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The IgE repertoire in PBMCs of atopic patients is characterized by individual rearrangements without variable region of the heavy immunoglobulin chain bias

      Background

      Patients with atopic diseases are characterized by high levels of specific IgE production. However, little is known about the composition of their B-cell repertoires.

      Objectives

      We sought to analyze the complete PBMC-derived IgE repertoire and to compare clonal expansions between different patients.

      Methods

      We have analyzed the IgE-bearing B-cell receptor repertoire in highly atopic patients (>1000 IU/mL) using quantitative RT-PCR, complementarity determining region 3 spectratyping, and sequence analysis. Three representative patients were additionally followed during anti-IgE therapy.

      Results

      Atopic patients exhibited 100 to 1000 times more IgE-specific transcripts than control individuals. These patients used a variable region of the heavy immunoglobulin chain (VH) ε repertoire highly similar to their IgM and IgG repertoires, with preference of VH3b, VH4, VH3a, and VH1 segments. Each patient harbored individual clonal expansions, most probably as correlation of allergen-specific IgE production. Common expansions within the complementary determining region 3 shared by several individuals with similar sensitization patterns were found in spectratyping analysis. However, these antigen-driven expansions showed differences on the sequence level. In omalizumab-treated patients the clinical improvement was paralleled by a clear increase in the ratio of IgG/IgE transcripts.

      Conclusion

      The IgE repertoire in atopic patients follows the VH use patterns seen for other immunoglobulins and seems to preferentially recruit individual rearrangements rather than public expansions.

      Clinical implications

      The detailed analysis of the IgE B-cell repertoire is highly suitable to follow changes in IgE uses during different therapy modalities.

      Key words

      Abbreviations used:

      CDR (Complementarity determining region), CH (Constant region of the heavy immunoglobulin chain), VH (Variable region of the heavy immunoglobulin chain)
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      a segment that is also used by only a minority of IgM and IgG clones.
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      It was therefore our interest to depict the entire IgE repertoire in the periphery of highly atopic patients and to apply this technique to selected individuals undergoing anti-IgE therapy.

      Methods

       Individuals

      All 14 recruited individuals were seen for atopic eczema consultation in the Department of Dermatology and Allergy, Biederstein, Technical University Munich. All patients were older than 18 years and had severe atopic eczema. A detailed list of the patients' characteristics is given in Table I. All patients provided informed consent for venous puncture and withdrawal of 50 mL of blood.
      Table IPatient characteristics
      Patient no.Sex/age (y)Total IgE (IU/mL)DiagnosisSpecific IgE levels (>3.5 kU/L)
      1M/6410,669AEHDM, HD, B, DD, CD, TP, MW
      2M/3913,674AE, RCA, ABTP, B, HDM, CD, N, P, W
      3F/4418,732AE, ABHDM, CD, HE, CF, W, C, TP, L, B, MW
      4F/476240AE, RCA, ABHDM, CD, TP, B, MW
      5F/6620,048AE, RCA, ABHDM, CD, TP
      6M/306262AEHDM, CD, W, C, TP, L, B
      7M/4234,957AE, RCA, ABHDM, CD, TP, CF, AF, W, L, B, MW, R, P, T, CH
      8F/4723,565AE, RCA, ABHDM, CD, HE, MP, CF, W, L, B, MW
      9F/383107AE, RCA, ABCD, W, TP, L, B, MW
      10F/634352AE, RCA, ABHDM, HD, DD, CD, TP, MW, B, L, N, W, P
      11F/482914AE, RCA, ABHDM, CD, TP, B, MW
      12F/211018AE, RCAC, TP, B
      13F/2413,970AE, RCA, ABHDM, AS, CH, HD, DD, CD, TP, MW, B, N, HE, MP, CF, W, P, L, C
      14M/4420,193AE, RCA, ABHDM, CD, P, N, T, W, R, C, TP, B, MW
      F, Female; M, male; AE, atopic eczema; RCA, rhinoconjunctivitis allergica; AB, asthma bronchiale; HDM, house dust mite; CD, cat dander; HE, hen egg; MP, milk protein; CF, codfish; W, wheat; L, latex; B, birch; MW, mugwort; HD, horse dander; DD, dog dander; TP, timothy pollen; N, hazelnut; P, peanut; AS, Aspergillus species; CH, Cladosporium herbarum; MP, milk protein; C, celery; R, rice; T, tomato.
      Included control patients were healthy adult volunteers displaying IgE levels of less than 75 IU/mL.
      All patients included in omalizumab therapy (Xolair; Novartis, Nürnberg, Germany) had bronchial asthma and allergic rhinoconjunctivitis in addition to atopic eczema and likewise provided informed consent to be followed by extensive documentation for the course of atopic eczema during treatment. All patients were informed of the off-label use of omalizumab for atopic eczema. For these patients, 150 mg of omalizumab was delivered subcutaneously in 2-week intervals, and total serum IgE levels, the extent of eczema (SCORAD
      • Kunz B.
      • Oranje A.P.
      • Labreze L.
      • Stalder J.F.
      • Ring J.
      • Taieb A.
      Clinical validation and guidelines for the SCORAD index: consensus report of the European Task Force on Atopic Dermatitis.
      ), and a follow-up photograph were documented. Omalizumab therapy was carried out for 10 cycles, after which a final evaluation was performed. B-cell repertoire analysis in PBMCs was performed before starting the therapy and between the sixth and eighth cycles of omalizumab administration. Additionally, a patient receiving cyclosporine (3 mg/kg body weight) therapy who responded clinically in a comparable way was included.

       Serum immunoglobulin analysis

      The anti-human IgG and IgM ELISAs were from Bethyl Laboratories (Montgomery, Tex). IgE levels in serum were measured by using the Immulite 2000 system (DPC Biermann, Bad Nauheim, Germany) and in a second approach by coating omalizumab directly as a capture antibody to the plastic wells and otherwise following a standard sandwich ELISA protocol. This approach carries the advantage that IgE/omalizumab complexes and free IgE can be distinguished, which is not possible with conventional techniques. All tests were evaluated by using human myeloma IgE (Calbiochem, San Diego, Calif) and myeloma IgE/omalizumab complexes in defined ratios.

       IgE, IgG, and IgM repertoire analysis with quantitative RT-PCR

      At first presentation and under the condition that patients had avoided any systemic treatment for the last 4 weeks, 50 mL of EDTA blood was withdrawn for PBMC preparations.
      RNA was prepared from the obtained cells by using the RNeasy kit from Sigma (Deisenhofen, Germany). RNA was converted into cDNA according to standard protocols by using Superscript II (Invitrogen, Carlsbad, Calif), oligo dT primers (Eurogentech, Paris, France), and RNAsin (Invitrogen).
      PCR reactions were carried out by combining a primer and a specific fluorophore-labeled probe for the constant region (CHε, CHμ, or CHγ), with one of 8 primers covering the different VH1 through VH7 chains. Reactions were carried out with the Taqman 7300 device (Applied Biosystems, Foster City, Calif) and standard reagents from Applied Biosystems. Because all primers were chosen to exhibit identical affinities, it was then possible to calculate the relative distribution of IgE, IgM, and IgG transcripts and the relative use of the different VH families. Details of the primers and the calculation of the relative transcriptions are filed as European and US patent (Repertoire determination of a lymphocyte B population, WO 2005/059176 A1) and can be obtained through the Pasteur Institute (Paris, France).

       Immunoscope analysis

      In a second approach PCR products were then subjected to runoff reactions with a nested fluorescent primer specific for the constant region (Fcε, Fcμ, or Fcγ) for 5 cycles. The fluorescent products were separated and analyzed on an ABI-PRISM 3730 DNA analyzer. The size and intensity of each band was analyzed with Immunoscope software.
      • Pannetier C.
      • Cochet M.
      • Darche S.
      • Casrouge A.
      • Zoller M.
      • Kourilsky P.
      The sizes of the CDR-3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments.
      Fluorescence intensity was plotted in arbitrary units on the y-axis, whereas the x-axis corresponds to complementary determining region (CDR) 3 length in amino acids.

       Sequence analysis of B-cell receptor transcripts

      PCR products were cloned into pCR4Blunt–TOPO vector (Invitrogen, Cergy Pontoise, France). Sequencing was carried out with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and analyzed on an ABI PRISM 3730 DNA analyzer. Sequences corresponding to the CDR-3 regions and VH mutations of the corresponding sequences were extracted and analyzed by using gcg software (version 10.3; Accelrys, Inc, San Diego, Calif) and Taps1.1 software (written by Emmanuel Beaudoing).

      Results

       Quantitative use of Fcε, Fcμ, and Fcγ transcripts and proportional use of variable segments within the heavy chains (VH segments)

      In control individuals the percentage of IgE transcripts among total transcripts of IgM, IgG, and IgE generally was found to be very low, accounting for less than 0.001% of all analyzed immunoglobulin-specific transcripts. In general, the vast majority of immunoglobulin transcripts were represented by IgM-specific transcripts (>98%) and, to a much lesser extent, IgG-specific transcripts (1% to 2%, Fig 1).
      Figure thumbnail gr1
      Fig 1All included atopic patients showed an enhanced transcription of IgE compared with that seen in nonatopic control subjects. Note the logarithmic scale on the y-axis, indicating that some patients yielded up to 4% of IgE among the combined totality of IgM, IgG, and IgE transcripts. IgM transcripts represented the majority of immunoglobulin transcripts, ranging from 84% to 99%, together with IgG percentages of between 1% and 15%. AE, Atopic eczema.
      Among the 8 different variable chains, we found a dominant use for the VH3b, VH3a, VH4, and VH1 segments together with lower use of VH2, VH5, VH6, and VH7, regardless of the type of immunoglobulin under investigation (Fig 2).
      Figure thumbnail gr2
      Fig 2Distribution of the various VH chains among the total immunoglobulin repertoire by means of quantitative PCR shows that IgE antibodies from atopic patients used VH chains in an almost identical percentage as IgM and IgG antibodies in healthy and atopic individuals. For each included individual, the dominant VH segment was VH3b, followed by VH4, VH3a, and VH1, with the latter varying between patients. AE, Atopic eczema.
      Analysis of IgE transcription in 15 highly atopic individuals revealed higher IgE transcription within their PBMCs, occasionally reaching up to 4% of all immunoglobulin-specific mRNA molecules (Fig 1). Among the VH segments used by atopic patients within their IgE molecules, we did not find particular preferences compared with the use of VH chains among IgM and IgG molecules. These preferentially used VH segments were VH3b, VH3a, VH4, and VH1 and thus did not differ from the distribution of VH chains in healthy individuals. Of note, we did not find use of either VH5 or VH6 above the ratio expected from healthy donors or from the distribution of the IgM or IgG rearrangements (Fig 2).

       CDR-3 spectra in CHε-VHε rearrangements

      Having established the differential use of VH chains for IgE expression, we sought to identify possible dominant expansions within the different CHε-VH combinations. This approach revealed multiple peak-like profiles within the CDR-3 length analysis suggestive of the expansion of clonal populations. A comparative analysis of VH1, VH3a, VH3b, and VH4 (the 4 preferentially used VH segments among CHε rearrangements in atopic subjects) is given in Fig 3. This analysis shows that the relative distribution of the preferred CDR-3 length is highly different between patients and does not suggest common patterns.
      Figure thumbnail gr3
      Fig 3IgE immunoscopy (CDR-3 spectratyping) for the preferentially used VH segments VH1, VH3a, VH3b, and VH4 is shown for all included patients, with individual expansions suggestive of antigen (allergen) stimulation. The line (CDR-3 size) marks the average size of 10 amino acids for any rearrangement. Although some common patterns are found (eg, the 10-amino-acid peak of the CHε-VH3a rearrangement between patients 1 and 12 or the 8-amino-acid peak of the CHε-VH3b rearrangement shared by patients 12 and 7), all patients display rather individual patterns of their CDR-3 profiles, arguing for individually selected repertoires.

       IgE repertoire in atopic patients is composed of individual expansions

      Interestingly, among several patients sharing the same pattern of sensitizations, we found rearrangements that were used with similar profiles between different patients, such as the CHε-VH3a rearrangement, which was compared between patients 3, 4, and 7.
      To identify possible public rearrangements between patients, PCR products were cloned and randomly sequenced. In each patient at least 45 different VH3a-CHε rearrangements were sequenced and compared (see Figs E1 and E2 in this article's Online Repository at www.jacionline.org). This experiment revealed intraindividual dominant sequences that were isolated several times, most probably as signs of clonal expansions (eg, the 8-amino-acid rearrangement in patient 4, which was isolated 21 times).
      However, although most of the obtained sequences shared common traits between each other and between different patients, we did not observe identical hypervariable regions, suggesting individual selection and affinity maturation patterns rather than public rearrangements.
      By comparing the sequences in detail, we found a high degree of somatic mutations together with affinity maturation. For example, of the 58 sequences of the VH3a-CHε rearrangement in patient 3, 54 were derived from the germline sequence of V3-73, of which 25 showed signs of mutation; 3 were derived from V3-49, which were all mutated; and 1 was derived from germline sequence V3-49, which was unchanged. In total, of 58 sequences, only 30 (51.7%) carried germline configuration. Within the VH region FR3, we found a frequency of somatic mutation of between 3% and 25% per 100 bp.

       Analysis of the quantitative IgE repertoire in atopic patients undergoing anti-IgE therapy

      To further apply the analysis of the B-cell repertoire, we have followed 3 patients during anti-IgE therapy. All 3 patients had very high total serum IgE levels before starting omalizumab therapy (>10,000 IU), and all patients had extended eczema, as documented by SCORAD values of greater than 50 (Fig 4, A). In all patients, 10 cycles of 150 mg of subcutaneous omalizumab injection were administered at 14-day intervals. Of the 3 patients, 2 (patients 13 and 7) responded rapidly to the treatment with a clear and lasting improvement of their clinical scores, whereas patient 2 remained unchanged with regard to his SCORAD value (Fig 4, A).
      Figure thumbnail gr4a
      Fig 4A, Images show the SCORAD values before and after 10 cycles of omalizumab. Clinical images are posted in the Online Repository in . B, Results for the immunoglobulin analysis for all 3 patients receiving omalizumab therapy. The graph for IgE represents the results expressed in micrograms per milliliter and represents unbound IgE. C, The 2 patients responding well to omalizumab therapy (13 and 7) clearly downregulated their relative IgE mRNA while in parallel upregulating their relative IgG mRNA. Patient 2 and the cyclosporine-treated patient 14 responded with a slight upregulation of IgE mRNA, together with either downregulated IgG transcripts and upregulated IgM (14) or no significant changes (2). Values are expressed as percentage transcription of the respective immunoglobulin among the combined total transcripts for IgM, IgG, and IgE. BT, Before therapy; DT, during omalizumab therapy. D, CDR-3 profiles for the predominately used rearrangement CHε-VH3b in omalizumab-treated patients. Whereas patient 2 showed virtually no changes in IgE and IgG repertoires, patients 13 and 7 showed broader profiles of their IgG transcripts, together with unchanged (13) or reduced (7) IgE repertoires for this rearrangement. IgM (with the exception of a single peak for patient 7) remained in a Gaussian-like distribution.
      Figure thumbnail gr4b
      Fig 4A, Images show the SCORAD values before and after 10 cycles of omalizumab. Clinical images are posted in the Online Repository in . B, Results for the immunoglobulin analysis for all 3 patients receiving omalizumab therapy. The graph for IgE represents the results expressed in micrograms per milliliter and represents unbound IgE. C, The 2 patients responding well to omalizumab therapy (13 and 7) clearly downregulated their relative IgE mRNA while in parallel upregulating their relative IgG mRNA. Patient 2 and the cyclosporine-treated patient 14 responded with a slight upregulation of IgE mRNA, together with either downregulated IgG transcripts and upregulated IgM (14) or no significant changes (2). Values are expressed as percentage transcription of the respective immunoglobulin among the combined total transcripts for IgM, IgG, and IgE. BT, Before therapy; DT, during omalizumab therapy. D, CDR-3 profiles for the predominately used rearrangement CHε-VH3b in omalizumab-treated patients. Whereas patient 2 showed virtually no changes in IgE and IgG repertoires, patients 13 and 7 showed broader profiles of their IgG transcripts, together with unchanged (13) or reduced (7) IgE repertoires for this rearrangement. IgM (with the exception of a single peak for patient 7) remained in a Gaussian-like distribution.
      Figure thumbnail gr4c
      Fig 4A, Images show the SCORAD values before and after 10 cycles of omalizumab. Clinical images are posted in the Online Repository in . B, Results for the immunoglobulin analysis for all 3 patients receiving omalizumab therapy. The graph for IgE represents the results expressed in micrograms per milliliter and represents unbound IgE. C, The 2 patients responding well to omalizumab therapy (13 and 7) clearly downregulated their relative IgE mRNA while in parallel upregulating their relative IgG mRNA. Patient 2 and the cyclosporine-treated patient 14 responded with a slight upregulation of IgE mRNA, together with either downregulated IgG transcripts and upregulated IgM (14) or no significant changes (2). Values are expressed as percentage transcription of the respective immunoglobulin among the combined total transcripts for IgM, IgG, and IgE. BT, Before therapy; DT, during omalizumab therapy. D, CDR-3 profiles for the predominately used rearrangement CHε-VH3b in omalizumab-treated patients. Whereas patient 2 showed virtually no changes in IgE and IgG repertoires, patients 13 and 7 showed broader profiles of their IgG transcripts, together with unchanged (13) or reduced (7) IgE repertoires for this rearrangement. IgM (with the exception of a single peak for patient 7) remained in a Gaussian-like distribution.
      Figure thumbnail gr4d
      Fig 4A, Images show the SCORAD values before and after 10 cycles of omalizumab. Clinical images are posted in the Online Repository in . B, Results for the immunoglobulin analysis for all 3 patients receiving omalizumab therapy. The graph for IgE represents the results expressed in micrograms per milliliter and represents unbound IgE. C, The 2 patients responding well to omalizumab therapy (13 and 7) clearly downregulated their relative IgE mRNA while in parallel upregulating their relative IgG mRNA. Patient 2 and the cyclosporine-treated patient 14 responded with a slight upregulation of IgE mRNA, together with either downregulated IgG transcripts and upregulated IgM (14) or no significant changes (2). Values are expressed as percentage transcription of the respective immunoglobulin among the combined total transcripts for IgM, IgG, and IgE. BT, Before therapy; DT, during omalizumab therapy. D, CDR-3 profiles for the predominately used rearrangement CHε-VH3b in omalizumab-treated patients. Whereas patient 2 showed virtually no changes in IgE and IgG repertoires, patients 13 and 7 showed broader profiles of their IgG transcripts, together with unchanged (13) or reduced (7) IgE repertoires for this rearrangement. IgM (with the exception of a single peak for patient 7) remained in a Gaussian-like distribution.
      As expected, the relatively low dose of anti-IgE had no substantial influence on total or free serum IgE levels at any point of the observation interval (Fig 4, B). However, in patients 13 and 7 we observed dramatic changes of the immunoglobulin repertoire on the mRNA levels. Both patients had strongly increased IgE mRNA transcripts before therapy, with almost 4% (IgG 4.6%) in patient 13 and 0.1% (IgG 3.4%) in patient 7 of all immunoglobulin-specific transcripts. In the course of anti-IgE treatment, however (between the sixth and the eighth injection of 150 mg of omalizumab), we observed a surprising increase of IgG transcripts, together with a decrease of IgE mRNA in both patients, which resulted in a percentage of IgG transcripts among all immunoglobulin-specific mRNA of 15.3% (IgE 0.4%) in patient 13 and 11.8% (IgE 0.03%) in patient 7. Patient 2, who clinically did not respond to anti-IgE treatment and who showed preclinical values of 0.08% IgE and 1.49% IgG transcripts, increased to 0.28% IgE and 2.5% IgG transcripts after the seventh omalizumab treatment cycle (Fig 4, C).
      As control for omalizumab-specific results, we included patient 14, who was treated with cyclosporine and who clinically responded in a comparable way (decrease in SCORAD value of 25). In PBMCs from this patient, we observed an increase in the percentage of IgE-specific transcripts from 0.2% to 0.4%, an increase of IgM-specific transcripts from 85.3% to 97.3%, and a decrease in IgG-specific transcripts from 14.5% to 2.4% (Fig 4, C).

       CDR-3 spectratyping during omalizumab therapy

      Analysis of CDR-3 profiles of the VH3b-CHε rearrangements in omalizumab-treated patients showed that for patient 2, no major differences occurred in both the VH3b-CHε and the VH3b-CHγ rearrangements, whereas patient 13 showed a broader use of rearrangements within the VH3b-CHγ segment, together with a virtually unchanged VH3b-CHε spectrum during therapy. Patient 7, in contrast, revealed a broader use of VH3b-CHγ rearrangements, together with a clearly reduced repertoire of the VH3b-CHε combination (Fig 4, D).

      Discussion

      In the present study we have analyzed the peripheral IgE heavy chain repertoire in highly atopic individuals. These patients not only showed strongly increased serum levels of IgE antibodies but also increased transcription of the CHε chain of the immunoglobulin locus. The percentages of IgE-specific transcripts were in general 100 to 1000 times higher in atopic than in control individuals and thereby reflected relatively accurately the increase of IgE molecules in the serum. Importantly, our technique is not suitable to differentiate between changes in immunoglobulin-specific mRNA caused by changes in the amount of mRNA per cell or changes caused by cell numbers expressing the respective immunoglobulin-specific mRNA. However, because FACS staining for absolute numbers of IgM+, IgG+, and IgE+ CD19+ B cells maximally differed by a factor of 5 between atopic and control individuals (data not shown), we think that the number of transcripts per cell plays a crucial role.
      Our approach revealed only a moderate quantitative association for immunoglobulin transcripts and serum immunoglobulins. This fact can be explained by several reasons. First, we analyzed percentages of immunoglobulin transcripts and not absolute values, which of course do not reflect the quantification of immunoglobulin in serum, for which absolute values are calculated. Second, we analyzed IgE mRNA–producing cells within PBMCs. A large part of the antibody production, however, is most probably caused by cells not circulating in the blood. These are long-living plasma cells, which are located in bone marrow,
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      Maintenance of serum antibody levels.
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      Stromal niches, plasma cell differentiation and survival.
      and plasma cell clones, which are induced in distinct body compartments (eg, mucosal tissue of various origin). For these cells, mRNA was not analyzed, although IgE secreted from these cells probably is found in serum.
      • Coker H.A.
      • Durham S.R.
      • Gould H.J.
      Local somatic hypermutation and class switch recombination in the nasal mucosa of allergic rhinitis patients.
      Third, the half-life of circulating IgE is highly variable, depending on the association to Fcε receptors and the rate of internalization after activation through the high-affinity receptor FcεRI.
      • Kinet J.P.
      The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology.
      Our analysis revealed that, at least in our adult collective, the distribution of VH chains followed the use seen for other immunoglobulins in the periphery. The preferential use of the families VH3b, VH4, VH1, and VH3a has already been described in IgM and IgG repertoires.
      • Guigou V.
      • Cuisinier A.M.
      • Tonnelle C.
      • Moinier D.
      • Fougereau M.
      • Fumoux F.
      Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.
      • Kohsaka H.
      • Carson D.A.
      • Rassenti L.Z.
      • Ollier W.E.
      • Chen P.P.
      • Kipps T.J.
      • et al.
      The human immunoglobulin V(H) gene repertoire is genetically controlled and unaltered by chronic autoimmune stimulation.
      These families have the highest number of submembers,
      • Guigou V.
      • Cuisinier A.M.
      • Tonnelle C.
      • Moinier D.
      • Fougereau M.
      • Fumoux F.
      Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.
      somehow arguing for a random selection. Because for patients with atopic eczema and patients with atopic rhinitis a preferential use of VH5 and VH6 has been described,
      • van der Stoep N.
      • van der Linden J.
      • Logtenberg T.
      Molecular evolution of the human immunoglobulin E response: high incidence of shared mutations and clonal relatedness among epsilon VH5 transcripts from three unrelated patients with atopic dermatitis.
      • Snow R.E.
      • Djukanovic R.
      • Stevenson F.K.
      Analysis of immunoglobulin E VH transcripts in a bronchial biopsy of an asthmatic patient confirms bias towards VH5, and indicates local clonal expansion, somatic mutation and isotype switch events.
      • Edwards M.R.
      • Brouwer W.
      • Choi C.H.
      • Ruhno J.
      • Ward R.L.
      • Collins A.M.
      Analysis of IgE antibodies from a patient with atopic dermatitis: biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3.
      we verified that the lack of VH5/VH6 amplification was not due to a lower affinity of the used primers, thereby neglecting substantial amounts of these IgE transcripts. This hypothesis can be excluded for 2 reasons. First, the quantitative approach was evaluated by means of thorough testing with recombinant plasmids of the various VH segments (including both VH5 families VH5-51 and VH5a), which showed highly similar affinity patterns for all used VH segment–specific primers. Second, our distribution analysis of VH segments in IgG and IgM molecules depicted the numbers given in the literature, with only minute percentages of VH5/VH6 transcripts.
      In line with our results, a recent publication on IgE transcripts in the PBMCs of allergic individuals showed use of the VH5 and VH6 segment in the expected range.
      • Andreasson U.
      • Flicker S.
      • Lindstedt M.
      • Valenta R.
      • Greiff L.
      • Korsgren M.
      • et al.
      The human IgE-encoding transcriptome to assess antibody repertoires and repertoire evolution.
      However, in the case of mucosa-based class-switch recombination, as reported for the nasal mucosa of individuals with pollen allergy, a bias toward VH5 was repeatedly seen, possibly as a result of selection by superantigens.
      • Coker H.A.
      • Durham S.R.
      • Gould H.J.
      Local somatic hypermutation and class switch recombination in the nasal mucosa of allergic rhinitis patients.
      Although a final conclusion cannot yet be drawn, it seems that the IgE repertoire in PBMCs shows less bias toward a certain VH family than compartment-specific repertoires. Because we had no access to B-cell populations in tissue, we were not able compare the PBMC repertoire with local IgE repertoires in our patients.
      Our findings were also complemented by the CDR-3 profiles for the preferentially used VHε segments VH3b, VH4, VH1, and VH3a, all of which showed highly individual patterns without signs of common imprints on the repertoire.
      All these results together argue for the concept that in atopic individuals there is no preferential VH segment for any of the immunoglobulins and that in principle, in response to a given antigen challenge, similar repertoires are used for IgM, IgG, and IgE repertoires.
      The analyzed sequences and additional 58 sequences for the VH3a-CHε rearrangement in patient 3 showed that IgE sequences in atopic individuals have multiple mutations, which is suggestive of affinity maturation during the process of antigen recognition. Type and level of mutations within the allergen contact site of the antibody further prompted us to look for common patterns within IgE rearrangements between different individuals. Such dominant public rearrangements have been found for T-cell receptor rearrangements between MHC-matched individuals but have never been described for human immunoglobulin molecules. To this end, the dominant VH3a-CHε rearrangements of patients 7, 3, and 4 were sequenced. However, despite an extensive analysis of altogether 177 sequences, we were not able to find matching results between the different patients. This fact rather argues for individual-associated preferences in the fine tuning of IgE antibody responses than for a preferential pattern that is induced by the allergen itself. Of note, the samples analyzed in our study were collected in the absence of a specific allergenic stimulus (eg, provocation tests); however, because the acquisition took place over the range of 6 months, we cannot rule out that spontaneous (eg, seasonal aeroallergens) challenges had an effect on our results.
      To apply the technique of the B-cell receptor repertoire analysis to a clinical setting, we followed 3 patients undergoing omalizumab treatment. These patients repeatedly showed IgE levels of greater than 10,000 IU/mL, and thus an appropriate dosage to remove the entire IgE molecules from the serum would have been unrealistic.
      • Corne J.
      • Djukanovic R.
      • Thomas L.
      • Warner J.
      • Botta L.
      • Grandordy B.
      • et al.
      The effect of intravenous administration of a chimeric anti-IgE antibody on serum IgE levels in atopic subjects: efficacy, safety, and pharmacokinetics.
      We therefore decided to apply a relatively low-dose regimen by administering 150 mg of omalizumab subcutaneously every 14 days. Surprisingly, this dosage led to a good clinical response in 2 of 3 patients, together with impressive changes in the IgE and IgG transcripts. In the 2 responding patients, transcripts for IgE were downregulated, together with a clear increase in IgG transcripts. The clinically unresponsive third patient showed a rather unchanged proportion of IgE- and IgG-specific transcripts. In parallel to the findings of overall transcription, we also observed accordant profiles in the CDR-3 spectratyping analysis, showing larger IgG repertoires in the responding patients together with unchanged profiles in the nonresponsive patient. Interestingly, total serum IgE, as well as total serum IgG and total serum IgM, showed no changes in all 3 patients during therapy. One explanation could be the persistence of antibody-producing plasma cells, which are not affected by omalizumab. A similar phenomenon is seen in patients receiving anti-CD20 therapy, who are completely devoid of B cells for 9 to 12 months but sustain their antibody levels in the periphery.
      • van der Kolk L.E.
      • Baars J.W.
      • Prins M.H.
      • van Oers M.H.
      Rituximab treatment results in impaired secondary humoral immune responsiveness.
      • Cutler C.
      • Miklos D.
      • Kim H.T.
      • Treister N.
      • Woo S.B.
      • Bienfang D.
      • et al.
      Rituximab for steroid-refractory chronic graft-versus-host disease.
      The excellent technical assistance of K. Holtz, B. Heuser, J. Grosch, and B. Halter and the great help in taking care of the included patients by M. Sbornik, N. Brandl, M. Ziai, and B. Belloni are gratefully acknowledged.

      Appendix. Supplementary data

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      • Correction
        Journal of Allergy and Clinical ImmunologyVol. 128Issue 3
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          With regard to the September 2007 article by Lim et al entitled “The IgE repertoire in PBMCs of atopic patients is characterized by individual rearrangements without variable region of the heavy immunoglobulin chain bias” (J Allergy Clin Immunol 2007;120:696-706), patient 10’s VH3a, VH3b, and VH4 rearrangements are incorrect. The revised figure below includes the corrected rearrangements.
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