal mucosa; lane 3, nona- liminary analysis of adjacent halves of a biopsy has suggested that topic subject GJ29 PBMC; lane 4, negative control (no DNA). B,VH-D- clonal families are highly localized within the mucosa (Coker et ⑀ J-C RT-PCR products from the individual VH gene classes (VH1-VH6) al., unpublished observations). Our inability to take more than a from the PBMC of nonatopic subject GJ29 were subjected to electrophore- single biopsy at any one time from a patient for ethical reasons sis on a 1% agarose gel. Lane 1, 100-bp DNA ladder (500 bp indicated);

implies that such clusters may be excluded by the nature of the by guest on September 23, 2021 lane 2,V 1 PCR products; lane 3,V 2 PCR products; lane 4,V 3 PCR H H H random sampling of ϳ0.1% of the inferior turbinate; each turbi- products; lane 5,VH4 PCR products; lane 6,VH5 PCR products; lane 7, nate is on average 10 g, and each nasal biopsy, on average, 10 mg VH6 PCR products; lane 8, all classes of VH PCR products; lane 9, neg- ative control (no DNA). C, GAPDH RT-PCR products were subjected to (L. Smurthwaite, unpublished observations). electrophoresis on a 1% agarose gel. Lane 1, 100-bp DNA ladder (500 bp Although this is the first study to investigate the local environ- indicated); lane 2, nonatopic subject GJ29 nasal mucosa; lane 3, nonatopic ment of the nasal mucosa in this number of patients, a previous subject GJ29 PBMC; lane 4, negative control (no DNA). study being restricted to just two lung biopsies from a single asth- matic patient (13), we suggest that it is highly significant that in ϩ many respects a consistent pattern of results is emerging in com- high level of serum IgE (from multiple different IgE B cell parison with that observed in the allergic asthmatic lung (13). Our clones) does not automatically result in the random migration of ϩ results additionally describe analysis of the V region repertoire of IgE cells to the nasal mucosa, although it is possible in allergic H the nasal mucosa of a healthy nonatopic patient with high systemic rhinitis patients that specific cell recruitment takes place. levels of IgE, but no detectable local mRNA-encoding IgE, sug- AID expression has been shown to be associated with both CSR gesting that IgEϩ B cells do not randomly migrate to the nasal and SHM (17, 18). The presence of AID in the nasal mucosa might mucosa from the circulation. We therefore conclude that the cells therefore be expected if the local microenvironment supported in the nasal mucosa of allergic rhinitis patients are stimulated with CSR and SHM. We detected AID mRNA transcripts in the nasal allergen, undergo class switch recombination and somatic hyper- mucosa of five of seven allergic rhinitis patients, demonstrating for mutation, and expand locally in the presence of AID. Our data the first time the presence of AID mRNA transcripts in local hu- strengthen the argument that the mucosa acts as a microenviron- man tissue and strengthening the suggestion of local CSR and ment in which cells from the direct, process, and SHM in the allergic nasal mucosa. Unfortunately, restricted sam- remove Ag in a localized manner, and we suggest that this is fun- ple size meant that we were only able to analyze AID mRNA ⑀ damental to the pathogenesis of allergic disease. expression from two of the samples used for VH-C analysis, CM10 and AP19. Interestingly, AID expression was clearly de- tected in CM10 (in which no reliable evidence of related clones Acknowledgments was detected), but was not detected from the nasal biopsy AP19 This work was conducted with the help and support of Duncan Wilson, ϩ Vicky Carr (Royal Brompton Hospital), and the Clinical Research Com- from which a family of related IgE B cell clones was detected. It mittee, Royal Brompton and Harefield Hospital’s National Health Service is therefore possible that the local activity in AP19 had ceased and Trust. We also thank Rebecca Beavil, Pooja Takhar, Lyn Smurthwaite, and that AID expression had subsequently been down-regulated, while Morgane Henry (King’s College London) for helpful discussions and prac- the related B cell clones remained. tical advice, and Kate Kirwan (King’s College London) for assistance with This investigation is the first to demonstrate the presence of graphics. We are also grateful for the advice provided by T. Honjo and T. local families of closely related B cell clones in the nasal mucosa Muto (Kyoto University, Kyoto, Japan). 5610 LOCAL SOMATIC HYPERMUTATION AND CLASS SWITCH RECOMBINATION

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