T Cell–Dependent Maturation of Pathogen-Specific Igs in the Antrum of Chronically Helicobacter pylori–Infected Patients Free

In humans, the vast majority of all Ig-secreting plasma cells (PC) is located in the gut mucosa (1). Most of the mucosal PC produce dimeric secretory IgA that is transported across the epithelial cell layer into the gut lumen mediating immune exclusion of luminal Ags by preventing attachment of microbes to the epithelium and clearing microbes that have crossed the epithelial barrier (2, 3).

One of the most frequent facultative pathogens of the human gut is H. pylori, a spiral-shaped, Gram-negative microaerophilic bacterium that predominantly colonizes the antral region of the stomach. Chronic H. pylori infection often results in ulceration, atrophy, carcinogenesis, or the development of an MALT B cell lymphoma (4). The mechanism of immune defense against H. pylori and subsequent malignant transformation of B cells is only partially understood, and the role of the induced specific local and systemic Ig response (5) has been discussed controversially. Although local IgA may limit metaplasia (6), IgA deficiency does not seem to influence the prevalence of H. pylori infection, and H. pylori–specific Ig in the serum or gastric mucosa may not protect efficiently the mucosal integrity (7, 8). We recently described a correlation of inducible NO synthase (iNOS) expression during H. pylori infection by B-lineage cells, in particular PC with clearance of experimental human infection and with expression of TNF and IFN-γ (9). Thus, iNOS⁺ PC might assist Th1 reactions that are important for the defense against H. pylori (5).

In contrast, expression of iNOS in H. pylori infection has been associated with gastric cancer and MALT B cell lymphoma (10), deregulated and exhaustive H. pylori–induced B cell activation seems to support low-grade B cell lymphoma, and enhanced Th1 reactions are supposed to favor peptic ulcers (11). The possible relatedness of mucosal H. pylori–reactive Ig-producing PC and MALT B cell lymphomas is unclear, also, because studies linking Ig specificity to respective rearranged VDJ gene sequences to allow a comparison with Ig expressed in MALT B cell lymphomas are missing.

The mucosal Ig repertoire is being characterized by sequencing their respective rearranged VDJ regions, and specific signatures during gastritis and of MALT B cell lymphomas begin to emerge (12–16). However, most studies on such Ig repertoires refer to sequencing data from whole tissues (12, 14–16), whereas data on Ag-specific cells, in comparison, are mainly based on ELISpot approaches without molecular characterization of the Ig genes (7). Approaches addressing both sequence and specificity of human Ig at single-cell level in the gut exist and identified polyreactive and Ag-specific intestinal Ig at steady-state (17). However, respective data in the context of a chronic infection are lacking.

Thus, in the current study, we aimed at characterizing the Ig repertoire of individual PC from the antral mucosa during chronic H. pylori infection at a single-cell molecular level to conclude about selection pathways, specificities, and relation to other conditions.

This study was approved by the Human Research Ethics Committee at the Charité Berlin (application numbers EA1/062/11 and 226-05a), and all participants gave written consent. For quantification of Ig, gastric antrum biopsy specimens were collected from randomly recruited patients undergoing upper endoscopy (eight H. pylori positive: three male, five female, mean age 60, range 20–78 and eight H. pylori negative: one male, eight female, mean age 54, range 29–73). For single-cell sorting of lamina propria lymphocytes (LPLs) and molecular characterization of Ig, antral biopsies of three H. pylori–positive, randomly recruited, consecutive patients that revealed obvious mucosal damage suspicious for infection with H. pylori during gastroscopy were collected (H. pylori–positive patients [HP]1–3, all women, mean age 48, range 34–75). HP1 presented with mild active H. pylori–associated gastritis of the antrum and corpus and small foci of intestinal metaplasia in the antrum. HP2 suffered from chronic gastritis of the antrum (grade 1–2) eradiating in the corpus, moderate reduction of glands, and low colonization with H. pylori. HP3 presented with severe erythematous gastritis of the antrum and the corpus (grade 2) with moderate follicular lymphatic hyperplasia and low colonization with H. pylori. H. pylori infection was confirmed by Warthin–Starry silver staining and the rapid urease test on antrum biopsies.

To determine production of Abs, biopsies from the antrum were weighted and cultivated at 37°C for 48 h in an atmosphere with 5% CO₂ and 80% O_2, as described before for duodenal biopsies, and supernatants were stored at −80°C until analysis (5).

IgA, IgM, and total IgG were quantified with BD Cytometric Bead Array (BD Biosciences, Heidelberg, Germany), and H. pylori–specific Abs were quantified with ELISA (RIDASCREEN Helicobacter IgG ELISA; R-Biopharm AG, Darmstadt, Germany) in plasma and antral biopsy supernatants according to the manufacturer’s protocols.

LPLs were isolated from antral biopsies of three H. pylori–infected patients (HP1–3, Table I) by collagenase type II (Sigma-Aldrich, Germany) digestion as described before (9).

To determine cell lineage, immediately following LPL isolation, unfixed LPLs were incubated at room temperature for 30 min with the following fluorochrome-labeled Abs, as previously described (9): anti-CD3–allophycocyanin-H7 (clone SK7; BD), anti-CD14–allophycocyanin-H7 (clone MφP9; BD), anti-CD19–allophycocyanin (clone HIB19; BD), anti-CD20–PerCP (clone 2H7; eBiosciences, San Diego, CA), anti-CD27–PE (clone O323; eBiosciences,), and anti-CD38–PE-Cy7 (clone HIT2; BD). For the detection of live NO-producing cells, 25 μg/ml 4-amino-5-methylamino-2’,7’-difluorescein diacetate (DAF-FM diacetate; Molecular Probes, Eugene, OR) was added to the staining solution (9).

After washing with PBS/0.2% BSA for 20 min at 4°C in the dark, DAPI (Molecular Probes) was added immediately before flow cytometric sorting of single living total (HP1) and iNOS⁺ and iNOS⁻ PCs (HP2, 3) (CD19⁺/CD27⁺⁺/CD38⁺⁺/CD3^–/CD14^–/CD20^–/DAPI^–/[DAF-FM^+/−]), respectively, into 96-well plates using a FACSAria II cell sorter (BD). Each well contained 30 μl of a modified 1× RT-PCR buffer (Titan One Tube kit (Sigma-Aldrich); 8.3 mM DTT, 0.5 μg of BSA, 0.4 ng of oligo-dT15 primer, 1.7% Triton X-100, and 20 U RNAsin). First-strand cDNA was generated at 50°C for 60 min following addition of 20 μl of a second mastermix containing 10 μl of Titan One Tube kit RT-PCR buffer, 0.5 mM dNTP, and 1 μl of Avian Myoblastosis Virus reverse transcriptase (Titan One Tube kit; Sigma-Aldrich).

Nested isotype-specific (IgA/IgG/IgM) PCRs were performed to amplify the rearranged IgH VHDJHCα/γ/μ cDNAs for sequence analyses and to amplify IgH and κ/λ IgL cDNA for cloning and reexpression of rIg, respectively. For external PCR, 5 μl of the amplified cDNA was used as template with a PCR mix containing AmpliTaq DNA polymerase kit components (Applied Biosystems by Life Technologies, Ober-Olm, Germany) and 0.2 mM dNTPs (Sigma-Aldrich), as previously described (18). The cycle program consisted of 7 min at 95°C, 1 min at 50°C, and 90 s at 72°C, followed by 50 cycles of 1 min at 94°C, 30 s at 50°C, 90 s at 72°C, and to finish, 1 min at 94°C, 30 s at 50°C, and 10 min 72°C. The internal PCR was performed in an identical manner with 5 μl of the reaction products from the external PCR as template and annealing at 58°C. External and internal isotype-specific and family-specific primers are listed in Supplemental Tables I–III. PCR products were separated by agarose gel electrophoresis, purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and sequenced at Eurofins MWG Operon (Martinsried, Germany).

For cloning of the PCR products into expression vectors [(19), kindly provided by H. Wardemann], IgH and IgL chain genes were amplified by using specific second PCR primers containing restriction sites (Supplemental Tables II–IV). The products were purified by QIAquick PCR Purification Kit (Qiagen) and eluted in 50 μl. Digests were carried out with the respective restriction enzymes AgeI, SalI, and XhoI (all from NEB, Ipswich, MA) in a total volume of 30 μl, and digested PCR products were separated by agarose gel electrophoresis and purified using a peqGOLD Gel Extraction Kit (VWR International, Darmstadt, Germany) before ligation into human Igγ1 (pγ1HC), Igκ (pκLC), and Igλ (pλLC) expression vectors containing a murine Ig gene signal peptide sequence and a multiple cloning site upstream of the human Igγ1, Igκ, or Igλ constant regions (provided by Hedda Wardemann, Heidelberg, Germany) (19). Transcription is under the influence of the human CMV promotor, and clones can be selected based on ampicillin resistance. Ligation of purified PCR product and linearized vector was performed with 400 U T4-Ligase (Invitrogen, Carlsbad, CA). Competent Escherichia coli TOP10 were transformed with the ligation product at 42°C. Colonies were screened by PCR using 5′absense as forward primer and 3′IgG internal, 3′Cκ494, or 3′Cλ as reverse primer, respectively (Supplemental Table IV). PCR products of the expected size (650 bp for Igγ1, 700 bp for Igκ, and 590 bp for Igλ) were sequenced to confirm identity with the original PCR products and resulted in nine Igλ and 23 Igκ and 22 IgH products, which formed 17 pairs (from 13 iNOS⁺ and 4 iNOS⁻ PC). Plasmid DNA was isolated from 12-ml bacteria cultures grown for 16 h at 37°C in LB medium (Difco Laboratories, Detroit, MI) containing 75 μg/ml ampicillin (Sigma-Aldrich) using peqGOLD Plasmid Miniprep I Kit (VWR International). From 6-ml bacteria cultures, on average, 14 μg of plasmid DNA was recovered after elution with 70 μl.

Human embryonic kidney (HEK)-293T cells (DSMZ, Braunschweig, Germany) were cultured in T25-mm culture flasks (BD) under standard conditions in HEK medium (DMEM medium [GibcoBRL, Darmstadt, Germany] supplemented with 7% heat-inactivated ultralow IgG FCS [Invitrogen], 5 g/l glucose; 25 mM HEPES; 2 mM l-glutamine; 1 mM Natriumpyruvate [all GibcoBRL]). Transient transfections were performed at 80% cell confluency as described previously (17, 19). In short, equal amounts (2.5 μg each) of IgH and corresponding IgL chain–encoding plasmid DNA and 25 μl of SuperFect Transfection Reagent (Qiagen) complemented to 150 μl with HEK medium were incubated for 10 min at room temperature. Subsequently, 1 ml of HEK medium was added and the mixture given to HEK-293T cells for 2.5 h at 37°C and 5% CO₂. The cells were washed with and cultured for 6 d in 25 ml of HEK medium before Ig were purified from supernatants with protein G beads (GE Healthcare, Berlin, Germany). Purified supernatants were analyzed by gel electrophoresis and ELISA for successful Ig production, as determined by the detection of both IgH and corresponding IgL and IgG₁ concentrations above 5 μg/ml in culture supernatants, which was observed in 12 of 15 preparations.

Expressed Ig were tested by ELISA for reactivity to H. pylori P12 wild type (wt) and for polyreactivity with dsDNA, ssDNA, and LPSs, as described previously (17). In brief, Costar ELISA plates (Sigma-Aldrich) were coated with 500 μg/ml H. pylori lysate and 500 μg/ml H. pylori lysate supernatant and each 10 μg/ml dsDNA, ssDNA, or LPS (all from Sigma-Aldrich) in PBS, respectively, overnight. Plates were incubated with 50 μl of purified Ig at 1 and 2 μg/ml in PBS. Reactive Abs were detected using HRP-coupled goat anti-human IgG (Jackson ImmunoResearch Laboratories, Cambridgeshire, U.K.) at a concentration of 0.8 μg/ml in PBS with 2 mM EDTA and 0.05% Tween-20 and HRP substrate TMB (BioLegend, Fell, Germany). ODs were measured at 620 nm. As a positive and negative control, control sera of the commercial RIDASCREEN Helicobacter IgG ELISA (R-Biopharm) were used.

The binding affinity to specific defined H. pylori proteins was tested with the recomLine Helicobacter IgG Kit (Mikrogen, Neuried, Germany).

Sequences with an average quality of >30 (corresponding to one error in 1000 sequenced bases) of HP1–3 were analyzed using the Chromas 2.33 sequence viewer (Chromas Technelysium, Helensvale, Australia) and the software JOINSOLVER (http://joinsolver.niams.nih.gov) by using the Kabat database. Sequences containing a stop codon or an out-of-frame rearrangement were considered as nonproductive, and sequences with an average quality of <30 were excluded from the analysis. In 19 of 124 sequences, a total of 69 among 26,745 bases (0.25%) showed an overlay, and in these cases of ambiguities the peak with the higher intensity was chosen. Ig isotypes and subclasses, V(D)J recombinations, CDR of the IgH (CDRH)3 lengths, frequencies of mutations, mutation rate of potential RGYW/WRCY motifs, and the ratio of replacement (R) to silent (S) mutations were determined by comparison with the respective germline sequences from the databases. Somatic hypermutations (SHM) within the VH genes were counted between framework region (FWR) 1 and FWR3, which include CDRH1 and CDRH2 but not CDRH3.

Quantitative parameters are presented as single measurements with medians and interquartile ranges. Prism version 5.0 and 6.0 (GraphPad Software, San Diego, CA) was used for statistical analyses. Mann–Whitney U tests were performed to analyze differences between two groups. The p values ≤0.05 were considered to be significant.

To characterize the clinical specimen and confirm increased systemic and local Ig production during chronic infection with H. pylori (7), we first quantified the major Ig classes in plasma and antral biopsy supernatants. In plasma of H. pylori patients and control subjects, concentrations of IgM, IgA, and total IgG were similar (Fig. 1A). In contrast, in supernatants from antral biopsies of H. pylori patients, IgA concentrations were elevated compared with controls, whereas concentrations of both IgM and total IgG were very low (Fig. 1B). However, significantly enhanced concentrations of H. pylori–specific Ig in plasma as well as biopsy supernatants of H. pylori patients compared with control subjects (Fig. 1C; p = 0.0011 and p = 0.0159, respectively) confirmed a specific systemic as well as mucosal reaction to the pathogen and validated our patient collective.

Inference on maturation mechanisms of antral PC can be made by analyzing molecular characteristics of individual PC isolated from cases of chronic infection with H. pylori. In this study, we attempted this approach for the first time, to our knowledge, and characterized IgH sequences of single PC from three H. pylori patients (HP1–3, Table I) with respect to isotype, VDJ recombination, CDRH3 length, and mutations to infer maturation paths and molecular relatedness. Genes of 53 total PC of donor HP1 and, in addition, 72 iNOS⁺ PC and 24 iNOS⁻ PC from HP2 and HP3 were analyzed, resulting in evaluable sequences from 124 IgH genes (Table I).

The distribution of Ig isotypes was similar for the three donors (Fig. 2). The most abundant subtypes were IgA₁, followed by IgA₂, IgG₁, IgG_2, and IgG₃, whereas IgM and IgG₄ were absent (Fig. 2). When analyzing iNOS⁺ and iNOS⁻ PC separately, the proportion of IgA₁ among all IgA-expressing PC was higher in iNOS⁺ compared with iNOS⁻ PC (Fig. 2).

Usage of V_H and J_H genes showed clear preferences in the selection of these Ig. We detected 35 different V_H gene segments. V_H3 and V_H4 were very prevalent in all three patients, in addition, V_H1 for HP1 and HP2. V_H6 was not found at all. The repeated presence of only seven V_H gene segments (V_H1-69, V_H3-11, V_H3-23, V_H3-30, V_H4-34, V_H4-59, V_H5-51) in all three H. pylori–infected donors was striking.

All known functional J_H families were found in similar distribution in HP1–3. J_H4 and J_H6 were the most frequent segments found in HP1–3 (J_H4: HP1: 23%, HP2: 76%, HP3: 39%; J_H6: HP1: 40%, HP2: 32%, HP3: 34%), whereas J_H1 and J_H2 were used only rarely (maximum: 13% J_H2 of iNOS⁻ PC of HP3). iNOS⁻ and iNOS⁺ PC revealed no differences concerning V_H and J_H family distribution. However, the prevalence of single V_H and J_H genes indicated that PC clones were not selected randomly from the complete genetic repertoire.

To evaluate possible T cell–driven selection processes during PC maturation, the frequency of SHM in the V_H regions (FWR1–FWR3) compared with the germline gene sequence was determined (Fig. 3A, 3B, Table I). The analyzed V_H regions encompassed in average 222 bp (216–228 bp, details see Table I), and the mutation frequency in this sequence was on average above 10% (Fig. 3A, 3B) and thus higher as described before for gut and stomach PC of healthy controls (15, 17) and in similar ranges as described for tetanus toxoid–specific PC (18).

When comparing mutation frequency in distinct V_H families, the only rarely found V_H2 and V_H5 sequences had a slightly reduced mutation frequency (mean HP1–3: 8.8 and 10.85%, respectively), whereas the more prevalent V_H1, V_H3, and V_H4 sequences revealed higher mutation frequencies (mean HP1–3: 12.8, 12.5, and 13.3%, respectively).

To infer the selection process of PC during germinal center (GC) reaction, the pattern of SHM in the FWR and in the CDRH were calculated. The ratio of R to S mutations was calculated using the accumulated number of R and S mutations because some sequences lacked S mutations, especially in the CDRHs. As expected for GC reactions, the ratios of CDRH R to S mutations were higher compared with the R/S values of FWR1-3 (Table I). As a characteristic of CD40/CD154–dependent, T cell–dependent affinity maturation, mutation frequency in RGYW/WRCY hotspot motifs (20, 21) was on average 48% for HP1–3 (median; HP1 50.56% [95% confidence interval 42.86–58], HP2 48% [95% confidence interval 41.67–52.63], and HP3 46.07% [95% confidence interval 42.42–52.63]) and thus higher than expected (22).

CDRH3 lengths of Ig genes of peripheral PC has been described to decrease during an Ag-driven selection process (23) but seems to be enhanced in mucosal compared with lymphoid PC and similar in small intestine and stomach (13, 15). Interestingly, in HP1–3 the number of bp forming CDRH3 was 45–49.5 bp and thus even longer, as described for gut and stomach PC of healthy individuals (Fig. 3C, 3D) (15, 17).

Mutation frequency (Fig. 3A, 3B), R/S values, mutation rate of RGYW/WRCY, and CDRH3 lengths (Fig. 3C, 3D) were independent of iNOS expression or Ig isotype. However, high mutation rate with especially high CDRH R/S values, in contrast to the FWR, and a high percentage of mutation in RGYW/WRCY hotspot motifs indicated continuous T cell–mediated, Ag-driven selection for some favored V_H families.

A high clonality of PC may reflect maturation of PC based on only few precursors with reactivity to only few selected major Ags. Thus, clones were defined by a common IgH sequence of the V region, by comparison of all V_HDJ_H recombinations and CDRH3 lengths (18). In this study, we detected only two pairs of clonally related PC sequences from 30 sequences obtained from HP1 and three pairs among 56 sequences for HP2 but none for HP3. All clonally related PC were of isotype IgA₁, and they differed concerning the accumulated mutations, indicating a diversified immune reaction.

To date, functional specificity has not been assigned directly to VDJ rearranged regions of individual Ig-producing cells in chronic H. pylori infection. Thus, we characterized specificities of Ig from the antral mucosa during chronic infection at the single-cell level. The V regions of IgH and the corresponding IgL of single cell–sorted antral PC of HP2 were cloned and expressed to enable analysis of binding specificities. A total of 47 IgH products and 77 IgL products, including 53 Igκ and 24 Igλ, were obtained, which corresponds to the expected average of 60% Igκ in humans (24). Functional V regions of both Igκ and Igλ were amplified in six cases that were excluded from further studies. Thus, for 40 of 47 IgH chain products, the corresponding IgL were amplified (10 Igλ and 30 Igκ) for expression vector cloning. Ig 12D was excluded from further analysis because no protein was detected (Fig. 4A) and thus, finally, cloning resulted in 14 pairs from 11 iNOS⁺ and 3 iNOS⁻ PC with molecular characteristics summarized in Table II. A functional Ig expression of IgH and IgL was shown by all specimens except 8A and 10D (data not shown), which both also revealed a low protein content (Fig. 4A).

Specificity could be demonstrated for 7 of the 14 (50%) cloned Ig, all originating from iNOS⁺ PC. Polyreactivity that is considered to contribute substantially to the intestinal Ab repertoire and to mediate regulatory functions by immune exclusion of luminal Ags (25, 26) was demonstrated for five (36%) rAbs: for 3E, 4A, 5A, and 6G, reactivity against LPS, dsDNA, and ssDNA was demonstrated, whereas 6H reacted to LPS and dsDNA only (Fig. 4D–F). Ig 4A, 5A, and 6H initially were IgA₁, 3E was IgA₂, and 6G, IgG₃ subtype. There was a cumulation of some gene segments (Table II): Abs 3E, 4A, 5A, and 6G used J_H4, whereas clones 3E and 4A used J_H4 combination with V_H3-23 and D 3-22.

Two Ig (15%) revealed a specificity to H. pylori: specificity of Ab 6A to a soluble H. pylori Ag was demonstrated by ELISA to Ag preparations of H. pylori P12 [soluble H. pylori lysate supernatant (Fig. 4B) and H. pylori whole cell lysate (Fig. 4C)], whereas the specificity of Ab 5C to VacA was shown by Western blot analysis (Fig. 5). Ig 6A originated from IgA₂ and 5C from IgA₁, both with Igκ, and interestingly, both clones used V_H1-69 in combination with J_H3.

Taken together, these data suggest that the majority of antrally produced Ig are either broadly directed to bacteria or specific to H. pylori. These Ig possess molecular features in their VDJ regions that have been repeatedly characterized previously in H. pylori–mediated gastritis tissue and MALT B cell lymphomas (12–14, 16).

Human PC responses are predominantly studied in the peripheral blood, and little is known about intestinal mucosal PC reactions or their specificity. A focus on the local response in the gastric mucosa in H. pylori is justified in view of the fact that local secretion of predominantly IgA in the antral mucosa was confirmed as a general feature of H. pylori infection (6, 7).

We therefore analyzed the mucosal Ig response and the IgH V sequence repertoire, focusing on tissue-resident PC from the antral mucosa in the context of a chronic infection with H. pylori and determined their antigenic specificity at a single PC level. An additional focus was on possible differences between iNOS⁺ and iNOS⁻ PC because we have previously found evidence that iNOS⁺ PC might play a role in the clearance of H. pylori infection (9). In mice, iNOS⁺ IgA⁺ PC of the lamina propria fulfill an important function in gut homoeostasis, as they support an effective production of IgA, influence the diversity of the microbiome, assist the defense against Citrobacter rodentium, and are long lived compared with iNOS⁻ PC (23, 27, 28).

An intriguing finding of our study was the detection of highly mutated IgH genes within the antral PC pool with long CDRH3 region lengths compared with Ig sequences from peripheral blood, stomach, and small intestine (13, 15, 17, 18, 23). A high frequency of mutation, especially targeting RGYW/WRCY hotspot motifs, is thought to be a clear sign of functional selection and indication of T cell–dependent affinity maturation (18, 21). The high proportion of V_H mutations found in IgH of PC of H. pylori–infected patients is consistent with this, as is the ratio of R/S mutation in FWR and CDRH because functional V-gene rearrangements of mucosal B cells are characterized by an R/S value of the FWRs between 1.0 and 1.6 (29), whereas the R/S value within the CDRH region should be increased (30). Clonal selection may continue after termination of the primary GC response in chronic infections such as H. pylori for which buildup of ectopic lymphatic structures with GC-like architectures have been described (31). Although the relative low number of iNOS⁻ PC analyzed limits our data, the expression of iNOS seems to have no influence on the maturation process of antral PC.

Our study is limited by the fact that in H. pylori–negative, healthy control persons, only few B-lineage cells infiltrate the gastric mucosa, which results in absence of CD19⁺CD38⁺CD27⁺ PC (9) and consequently unsuccessful PCR to detect VDJ IgH rearrangements (32). Sex, chronic infection, and immune senescence are known to shape the immune response to H. pylori (33), and these factors may theoretically also have an effect on the specific Ig gene repertoire generated. However, they may not constitute major selective forces because V_H usage, mutation rate, and distribution of SHM to FWR and CDRH seem to be similar in mucosal tissues from various patient collectives (12, 13, 15). For example, the frequency of SHM, the main indicator of Ag-driven, T cell–dependent B cell maturation (20, 21, 34), remains at a stable level following a rapid increase in early childhood (35). Thus, the frequency and location of SHM and R/S ratios of the Ig repertoire of single cell–sorted antral PC that is reported in this study (arguably a small subset) are in line with the idea of Ag-specific GC-dependent maturation of B cells and PC that ultimately localize to the intestinal and gastric tissues, which may also be true for MALT B cell lymphoma cells based on findings from whole tissue analysis (13, 15, 17, 34, 36).

We confirmed a relative overrepresentation of V_H1-69, V_H3-11, V_H3-23, V_H3-30, V_H4-34, V_H4-59, and V_H5-51, indicating a strong selection operating to shape the repertoire of tissue-resident PC in H. pylori–mediated gastritis (12). With the exception of V_H1-69, the data are in good agreement with PC-repertoire analyses from antral-derived IgA⁺ PC and from peripheral B cell populations of healthy subjects based on which accumulation of V_H3 and V_H4 (found for all H. pylori patients) and of V_H1 (found for two of the three patients) was expected (15, 17, 37, 38). Overall, the most frequent combination found was V_H3-23 with J_H4 that was detected in both iNOS⁺ and iNOS⁻ PC. In addition, we noted a striking dominance of J_H6 among the J_H elements of H. pylori patients. Furthermore, V_H5-51 in combination with J_H4 and V_H1-69 with J_H6 both were described in gastritis patients with and without infection with H. pylori (12) and were also prevalent in our patients. V_H1-69, in addition, is described in the context of MALT lymphoma (12–14, 16), whereas V_H5-51/J_H4 is supposed to be often selected by autoantigens during the early phase of an immune response (39).

For functional testing, the complete Ig of 11 iNOS⁺ and two iNOS⁻ PC of one H. pylori patient could be reexpressed. For two of these 13 Ig we could assign the H. pylori specificity, one recognizing an as yet unidentified soluble protein and the other recognizing VacA. Both H. pylori–specific Ig originated from iNOS⁺ PC, used the κ IgL, shared the bespoken V_H1-69 in combination with J_H3, and both revealed a V region length of 216 bp. The malignant transformation of B-lineage clones present at the beginning of the infection with H. pylori to MALT lymphoma has been discussed for a long time (11, 40), and our data support the idea that deregulated and exhaustive H. pylori–induced T cell–dependent B cell activation can promote the onset of low-grade B cell lymphoma by linking for the first time, to our knowledge, the usage of V_H1-69 with specificity to H. pylori. Chronic and ineffective activation may originate from immunological niches where H. pylori is protected from the mucosal immune response (41). In addition, the frequency with which we could assign specificity was surprising and corresponded to a 7–10-fold enriched, Ag-specific, Ab-producing cell population when compared with frequencies of similar reactivities in PBMCs of experimentally infected human volunteers (5) and frequencies of gastric H. pylori–specific IgA⁺ cells among all IgA⁺ cells as documented by ELISpot (7).

Additionally, for another five of the reexpressed Ig (38%) from iNOS⁺ PC, a polyreactive but specific recognition of the polyanionic Ags LPS and dsDNA/ssDNA could be demonstrated. Four of the polyreactive Abs used JH4, two in combination with VH3-23, a combination that has been described before in patients with gastritis with and without H. pylori infection (12), and our functional data suggest that these likely reflected polyreactive Ig, too. Polyreactivity is a common feature of MALT lymphoma (13), more often observed in mucosal Ig, and is thought to be caused by a certain degree of flexibility of the Ag-binding pocket that has been suggested to be enabled by longer CDRH3 regions (42).

Polyreactive IgA participate in the defense against commensals, and in the initiation of regulatory mechanisms by immune exclusion of luminal Ags (25, 43), they often possess a weak affinity and broad specificity, and they are important for intestinal homoeostasis by assisting immune exclusion of luminal Ags before the generation of specific immune responses (25, 26, 44). Based on mouse experiments, it is hypothesized that polymeric mucosal Ig use germline-encoded V_H genes but are also generated by T cell–independent endogenous mechanisms of selection in Peyer patches that enrich polyreactive specificities (25, 26). Although T cell help and GC formation seem to be dispensable for generation of polyspecific IgA, the presence of microbiota is essential (25). The IgA₁ isotype was proposed to be stimulated rather by protein Ags (45), whereas IgA₂ has been associated with polyreactivity against (e.g.) LPS (17, 45). Clearly, our data do not support such a categorization; the polyreactive Ig also exhibit high rates of SHM and signs of T cell–dependent maturation. Moreover, three polyreactive Ig were IgA₁, one IgA₂, and one IgG₃. Thus, albeit only a few sequences exhibit polyreactivity, it did not seem to depend on the Ig isotype, and reactivity to LPS was not limited to IgA₂.

For seven clones (50%), no reactivity was defined, and one can only speculate about possible targets. Although some may be of too low affinity to H. pylori, LPS, or ss- or dsDNA to detect it, a proportion of these Ig may be self-reactive and reactive to other commensal or pathogenic microorganisms with previous contact to the intestinal mucosa (17).

In conclusion, mucosal iNOS⁺ PC producing H. pylori–specific Ig are the product of Ag-driven, T cell–dependent B cell differentiation and enrich in the pathogen-colonized tissue. They express rearranged V-(D)-J genes that are reminiscent of characteristics of gastric MALT B cell lymphoma–expressed Ig, and, as shown in this study, such rearrangements can encode H. pylori specificity or specificity for polyanionic Ags such as LPS or DNA. This new information on shared sequence features between pathogen-specific Ig to Ig types associated with MALT B cell lymphomas creates the basis for generating much-wanted models to study MALT lymphoma formation (e.g., by transgenic expression of the respective Ig in mice to be infected with H. pylori).

The authors thank Martina Seipel and Diana Bösel for technical assistance. The authors are grateful to Frank Heller (Gastroenterologische Praxis am Rathaus Steglitz, Berlin, Germany) and all patients and control subjects for help in obtaining samples, Christoph Loddenkemper (Pathotres, Berlin, Germany) for histological evaluation of the H. pylori–infected biopsies, and Hedda Wardemann and Christian Busse (both from Division of B Cell Immunology, German Cancer Research Center, Heidelberg, Germany) for supply of expression vectors and consultation on recombinant expression.

The authors have no financial conflicts of interest.