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Impact of IL-6 receptor inhibition on human memory B cells in vivo: impaired somatic hypermutation in preswitch memory B cells and modulation of mutational targeting in memory B cells
  1. Khalid Muhammad1,
  2. Petra Roll1,
  3. Thomas Seibold1,
  4. Stefan Kleinert1,
  5. Hermann Einsele1,
  6. Thomas Dörner2,3,
  7. Hans-Peter Tony1
  1. 1Medizinische Klinik und Poliklinik II, Division of Rheumatology/Clinical Immunology, University of Würzburg, Würzburg, Germany
  2. 2CC12, Department of Medicine/Rheumatology and Clinical Immunology, Charite Universitätsmedizin, Berlin, Germany
  3. 3DRFZ Berlin, Berlin, Germany
  1. Correspondence to Dr Hans-Peter Tony, Medizinische Klinik und Poliklinik II, Division of Rheumatology/Clinical Immunology, Oberdürrbacher Strasse 6, 97080 Würzburg, Germany; tony_h{at}medizin.uni-wuerzburg.de

Abstract

Objective Interleukin 6 (IL-6) receptor (IL-6R) inhibition by tocilizumab is a novel anti-inflammatory therapy for rheumatoid arthritis (RA) patients. As IL-6 is a late differentiation factor of B cells the authors asked if IL-6R inhibition impacts on the mutational differentiation of human memory B-cell antigen receptors in vivo.

Methods 1733 immunoglobulin receptors (IgR) of single cell sorted preswitch and postswitch memory B cells were prospectively analysed from 11 RA patients under IL-6R inhibition (7 patients) or tumour necrosis factor (TNF) inhibition (4 patients).

Results The results show a reduced mutational frequency in IgR of preswitch memory B cells (p=0.0001) during week 12, week 24 and 1 year of tocilizumab therapy. Mutational hotspot RGYW/WRCY motifs indicated significantly decreased targeting (p<0.05) in preswitch and postswitch memory B cells. Anti-TNFα therapy had no effect on mutational frequency and mutational hotspot targeting motifs in memory B-cell subsets.

Conclusions These data suggest that preswitch and postswitch memory B cells are susceptible to IL-6R inhibition in vivo. Acquisition of mutations was substantially altered in preswitch memory B cells, while targeting of mutational hotspots affected preswitch and postswitch memory B cells. The results indicate that preswitch and postswitch memory B cells have a differential dependence on the IL-6/IL-6R system for differentiation, which can be influenced by tocilizumab in vivo.

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Interleukin 6 (IL-6), originally identified as a B-cell differentiation factor, is a pleiotropic cytokine involved in the initiation and maintenance of inflammatory and immune responses.1 It is produced by a variety of cells including antigen-presenting cells, macrophages, B cells, dendritic cells and non-haematopoietic cells (including epithelial cells, astrocytes and fibroblasts).2 In-vitro studies document the direct influence of IL-6 on B-cell differentiation.3 Its excessive activity in vivo results in polyclonal B-cell activation and contributes to the pathophysiology of various autoimmune and inflammatory diseases.4

Tocilizumab is a humanised monoclonal antihuman IL-6 receptor (IL-6R) antibody. Clinical studies have shown that inhibition of IL-6R by tocilizumab is therapeutically effective in different stages of rheumatoid arthritis (RA).5 6

We have recently shown decreasing frequencies of peripheral preswitch and postswitch memory B cell cells along with reductions in serum immunoglobulin levels in RA patients treated with therapeutic IL-6R inhibition using tocilizumab.7 Here we extend these studies to the molecular modulation of B-cell receptors that shape the immunoglobulin receptor (IgR) gene repertoire. In particular, we examined the pattern of mutations in single rearranged B-cell immunoglobulin genes before and during 1 year of tocilizumab treatment to explicate the therapy-mediated changes in mutated IgR in different memory B-cell subsets.

Materials and methods

Informed consent was obtained from all patients according to the protocol approved by the ethics committee of the University of Würzburg, Germany. Patient characteristics and all methods are described in a supplementary file (available online only).

Results

Modulation in somatic hypermutation of IgVH gene rearrangements of preswitch and postswitch memory B cells during tocilizumab therapy

Individual preswitch memory B cells (CD19/CD27/IgD+) were sorted and Ig-VH3/VH4 gene rearrangements were analysed at four different time points: baseline, week 12, week 24 and 1 year during the administration of tocilizumab. Baseline analysis of Ig-VH3 and VH4 gene sequences from seven RA patients revealed a diversified mutational pattern with an expected mutational frequency of 4.1±0.2% (mean±SEM) in preswitch memory B cells comparable to healthy controls (see supplementary figure S1A, available online only). At week 12, week 24 and 1 year during tocilizumab significantly reduced mutational frequencies (baseline 4.1±0.2% to 2.9±0.2%, 2.5±0.2% and 2.1±0.1% at week 12, week 24 and 1 year; p=0.0001) were observed (figure 1A). Patients' individual data are shown in supplementary figure S2 (available online only) with significant reductions in mutational frequency in every single patient.

Figure 1

Mutational frequency and hotspot targeting in individual preswitch memory B cells during tocilizumab therapy. (A) Data indicate significant reduction in mutational frequency during interleukin 6 receptor inhibition in preswitch memory B cells (n, number of individuals; S, number of analysed sequences; ***p=0.0001 in comparison with baseline data). (B) A significantly reduced RGWY/WRCY mutational targeting is identified in preswitch memory B cells during tocilizumab therapy (***p<0.025).

Compared with the preswitch B cells, postswitch memory B cells (CD19/IgD/CD27) at baseline showed a higher mutational frequency of 6.0±0.3% similar to healthy controls (supplementary figure S1B, available online only). In these cells mutational frequency was not modulated under tocilizumab during 24 weeks of treatment (figure 2A). The results indicated that tocilizumab particularly influenced the process of somatic hypermutation of preswitch memory B cells.

Figure 2

Mutational frequency and hotspot targeting in postswitch memory B cells during tocilizumab treatment. (A) Mutational frequency obtained from postswitch memory B cells is not affected during tocilizumab treatment. (B) Mutational hotspot targeting (RGYW/WRCY) is significantly decreased at week 12 and week 24 follow-up (**p<0.05) under tocilizumab.

Reduced targeting of mutational hotspot motifs in preswitch and postswitch memory B cells following tocilizumab treatment

Somatic hypermutation favours defined mutational hotspots. In particular, RGYW/WRCY motif mutations (R, purine; Y, pyrimidine; W, A/T) are preferentially targeted by T-cell-dependent signals including CD40–CD40 ligand interactions.8 The frequency analysis of targeted mutations (figures 1B and 2B) were found to decrease significantly during tocilizumab therapy in preswitch memory B cells (mean 25.0±1.2% before therapy vs 18.7±1.3% at week 12, 18.5±1.3% at week 24 and 17.3±1.2% at 1 year, p<0.05) and postswitch memory B cells (mean 30.2±3.2% before therapy vs 18.7±2.5% at week 12 and 17.6±0.8% at week 24, p<0.05) in all patients.

Anti-TNFα therapy did not provide a significant effect on mutational frequency and mutational hot spot targeting of IgV gene rearrangements

As a control for effective anti-inflammatory therapy and IL-6-specific changes we studied patients who were treated with tumour necrosis factor (TNF) inhibition using etanercept (n=4) (figure 3). Notably, the mutational frequency and mutational hotspot targeting analysis revealed no change in both preswitch (figure 3A,B) and postswitch memory B cells (figure 3C,D).

Figure 3

Mutational frequency and hotspot targeting in preswitch and postswitch memory B cells during anti-tumour necrosis factor alpha therapy. The mutational pattern and RGYW/WRCY motif targeting of (A and B) preswitch and (C and D) postswitch memory B cells are not substantially influenced by etanercept therapy.

Discussion

IL-6R inhibition by tocilizumab is used to reduce signs and symptoms5 as well as radiological progression6 in RA. So far it has not been determined which of its pleiotropic effects impact on the observed clinical response. The in-vitro effects of IL-6 on late stages of B-cell differentiation are well documented.3 In vivo IL-6 overexpression is associated with B-cell hyperactivity, autoantibody production and immunopathology.9 The aim of the current study was to evaluate the in-vivo effect of IL-6R inhibition on the mutational activity of antigen receptors of peripheral memory B cells, a hallmark of adaptive immunity. Our results show clear evidence of immunoglobulin gene modulations in peripheral preswitch and postswitch memory B cells induced by tocilizumab already at week 12 and continuously during week 24 and 1 year. In the preswitch memory pool we observed a significant reduction of somatic hypermutation in IgR (figure 1A) during tocilizumab treatment. The reduced mutational frequency in the preswitch memory compartment is due to a reduction of individual B cells with a highly mutated IgR and a relative increase of B cells with low or unmutated IgR (see supplementary figure S3, available online only). These effects seem to be directly affected by IL-6R inhibition because blocking TNFα using etanercept did not modulate the mutational status of preswitch memory B cells despite similar effects on the clinical reduction of inflammatory disease activity (figure 3 and supplementary table S1, available online only). Postswitch memory B cells revealed a different dependence on IL-6 signals in vivo. Even over a period of 24 weeks of ongoing IL-6R inhibition the mutation rate in single IgR was not modulated (figure 2A).

The nature and origin of peripheral preswitch memory Bcells is still debated. Nevertheless, there is considerable evidence for these B cells to be related to splenic marginal zone B cells based on similar phenotypic markers and different requirements for IgR mutation.10 In New Zealand black/white F1 lupus mice treated with anti-IL-6 monoclonal antibody therapy, a numeric decrease of marginal zone B cells is observed.11 Studies of splenectomised patients attribute a shorter lifespan compared with postswitch memory B cells.12 In our study the mutational differentiation of preswitch memory B cells seems to be particularly sensitive to IL-6R inhibition in vivo, which adds more evidence to the idea that preswitch memory B cells have different maturation requirements12 compared with postswitch B cells, with probably less dependency on germinal centre reactions.

So far the prime target cells for IL-6R inhibition relating the observed effects have not yet been determined. In animal studies somatic hypermutation is promoted by IL-6 produced from immune complex-bearing follicular dendritic cells and is impaired in IL-6 knockout mice.13 14 These studies substantiate the dependency of somatic hypermutation on IL-6 and might indicate that IL-6 provided by immune cells that facilitate or trigger somatic hypermutation is inhibited by tocilizumab therapy.

It has previously been shown that mutational frequency and sequence-specific aspects of somatic hypermutation are not interlinked.15 Somatic hypermutation is a tightly regulated multistep process and activation-induced cytidine deaminase plays an essential role in which increased G/C mutations have been recognised as a target of activation-induced cytidine deaminase.16 Therefore, we determined dynamic changes in somatic hypermutations in specific DNA motifs, RGYW/WRCY, which were previously identified to be targeted8 following T-cell-dependent CD40–CD40 ligand interactions.17 We found significantly decreased mutational targeting in RGYW/WRCY motifs in both preswitch and postswitch memory B cells during IL-6 inhibition (figures 1B and 2B) but not TNF inhibition (figure 3). There was also a trend in reduced R/S mutations; however, without reaching statistical significance. It can be hypothesised that the reduced hotspot targeting particularly reflects an impaired T-helper cell activity under tocilizumab. In fact, a reduction in helper T cells under anti-IL-6R antibody has been described by other studies.18

It is not currently clear how the described effects of IL-6 inhibition on memory B cells relate to the mechanism of action of tocilizumab in RA. Nevertheless, the influence on B-cell memory may contribute to the action of IL-6 R inhibition in RA. In studies using temporal B-cell depletion after rituximab the number of persisting memory B cells19 has been linked to response. Current data on tocilizumab treatment in systemic lupus erythematosus patients also indicate in-vivo effects on B-cell homeostasis, with a marked reduction in recirculating plasma cells.20 Therefore, modulation of memory B cells in RA may be one possible target for tocilizumab. In addition, regarding the described effects of IL-6 inhibition on memory B cells, future studies will also have to address the effect of IL-6 inhibition on the development of protective immunity.

In summary, this is the first study to demonstrate an in-vivo effect of IL-6 inhibition by tocilizumab on the mutational differentiation of memory B cells, with significantly decreased somatic hypermutation in VH gene rearrangements in preswitch memory B cells. In addition, mutational hotspot targeting in RGYW/WRCY motives was reduced in both preswitch and postswitch memory B cells. These effects appeared to be directly dependent on IL-6 inhibition because TNF inhibition, despite a similar reduction in clinically observed inflammation, had no effect on IgR mutations. The study provides further molecular insight into the differential effect of IL-6R inhibition on preswitch and postswitch B cells, and clearly demonstrates that anti-IL-6R therapy has complex effects on memory B cells in vivo.

Acknowledgments

The authors thank Christian Linden and Yvonne Gold for their invaluable help in single cell sorting. The authors are grateful for the cooperation of the patients for their participation in the study.

References

Supplementary materials

Footnotes

  • Funding This work was supported by Wilhelm Sanderstiftung 2006.037.2 and project 16, SFB 650.

  • Competing interests Drs. Kleinert and Tony have received consulting fees, speaking fees, and/or honoraria from Roche, Abbott, Chugai and Wyeth (less than $10,000 each). Dr. Dörner has received consulting fees and/or honoraria from Roche, Genentech, Immunomedics, and UCB (less than $10,000 each). All other authors declare no competing interest.

  • Patient consent Obtained.

  • Ethics approval This study was conducted with the approval of the ethics committee of the University of Würzburg, Germany.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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