Effect of ATG-F on B-cell reconstitution after hematopoietic stem cell transplantation.

Antithymocyte globulin Fresenius (ATG-F) is used before hematopoietic stem cell transplantation to prevent graft rejection and graft-versus-host disease in patients with HLA-matched unrelated donors or mismatched volunteers. However, little is known about the effect of ATG-F on the reconstitution of B-cell subsets. Sixty-seven patients were longitudinally studied at day 15, day 30, and then monthly after hematopoietic stem cell transplantation. Conditioning regimes included ATG-F, which was infused at days 3, 2 and 1 at a dosage of 10 mg/kg/d. Twenty-seven patients received conditioning regimes without ATG. ATG-treated patients showed a significant delay of CD19+ B cells in the early recovery period. The absolute numbers of circulating CD19+ B cells were significantly lower (P < 0.05) up to 5 months post-transplantation compared to non-ATG patients. The recovery of the memory compartment was delayed in both groups and did not reach normal values 1-year post-transplantation. ATG-treated patient showed significantly lower absolute numbers of circulating CD27+ memory B cells in the first-month after transplantation compared to non-ATG patients. In conclusion, treatment with ATG in the conditioning regime of patients undergoing allogeneic hematopoietic stem cell transplantation leads to a significant delay of CD19+ B cells. Thus, ATG seems also to negatively influence B-cell immune reconstitution.

Long-term repopulation of peripheral B-cell subsets after single and repeated rituximab infusions in patients with rheumatoid arthritis.

OBJECTIVES: B-cell depletion using rituximab (RTX) has proven efficacy in patients with RA. Long-term effects on the B-cell system after single and repeated treatments are sparse. Our aim was to study the effect of multiple courses of rituximab to evaluate its impact on repeated B-cell re-population capacity. METHODS: Two cohorts, altogether 20 patients with RA were included in an open label extension study with RTX. Cohort 1 received one cycle RTX and was followed for up to 7 years. In cohort 2 patients were studied under up to 5 cycles of RTX. Immunophenotyping was performed before therapy and during follow-up. RESULTS: After a single therapy with RTX (cohort 1) the frequency of pre-switch (MZ-like) B cells were significantly reduced during the follow-up of 7 years and absolute numbers slowly repopulated to nearly 50% of baseline value without numerical normalisation. The acquisition of mutations in Ig receptors of pre-switch (MZ-like) memory B cells was also significantly reduced 10 years after one course. In contrast, absolute numbers of (classical) post-switch B cells tended to normalise to baseline values after 7 years. Analysing B-cell repopulation capacities after multiple cycles revealed (cohort 2) a comparable repopulation pattern after each cycle with no substantial further impact on memory B cells. CONCLUSIONS: A single therapy with RTX leads to long-term changes in the memory B-cell compartment particularly in pre-switch memory B cells. Multiple cycles of RTX show a comparable repopulation pattern after each cycle with no additional cumulative effect on memory B cells.

In vivo effects of the anti-interleukin-6 receptor inhibitor tocilizumab on the B cell compartment.

OBJECTIVE: Interleukin-6 (IL-6) receptor inhibition by tocilizumab was recently licensed for the treatment of rheumatoid arthritis (RA). IL-6 induces in vitro differentiation of B cells into antibody-forming cells; however, the in vivo effects of IL-6 inhibition on the B cell compartment are currently not known. The purpose of this study was to examine this feature. METHODS: Sixteen patients with active RA were treated in an open-label study with tocilizumab (8 mg/kg every 4 weeks). Immunophenotyping was performed at baseline, week 12, and week 24. RESULTS: Memory B cell subsets declined significantly during tocilizumab therapy. Preswitch memory B cells decreased from a median of 19.6% to 12.3% at week 24 and postswitch memory B cells declined from a median of 18.6% to 15.0% at week 24 (P = 0.04). In parallel, CD19+IgA+ and CD19+IgG+ B cells decreased significantly. The proportion of IgA-expressing B cells fell from a median of 9.2% at baseline to 4.3% at week 12 and to 3.6% at week 24 (P = 0.01). IgG+ B cells declined from a median of 6.7% at baseline to 4.9% at week 12 (P = 0.007) and 2.8% at week 24 (P = 0.01). In parallel, serum levels of IgA and IgG were significantly diminished at week 24 (P < 0.05). There was a good correlation between relative and absolute numbers of IgA+ B cells with serum IgA at week 24. CONCLUSION: Tocilizumab induced a significant reduction in the frequency of peripheral preswitch and postswitch memory B cells. In addition, the number of IgG+ and IgA+ B cells declined and correlated well with reduced serum immunoglobulin levels. The data indicate that IL-6 blockade affects the B cell hyperreactivity in RA patients.

Vaccination survey in patients with rheumatoid arthritis: a cross-sectional study.

The objective of this study is to evaluate the vaccination status in rheumatoid arthritis (RA) patients during routine clinical practice, data from a German non-interventional cross-sectional study. In this prospective study, patients with rheumatoid arthritis were interviewed using a standardized questionnaire focusing on vaccination. Available vaccination documents were evaluated, and titers for common vaccination antigens (hepatitis B, rubella, mumps, measles, diphtheria, tetanus) were analyzed with special regard to the underlying treatment and age of patients. A total of 301 RA patients treated with conventional DMARDs alone (cohort I, n = 125), TNF-blocking agents (cohort II, n = 117), or B-cell depletion with rituximab (cohort III, n = 59) have been studied. Significantly more patients in the biologic cohorts II and III were aware of an increased risk of infections (I: 67.7%, II: 83.8%*, III: 89.9%*, P < 0.05). Pneumococcal vaccination rate was significantly higher (I: 20.2%, II 36.8%* and III: 39.0%*, P < 0.05) compared with cohort I. Differences were less evident for influenza. Significantly more patients ≥60 years of age have been vaccinated against Streptococcus pneumoniae and influenza. An obvious discrepancy existed between vaccination awareness and actual vaccination rates for all cohorts. No significant differences in vaccination titers could be seen between the three cohorts. Awareness of infectious complications was more present in patients treated with biologicals, and also, the rate of patients vaccinated against Streptococcus pneumoniae increased significantly depending on the underlying treatment. Nevertheless, there was a discrepancy between vaccination awareness and actual vaccination rates for all cohorts.

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.

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.

CD27-IgD- memory B cells are modulated by in vivo interleukin-6 receptor (IL-6R) blockade in rheumatoid arthritis.

INTRODUCTION: Enhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ). METHODS: DN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, VH gene rearrangements and specific isotypes. RESULTS: Phenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (P <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05 to 2.06). CONCLUSIONS: In RA, the heterogeneous DN B cell compartment is expanded and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.

Rituximab therapy leads to reduced imprints of receptor revision in immunoglobulin κ and λ light chains.

OBJECTIVE: Transient B cell depletion by rituximab (RTX) has become a specific treatment of rheumatoid arthritis (RA). Although phenotypic repopulation kinetics of B cell subsets are well documented, precise molecular analyses of the reconstituting immunoglobulin (Ig) genes encoding the B cell receptor in RA are sparse. METHODS: A total of 708 individual CD19+CD27+ (memory) and CD19+CD27- (naive) B cells from 2 patients with RA were analyzed at baseline and 7 months after RTX at B cell repopulation. Ig light chain variable kappa (Vκ) and lambda (Vλ) light chain gene rearrangements were amplified, sequenced, and analyzed with a focus on receptor revision. RESULTS: The naive as well as the memory repertoire repopulated polyclonally with diverse use of variable light chain gene families and minigenes. During the reconstitution phase, B cells used significantly fewer Jκ distal Vκ genes (p = 0.0006), with a higher frequency of somatic hypermutation of rearrangements employing Jκ5 compared to baseline in memory B cells. The use of Vλ rearrangements in regenerating B cells was also biased toward use of Vλ genes of the proximal cassette. In general, reemerging CD27+ Ig light chain genes were substantially more highly mutated than before RTX therapy (p < 0.0001, baseline vs during reconstitution). CONCLUSION: Our data indicate that RTX therapy leads to generation of distinct Vκ/Jκ and Vλ/Jλ gene repertoires consistent with replenishment of antigen-experienced B cells by germinal centers. At baseline, the imprints of receptor revision appeared to be more striking, which indicates that receptor revision is active in patients with RA and can be reduced by RTX.

Renal perfusion in scleroderma patients assessed by microbubble-based contrast-enhanced ultrasound.

OBJECTIVES: Renal damage is common in scleroderma. It can occur acutely or chronically. Renal reserve might already be impaired before it can be detected by laboratory findings. Microbubble-based contrast-enhanced ultrasound has been demonstrated to improve blood perfusion imaging in organs. Therefore, we conducted a study to assess renal perfusion in scleroderma patients utilizing this novel technique. MATERIALS AND METHODOLOGY: Microbubble-based contrast agent was infused and destroyed by using high mechanical index by Siemens Sequoia (curved array, 4.5 MHz). Replenishment was recorded for 8 seconds. Regions of interests (ROI) were analyzed in renal parenchyma, interlobular artery and renal pyramid with quantitative contrast software (CUSQ 1.4, Siemens Acuson, Mountain View, California). Time to maximal Enhancement (TmE), maximal enhancement (mE) and maximal enhancement relative to maximal enhancement of the interlobular artery (mE%A) were calculated for different ROIs. RESULTS: There was a linear correlation between the time to maximal enhancement in the parenchyma and the glomerular filtration rate. However, the other parameters did not reveal significant differences between scleroderma patients and healthy controls. CONCLUSION: Renal perfusion of scleroderma patients including the glomerular filtration rate can be assessed using microbubble-based contrast media.

Efficacy and safety of rituximab treatment in patients with antineutrophil cytoplasmic antibody-associated vasculitides: results from a German registry (GRAID).

OBJECTIVE: Rituximab (RTX) therapy is a treatment option in patients with refractory antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We investigated the tolerability and clinical efficacy of RTX in a cohort of patients with refractory AAV. METHODS: Clinical and safety data of patients with AAV treated with RTX were retrospectively assessed from the data of a German national registry. RESULTS: In total, 58 patients were included in this analysis (50/58 with granulomatosis with polyangiitis; 8/58 with microscopic polyangiitis who received at least 1 cycle, 17 patients who received 2 cycles, and 3 patients who received 3 cycles of RTX). Response was classified as complete and partial in 22 (40%) and in 29 cases (52.7%), respectively. Four patients (7.3%) were classified as nonresponders. CONCLUSION: RTX was well tolerated with good clinical efficacy in patients with refractory AAV.

Sustained Remission After Combination Therapy with Rituximab and Etanercept in Two Patients with Rheumatoid Arthritis After TNF Failure: Case Report.

OBJECTIVES: Approximately up to 40% of patients with rheumatoid arthritis (RA) fail to respond to tumor necrosis factor (TNF) inhibitors, lose response over time or are unable to tolerate treatment. MATERIALS AND METHODOLOGY: We report two female patients suffering from active, refractory rheumatoid arthritis despite TNF blocking agents who have been treated with rituximab added to ongoing therapy with etanercept. RESULTS: Combination therapy was tolerated without any acute side effects. Both patients improved with a significant, long lasting reduction of disease activity (DAS28, CRP). Evaluation of the immunological parameters showed the expected B-cell depletion and a transient reduction of immunoglobulin-levels. One patient developed four serious infections requiring antibiotic treatment (1 pneumonia, 3 exacerbations of her pre-existing chronic bronchitis) within follow up of 45 months. CONCLUSION: Combination therapy of rituximab and etanercept lead to a significant improvement of clinical disease activity and inflammatory parameters in two RA patients refractory to anti-TNF treatment.

Delayed acquisition of somatic hypermutations in repopulated IGD+CD27+ memory B cell receptors after rituximab treatment.

OBJECTIVE: Transient B cell depletion by rituximab has been used with clinical efficacy in the treatment of patients with rheumatoid arthritis (RA). Previous studies of B cell repopulation have shown long-term numerical reduction in memory B cells. Non-class-switched IgD+CD27+ memory B cells, in particular, repopulate slowly. This study was undertaken to determine whether mutational acquisition in individual B cell receptors in repopulating class-switched and non-class-switched memory B cells is affected by rituximab. METHODS: Cells obtained from 16 RA patients, 4 healthy donors, and 3 patients who underwent allogeneic stem cell transplantation (ASCT) were analyzed using single B cell sorting followed by nested polymerase chain reaction and Ig V(H)3 sequencing. RESULTS: There was a delayed acquisition of mutations in Ig receptors of IgD+ memory B cells over a period of 6 years after a single course of rituximab. One year after rituximab treatment, 84% of single repopulating IgD+CD27+ B cells were unmutated, and no highly mutated Ig receptors were found (compared with 52% before therapy). Over time, increasing numbers of mutations were detected. Even 6 years after rituximab treatment, however, mutations in IgD+ memory B cells were still significantly reduced. In contrast, class-switched memory B cells repopulated with quantitatively normal mutations. In comparison, in patients undergoing ASCT, IgD+ memory cells repopulated earlier with higher mutations in Ig receptors. CONCLUSION: Our data suggest that IgD+ memory B cells are particularly susceptible to the effects of rituximab, with delayed acquisition of mutations in their Ig receptors still evident 6 years after a single course of rituximab. Our findings indicate that these cells have different requirements for mutational acquisition compared with class-switched memory B cells.

Anti-CD20 therapy in patients with rheumatoid arthritis: predictors of response and B cell subset regeneration after repeated treatment.

OBJECTIVE: B cell depletion with the anti-CD20 antibody rituximab has proven efficacy in patients with rheumatoid arthritis (RA). The effects on B cell homeostasis after repeated treatments and the relationship of certain B cell subsets to clinical response or relapse are currently not known. METHODS: In this open-label study, 17 patients with RA refractory to standard therapy were treated with 1 cycle of rituximab. Of these 17 patients, 11 received a second cycle of rituximab therapy. Immunophenotyping was performed before therapy and during B cell recovery. RESULTS: Twelve of 17 patients showed a good European League Against Rheumatism response after receiving 1 cycle of rituximab therapy. At the time of B cell recovery, the IgD+,CD27+ memory B cell subset was significantly larger (P = 0.019) in the nonresponder group. Within the group of 12 responders, 6 patients, whose disease was characterized by a significantly higher proportion of overall CD27+ memory B cells before therapy, experienced an early relapse (weeks 24-40 posttreatment). Eleven patients were re-treated, again resulting in a good clinical response. B cell reconstitution followed a similar pattern after each cycle. The early reconstitution phase was characterized by immature CD38++,IgD+,CD10+ B cells, whereas the number of naive B cells increased continuously thereafter. The number of memory B cells was still reduced at the time of the second depletion but recovered to levels similar to those following the first cycle of therapy. CONCLUSION: Data derived from repeated B lymphocyte depletion with rituximab in patients with RA suggest that analysis of certain memory B cell subsets provides information on efficacy, response, and late as well as early relapse, consistent with the conclusion that targeting memory B cells is a key to its mechanism of action.

Modulation of molecular imprints in the antigen-experienced B cell repertoire by rituximab.

OBJECTIVE: Transient B cell depletion by rituximab has recently gained more importance in the treatment of rheumatic disorders. Nevertheless, little is known about the reemerging B cells. We analyzed dynamic changes in the repopulating B cells, particularly the postswitch B cells, and studied the mutational patterns of Ig genes in antigen-experienced B cells. METHODS: Five patients with active rheumatoid arthritis (RA) were treated with rituximab. In 3 patients, B cell receptor (BCR) gene analysis was performed before treatment and during B cell recovery using genomic DNA. In 2 patients, B cell subsets were studied during the early recovery phase using single-cell technology. For comparison, immunophenotyping of B cell subsets was performed. RESULTS: Early B cell recovery was marked by a relatively expanded population of highly mutated B cells, which were correlated with B cells with a plasmablast phenotype on comparative immunophenotyping. Analysis of the mutational pattern in these cells revealed increased RGYW/WRCY (where R = A/G, Y = C/T, and W = A/T) hotspot targeting (44% before rituximab versus 59% after) and elevated ratios of replacement to silent mutations within the complementarity-determining regions in Ig genes (1.87 before rituximab versus 2.67 after; P < or = 0.0025). CONCLUSION: Our findings show that rituximab leads to qualitative changes in the imprints of highly mutated, antigen-experienced BCRs, representing the result of selection, whereas molecular processes such as Ig V rearrangements are not affected by this treatment.

Frequency of regulatory T cells is not affected by transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis.

OBJECTIVES: Transient B cell depletion with the monoclonal anti-CD20 antibody rituximab has shown favourable clinical responses in patients with rheumatoid arthritis (RA). Recently a characteristic regeneration pattern of B cell subpopulations has been reported. However, little is known about the impact of B-cell depletion on peripheral T cells in particular regulatory T cells. MATERIALS AND METHODOLOGY: 17 patients with RA having failed anti-TNF were treated with rituximab. Four colour staining was performed using CD19, CD3, CD4, CD8, CD16, CD56, CD25, HLA-DR, HLA-G and intracellular Foxp3 at five time points spanning up to 12 months after rituximab. In addition, quantification of the soluble form of the HLA class I molecule HLA-G by ELISA has been performed. RESULTS: Peripheral B cell depletion lasted 6 to 9 months. The absolute number of CD3+, CD4+ and CD8+ lymphocytes showed no significant changes up to 1 year after B-cell depletion compared to before therapy. Only the relative frequency for CD3 and CD4 showed a significant increase (p < 0.05). In particular, CD4+CD25++ and Foxp3 positive regulatory T cells remained constant. The percentage of HLA-G positive cells in the CD4+ or CD8+ population did not change significantly either. The amount of sHLA-G remained without significant changes. CONCLUSION: Absolute T cell counts showed no significant changes after rituximab compared to the time point before therapy.In particular, the frequency of regulatory T cells with a CD4+CD25++ phenotype as well as positive Foxp3 expression were numerically stable. Additionally, HLA-G positive regulatory T cells and soluble levels of HLA-G showed no significant changes.

Regeneration of B cell subsets after transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis.

OBJECTIVE: Transient B cell depletion with the monoclonal anti-CD20 antibody rituximab has resulted in favorable clinical responses in patients with rheumatoid arthritis (RA). However, little is known about the regeneration profile of different peripheral B cell subpopulations. The aim of this study was to delineate the regeneration profile of different B cell subsets in the peripheral blood after selective anti-CD20-mediated B cell depletion. METHODS: Seventeen patients with RA refractory to standard therapy were treated with rituximab. Patients 1-6 received 4 weekly infusions of rituximab at a dose of 375 mg/m2, and patients 7-17 received 2 infusions of rituximab (1,000 mg), 2 weeks apart. Four-color staining was performed at several time points, using CD38, IgD, and CD27 in addition to other cell surface markers. In one patient, the mutational status of the immunoglobulin receptor was examined. RESULTS: The analysis revealed a distinct pattern of B cell regeneration. The first wave of repopulating B cells were immature B cells (CD38high,IgD+,CD10+,CD24high), the immunoglobulin receptors of which were not yet somatically mutated. In parallel, a recirculation of plasma cells was observed. Later, the number of naive B cells increased, and these cells predominated in the peripheral blood B cell pool. CD27+ memory B cells showed a slow and delayed repopulation, and the level of these cells stayed significantly reduced (<50%) compared with baseline values, for more than 2 years. CONCLUSION: Our findings provide evidence for a characteristic regeneration pattern of B cell subpopulations, with long-lasting modulation of B cell subset composition, after selective anti-CD20-mediated B cell depletion.