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. 2015 Feb 16;179(3):414–425. doi: 10.1111/cei.12472

Effect of rituximab on B cell phenotype and serum B cell-activating factor levels in patients with thrombotic thrombocytopenic purpura

E Becerra *, M A Scully †, M J Leandro *, E O Heelas †, J-P Westwood †, I De La Torre ‡, G Cambridge *,✉
PMCID: PMC4337674  PMID: 25339550

Abstract

Autoantibodies inhibiting the activity of the metalloproteinase, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), underlie the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Rituximab (RTX) combined with plasma-exchange (PEX) is an effective treatment in TTP. Patients can remain in remission for extended periods following PEX/RTX, and this is associated with continuing reduction in antibodies to ADAMTS13. Factors controlling B cell differentiation to autoantibody production, including stimulation through the B cell receptor and interactions with the B cell-activating factor (BAFF), may thus impact length of remission. In this cross-sectional study, we measured naive and memory B cell phenotypes [using CD19/immunoglobulin (Ig)D/CD27] following PEX/RTX treatment in TTP patients at B cell return (n = 6) and in 12 patients in remission 10–68 months post-RTX. We also investigated relationships among serum BAFF, soluble CD23 (sCD23– a surrogate measure of acquiring B memory (CD27+) phenotype) and BAFF receptor (BAFF-R) expression. At B cell return after PEX/RTX, naive B cells predominated and BAFF-R expression was reduced compared to healthy controls (P < 0·001). In the remission group, despite numbers of CD19+ B cells within normal limits in most patients, the percentage and absolute numbers of pre-switch and memory B cells remained low, with sCD23 levels at the lower end of the normal range. BAFF levels were correlated inversely with BAFF-R expression and time after therapy. In conclusion, the long-term effects of RTX therapy in patients with TTP included slow regeneration of memory B cell subsets and persistently reduced BAFF-R expression across all B cell subpopulations. This may reflect the delay in selection and differentiation of potentially autoreactive (ADAMTS13-specific) B cells, resulting in relatively long periods of low disease activity after therapy.

Keywords: BAFF, BAFF-R, B cells, rituximab, TTP

Introduction

Thrombotic thrombocytopenic purpura (TTP) is an acquired, life-threatening disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia and signs of organ dysfunction, including neurological, cardiac, renal and abdominal symptoms 1. It is due to deficiency or dysfunction of the metalloproteinase ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves von Willebrand factor, with resulting increased platelet adhesion and thrombus formation in small blood vessels 2,3. Most cases are associated with the production of autoantibodies [immunoglobulin (Ig)M/IgG/IgA] to ADAMTS13, of which the most predominant subclasses are IgG4, followed by IgG1 4. Plasma exchange (PEX) and corticosteroids are the standard therapies, but many patients require additional immunosuppression 5.

B cell depletion therapy based on rituximab (RTX; a chimeric monoclonal antibody against the pan-B cell marker, CD20) was first used to treat patients with non-Hodgkin's lymphoma 6, and is now also used widely in autoimmune diseases such as rheumatoid arthritis (RA) 7 and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis 8. The inclusion of RTX in the treatment of acute TTP has significantly improved clinical outcome in these patients, who achieve higher response rates and greatly increased median times to relapse compared with PEX alone 9–13. In addition, consideration of pre-emptive rituximab treatment (as monotherapy) has been suggested in cases where severe ADAMTS13 deficiency persists during TTP remission in order to prevent subsequent flare 13,14.

The development of the normal B cell repertoire through to antibody secretion by daughter plasma cells is largely dependent upon the concerted expression of the three different receptors for cytokine B cell activating factor BAFF, with levels of expression varying over the different stages of B cell differentiation 15. BAFF-R (BR3), however, is expressed on all circulating B cell subsets. Transitional (early naive) human B cells are most sensitive to prosurvival signals delivered by BAFF through BAFF-R prior to activation through the B cell receptor (BCR) 16. Low or faulty expression of BAFF-R in primary immunodeficiency results in low levels of serum IgM and IgG, increased numbers of transitional B cells and strongly reduced T independent immune responses 17. Serum BAFF levels before RTX are reportedly increased in some TTP patients at presentation, and to rise following treatment with RTX 18. This is similar to what has been described for other autoimmune diseases 19–21.

Immunophenotyping of B cells using monoclonal antibodies to CD19 (a pan-B cell marker), IgD and CD27 allows identification of naive and antigen-experienced B cell subpopulations, namely naive (IgD+CD27–), pre-switch memory (IgD+CD27+), switched memory (IgD–CD27+) and IgD-CD27– resting memory B cells 22,23. The maturation of naive B cells to memory status (often accompanied by class-switch recombination), is usually associated with gain of CD27 expression 24 and is largely dependent upon interactions with T cells and with Toll-like receptors (TLR) 25.

Remission after PEX/RTX can last for long periods of time after B cell return in TTP patients and is related to the continuation of a reduction in the production of IgG anti-ADAMTS13 antibodies 26. It is therefore likely that factors impacting B cell survival and maturation, and differentiation into immunoglobulin secretion are responsible for such extended remissions. In this retrospective study, we explored the dynamics of the BAFF/BAFF-R system in patients with TTP undergoing therapy with RTX by following B cell phenotypes, BAFF-R expression and relationships with serological parameters. In this cross-sectional study, we utilized samples from two time-points after RTX-induced depletion: (i) B cell return and (ii) in patients maintaining long-term remission. Laboratory parameters, serum BAFF levels and circulating soluble CD23 (sCD23) were also measured. CD23, the low-affinity Fc epsilon receptor II, is expressed on mature naive B cells. Following antigen encounter or activation through TLR, surface expression of CD27 induces cleavage of CD23 and the soluble receptor, sCD23, is released into the circulation 27. Levels of sCD23 were therefore used as a surrogate marker of B cell maturation to a CD27+ phenotype following RTX therapy, as described previously 28.

Methods

Patients

Blood samples were obtained from a total of 20 patients with TTP (age range 29–79 years) and 12 healthy controls (HC) (age range 22–59 years). Clinical data are summarized in Table 1. Blood samples were available from patient 1 both at acute admission and at B cell return. Patients were all attending the Department of Haematology at University College London Hospital (UCLH) and treated on the basis of clinical need. In acute presentations of TTP, PEX and corticosteroids were given. At UCLH, RTX is added following confirmation of an autoimmune phenotype, as described previously 1. RTX was delivered in at least four infusions of 375 mg/m2 but up to eight infusions, if required, to reduce IgG anti-ADAMTS13 levels and to normalize ADAMTS13 activity 10. Time after RTX was defined as time since first rituximab infusion. The study had ethical approval and all patients gave informed consent before entering the study (MREC: 08/H0716/72). ADAMTS13 activity levels were documented and values shown in Table 1 were on the day of PBMC collection.

Table 1.

Clinical and laboratory characteristics of thrombotic thrombocytopenic purpura (TTP) patients

Age Gender Platelet count (× 109/l) ADAMTS13 (% normal value) BAFF (ng/ml) Months post-RTX sCD23 (pg/ml) CD19+ absolute count (× 106)
Acute TTP
 1 65 M 16 26 1·01 3876 n.a.
 2 52 M 9 <5 1·11 3284 n.a.
 3 53 F 12 <5 1·69 4103 n.a.
B cell return
 1* 65 M 215 96 3·12 10 1026 30
 4 64 M 259 40 2·28 7 250 7
 5 26 M 299 102 1·69 8 946 143
 6 79 F 204 >100 4·30 5 315 2
 7 36 F 335 88 4·46 6 2354 7
 8# 75 M 35 <5 2·42 5 3564 218
Remission
 9 41 M 174 34 1·56 32 1143 323
10 43 F 265 >100 1·58 44 950 717
11 53 F 288 75 1·17 68 1517 178
12 16 F 277 37 1·42 18 1086 154
13 72 M 475 91 2·86 14 1184 69
14 52 F 289 >100 1·58 29 1382 335
15 63 M 216 58 1·28 31 1001 82
16 45 M 246 <5 0·91 51 1740 1326
17 44 M 191 115 1·30 33 1162 198
18 42 F 274 109 2·35 54 1407 185
19 29 F 544 99 3·26 10 1257 141
20 57 F 279 16 1·01 60 1210 398
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(a) Patients 1 and 1* (shown in bold type) serial samples from same individual. (b) #Patient 8: was relapsing at B cell return. (c) Normal ranges given in the Methods. ANAMTS13 = a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; RTX = rituximab; BAFF = B cell activating factor; n.a. = not applicable.

Patients with TTP were divided into three groups, as follows.

  • Acute TTP before RTX (n = 3).  Patients with a de-novo acute presentation of TTP associated with significantly decreased ADAMTS13 activity and positive IgG anti-ADAMTS13 antibodies.

  • B cell return (first documented) in TTP patients who had achieved clinical remission (sustained normal platelet counts >150 × 109/l) following RTX therapy (n = 6).  After adequate depletion of B cells in peripheral blood (<5 × 106 CD19+ B cells/l), first B cell return was defined as when B cell count became equal to or exceeded 5 × 106 CD19+ B cells/l or rose to ≥0·5% of the total lymphocyte count. The word ‘documented’ repopulation was used because although the patients had >5 × 106 CD19+ B cells/l present at that particular clinic visit, the exact time that B cells started repopulating could have occurred at any time between routine visits.

  • TTP patients in remission (n = 12).  TTP patients who remained in clinical remission (platelet count >150 × 109/l) following B cell return, as defined above.

Assessment of B cell depletion

The normal range for CD19+ B cells used by our pathology laboratory was 0·03–0·40 × 109/l (5–15% of lymphocytes). Adequate depletion of B cells in the peripheral blood was deemed to have occurred when CD19+ cells <5 × 106/L.

Peripheral blood mononuclear cells (PBMC) isolation and staining

PBMC were isolated from 10 ml heparinized whole blood from patients and healthy controls and frozen in liquid nitrogen. For fluorescence activated cell sorter (FACS) analysis, PBMC (1 × 106/sample) from patients and at least one healthy control were thawed and incubated with conjugated antibodies for 20 min in the dark at 4°C. Cells were washed, fixed with paraformaldehyde 2% for 5 min and 300 000 events per sample gated on total lymphocytes acquired with a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analysed with FlowJo (TreeStar, Stanford University, CA, USA). Absolute cell counts were calculated from routine lymphocyte counts. Acute TTP patient samples were taken before RTX therapy, but patients had received PEX and steroids on admission.

Phenotypical analysis

Immunophenotyping of PBMC was performed using matched combinations of anti-human murine monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein cyanin (PerCP-Cy5·5) or allophycocyanin (APC). Combinations of anti-CD19 PerCP-Cy5·5, anti-IgD-FITC and anti-CD27-APC were used to define B cell subsets. Expression of BAFF-R on each subset was analysed using anti-BAFF-R-PE (11C1). All antibodies were purchased from BD Biosciences (San Jose, CA, USA) or eBioscience (San Diego, CA, USA).

Measurement of BAFF

Serum BAFF levels were quantified using the Human Quantikine® BAFF/BLyS immunoassay ELISA kit (R&D Systems Europe Ltd, Abingdon, UK). The mean ± 3 standard deviations for normal sera given by the manufacturer was 1·17 ± 0·84 ng/ml (2·01 ng/ml). This was therefore used as the normal range upper-level cut-off.

Soluble CD23

Commercial ELISAs were used to measure serum sCD23 (R&D Systems Europe Ltd). The normal range for sCD23 given by the manufacturers was 1235–5024 pg/ml.

ADAMTS13 assays

Plasma ADAMTS13 activity was measured by a fluorescence resonance energy transfer assay 29 and expressed as a percentage relative to the activity in pooled normal plasma (normal range 60–123%; lower limit of detection 5%). IgG antibodies to ADAMTS13 were measured to confirm diagnosis, as described previously (normal range <6·1%) 10.

Statistical analysis

The proportions (%) and absolute number of B cell subsets within the CD19 gate and of CD19 cells expressing BAFF-R [% and mean fluorescence intensity (MFI)] in patient and control groups were compared using the non-parametric Mann–Whitney U-test or, for, normally distributed data, the unpaired t-test with Welch's correction. Correlation statistics were used to determine relationships between serum BAFF levels, percentage of BAFF-R-positive cells and expression (MFI), with length of time after RTX and with laboratory parameters. Analyses were performed using GraphPad Prism 6 (GraphPad, San Diego, CA, USA).

Results

Clinical characteristics

Table 1 summarizes the clinical characteristics of patients with TTP. Due to the difficulty of collecting samples from these patients before instituting (often emergency) treatment, only three de-novo presentations were collected, and therefore these cases have been included in the descriptive, but not the statistical, analyses. All three cases had received PEX and corticosteroids before blood sampling. Of the six TTP patients studied at B cell return (5–10 months after RTX), one patient was undergoing clinical relapse (patient 8). This patient had the highest CD19 absolute count and level of sCD23. In all 12 patients in remission, B cell return was confirmed in samples taken between 10 and 68 months after RTX, with all having CD19 counts within or even exceeding the normal range (Table 1; Fig. 5c).

Fig 5.

Fig 5

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Serum B cell activating factor (BAFF) levels and relationships with B cell return, time after rituximab (RTX) and B cell numbers during remission. In (a) serum BAFF levels in healthy controls (HC) and in thrombotic thrombocytopenic purpura (TTP) patients at acute presentation and at B cell return are shown. Box indicates median, 25th and 75th percentiles and the whiskers indicate ranges of values for each group. Comparisons were made using the Mann–Whitney U-test with significance levels indicated (**P < 0·001). In (b) and (c), respectively, the relationship between serum BAFF levels with time after plasma-exchange (PEX)/RTX and with number of CD19+ B cells, respectively, in patients remaining in long-term remission are shown. The solid lines indicate the calculated linear regression and correlation statistic (Spearman's rank) in each graph. Dashed lines show upper limit of normal range for serum BAFF. Dotted line in (b) indicates cut-off level for B cell return (>5 CD19+ cells/μ).

B cell phenotype in TTP patients after RTX compared with healthy controls

Figure 1a is a representative plot showing B cell phenotypes in CD19-gated PBMC from an HC as defined by the combination of IgD/CD27. Figure 1b shows the distributions of the same B cell subpopulations in a sample taken from a TTP patient at B cell return. In cross-sectional analyses (Fig. 1c,d) the distribution of B cell subpopulations at B cell return after RTX is compared with HC. Absolute numbers of cells within each B cell subpopulation are plotted in Fig. 1c, percentage of CD19+ B cells, and in Fig. 1d. Naive B cells (IgD+CD27–; Fig. 1b) predominated at B cell return, with their percentage significantly higher than in HC; pre-switch memory (IgD+CD27+) populations were reduced significantly (Fig. 1c). In Fig. 1d the absolute numbers of B cells at B cell return are shown. The TTP patient relapsing at B cell return (indicated with the crossed symbol) had the highest absolute numbers of post-switch CD27+ and CD27– memory B cells and also the highest value of sCD23 at B cell return (Table 1), but percentages of each B cell subpopulation were similar throughout.

Fig 1.

Fig 1

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Examples of immunochemical stainings for B cell subpopulations from a healthy control and from a patient with thrombotic thrombocytopenic purpura (TTP) at B cell return. Representative plots showing B cell subpopulations in CD19-gated peripheral blood mononuclear cell (PBMC) sample as defined using combinations of immunoglobulin (Ig)D and CD27 in a healthy control in (a) and (b) using PBMC taken from a patient with TTP at B cell return after rituximab (RTX). (c) Relative proportions of each B cell subpopulation (% total CD19+ cells) in each cohort of TTP patients at key points over the course of RTX are compared with healthy controls (HC). Comparisons were also made between median values in at key points, namely B cell return and remission. (d) Absolute numbers of B cells within each subpopulation are shown. Results were compared using Mann–Whitney rank sum analysis and significance levels indicated as *P < 0·05; **P < 0·01; ***P < 0·001.

In Fig. 2, B cell subpopulations in the remission group of TTP patients are shown in relation to time after RTX. The decrease in the percentage of naive (IgD+CD27–) B cells and the increase in percentages of CD27+ and CD27– memory B cells were related significantly to time after treatment (Fig. 2a,c,d; P < 0·01 for all). There was no relationship between time after PEX/RTX and the rate of recovery of the pre-switch (IgD+CD27+) B cells, which was highly variable between individuals. (Fig. 2b). The resting memory population (IgD–CD27–) (Fig. 2d) appeared the most robust, with values approximating those of HC in most patients even at points close to B cell return.

Fig 2.

Fig 2

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Thrombotic thrombocytopenic purpura (TTP) patients in long-term remission: relationships between B cell subpopulations with time after rituximab (RTX). Percentages of each B cell subpopulation (CD19+) in the 12 TTP patients in remission for >10 months following treatment are plotted against time in months after plasma-exchange (PEX)/RTX in (a–d). The solid line in each plot depicts the calculated linear regression with correlation statistics also given (Spearman's rank and correlation statistic) for each B cell phenotype. Shaded areas indicate the normal range of each B cell phenotype found in corresponding B cell subpopulations in healthy controls (HC). The decrease in the percentage of naive (IgD+CD27–) B cells and the decrease in percentages of CD27+ and CD27– memory B cells with time after treatment were significantly related (P > 0·01 for all) but there was no relationship between time after PEX/RTX and recovery of the pre-switch B cell subpopulation.

BAFF-R expression (MFI) at B cell return

In Fig. 3a, BAFF-R expression in PBMC in an HC shows that only CD19+ cells express BAFF-R. After gating on the CD19+ population, the majority (>99%) of CD19+ cells express BAFF-R. Figure 3b shows a representative plot of BAFF-R expression on PBMC from a patient with TTP at B cell return, showing that BAFF-R expression was again restricted to CD19+ B cells but was greatly reduced.

Fig 3.

Fig 3

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CD19+ B cells in thrombotic thrombocytopenic purpura (TTP) patients at B cell return after rituximab (RTX). The representative plot in (a) shows B cell activating factor (BAFF)-R expression on CD19+ B cells from a healthy control within the lymphocyte gate and also the derived histogram showing relative expression of BAFF-R within the gated CD19+ population. (b) The %CD19+ B cells positive for BAFF-R are shown for peripheral blood mononuclear cells (PBMC) taken close to B cell return from TTP patients. In (c), % CD19+ cells positive for BAFF-R and (d), mean fluorescence intensity (MFI) of BAFF-R expression, are compared at B cell return with values for B cell subpopulations from healthy controls (HC). Box-and-whiskers indicate median, 25th, 75th percentiles and ranges in all four B cell subpopulations, as defined by CD19/IgD/CD27. Significance levels comparing HC with TTP patient groups at B cell return were determined using the Mann–Whitney rank sum test (***for P < 0·001; **P < 0·01; *P < 0·05).

Virtually all peripheral B cells from normal individuals express BAFF-R, as can be seen in Fig. 3c. At B cell repopulation in TTP patients, the percentage of B cells in each subpopulation expressing BAFF-R at B cell return was found to be significantly lower than HC in all subpopulations. The mean fluorescence intensity, which reflects the relative number of BAFF-R per cell, was also reduced markedly compared with HC, again across all B cell subpopulations (Fig. 3d).

BAFF-R on B cell subpopulations in patients in remission: relationship with serum BAFF

Although the percentages of BAFF-R+ B cells within all subpopulations were reduced compared with HC at B cell return, BAFF-R+ B cells increased to within the normal range in patients during remission following B cell return (Fig. 4a). However, despite a time-dependent increase in BAFF-R expression (MFI), most notably in naive and pre-switch B cells (Fig. 4b) the numbers of BAFF-R per cell remained well below the normal limits for healthy individuals for many months after PEX/RTX.

Fig 4.

Fig 4

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Changes in percentages positive and relative expression [mean fluorescence intensity (MFI)] of B cell activating factor (BAFF)-R on B cell subpopulations with time after rituximab (RTX). The panel in (a) shows the percentages of CD19+ B cells positive for BAFF-R in B cell subpopulations defined by immunoglobulin (Ig)D/CD27 for TTP patients in long-term remission after plasma-exchange (PEX)/RTX in relation to time after treatment. Similarly, in (b) the mean fluorescence intensity (MFI) of BAFF-R expression on CD19+ B cells in each subpopulation are shown. The normal ranges of values given by respective B cell subpopulations in healthy controls (HC) are indicated by shaded areas in both (a) and (b). In (b) the MFI of BAFF-R expression in all four B cell subpopulations were plotted against serum BAFF levels. The solid lines in all graphs depict the calculated linear regression and correlation statistics (Spearman's rank test). Vertical lines in (c) indicate upper limit of normal range for serum BAFF.

Relationships among serological parameters

It has been suggested that total available BAFF-binding receptors may regulate serum BAFF levels 30. We therefore investigated whether there was a relationship between serum BAFF levels and BAFF-R expression (MFI) in repopulated TTP patients, and found a statistically significant inverse correlation over all B cell subpopulations (Fig. 4c) (r2 > 0·45; P < 0·01 for all). BAFF levels in acute TTP patients and at B cell return were compared with HC and are shown in Fig. 5a. In the three acute TTP patients studied here BAFF levels were within normal limits, but may have been influenced by PEX/corticosteroids given before sampling. Following RTX, median BAFF levels remained significantly raised at B cell return compared to HC (P < 0·05), and most were above the normal upper-limit range (Table 1). Serum BAFF levels showed a significant negative correlation with time after treatment in the remission group, but most had fallen to levels within the normal range after less than 1 year in remission (Fig. 5b). In Fig. 5c, serum BAFF levels are plotted against numbers of CD19+ B cells during remission; no correlation was found, but notably most patients' B cell counts had shown a robust recovery after PEX/RTX to well within the normal range.

ADAMTS13 levels showed a weak but significant negative correlation with both absolute numbers of memory B cells and serum total IgG levels (Spearman's rank correlation; r2 > 0·36; P < 0·05) (data not shown). Although sCD23 levels remained at the lower end of the normal range they were associated strongly with absolute numbers of IgD+CD27+ and IgD–CD27+ pre- and post-switched memory B cells in patients in remission (Spearman's rank correlation; r2 = 0·57 and 0·53, respectively, P < 0·01 for both). There was also a significant negative correlation between soluble CD23 levels with the percentage of naive B cells (IgD+CD27–) and a positive correlation with the percentage of post-switch memory B cells (both r2 > 0·52; P < 0·01) (Supporting information, data 1).

Discussion

In patients with TTP, therapy based on RTX has established that autoreactive B cells play key roles in disease pathogenesis 7,11. One of the first observations with respect to B cell kinetics after B cell-depleting therapy in autoimmune diseases was that relapse occurred at the time of or after B cell return, and that B cell return mirrored ontogeny 31,32. The relative number of returning B cells, however, has not been a reliable indicator of the likelihood of return of symptoms either in patients with RA or in TTP 26,33. Analysis of B cell phenotype and serological studies following RTX-based therapy in patients with autoimmune rheumatic diseases has shown that the most consistent indicator of disease resumption is rapid progress towards differentiation into CD27+ memory B cells and immunoglobulin secretion 31,32,34. As ADAMTS13 autoantibodies are associated strongly with both the presence and activity of disease, it is likely that the same applies to patients with TTP.

B cells re-expand into an otherwise mainly intact immune environment of antigen processing and presenting cells and memory T cells after RTX. Resumption of pathogenic autoantibody production and subsequent relapse may be triggered by clonal expansion of autoreactive B cells either following selection from new naive B cells or from residual memory populations. The qualitative element for selection would thus be in the specificity of particular B cell receptors (BCR) on autoreactive B cells. Differentiation to autoantibody production would necessarily also imply that the parent B cells had successfully escaped tolerogenic checkpoints and were able to recruit appropriate T cell help.

In this cross-sectional study, we found that following the return of predominantly naive B cells to the periphery after RTX, the levels of pre- and post-switched memory B cells in TTP patients remained significantly lower than in healthy controls and resembled a developmental stage analogous to that in young children 35. During the months following B cell return, there was a gradual rise in memory compartments, but even in patients with up to 5·5 years of follow-up, all B cell subpopulations failed to reach levels seen in (adult) HC, despite the CD19 count being well within the normal range (Fig. 2; Table 1 and Fig. 5c). The finding that sCD23 levels remained towards the lower end of the normal range supported the phenotype studies, but was linked with both percentage and absolute numbers of CD27+ B cells in vivo, may prove a useful predictive marker of reconstitution of the memory B cell compartment. Without further markers, it was not possible to discern whether the poorly recovering pre-switch memory B cell subpopulation was representative of splenic marginal zone (IgM+IgD+CD27+) or IgM–IgD+CD27+ memory B cells 36–38. Poor memory B cell reconstitution may reflect long-term indirect effects of RTX on B cells, described both after organ transplantation 39 and in other autoimmune diseases 32,40. The reasons are not understood fully, but it has been suggested that productive germinal centre reactions may be disturbed due to microenvironmental changes caused by the absence of, or reduction in, mature B cells 40–42.

Our results also suggested that slow memory B cell reconstitution in TTP patients was linked with extended periods of remission. As relapse is associated most strongly with return or rises in autoantibodies to ADAMTS13, the selection and differentiation of specifically autoreactive B cell clones from naive and/or memory B cell populations must also play a key role. If derived from newly generated naive B cells, the production of anti-ADAMTS13 may additionally involve a change in affinity through clonal expansion and somatic hypermutation. The chances of sufficient autoreactive clones being selected, expanded and differentiating into immunoglobulin secreting cells may thus be low after RTX in TTP patients. The relative frequencies of autoreactive (ADAMTS13) B cell clones and the availability of specific T cells may therefore affect the relative lengths of remission in TTP, which may be distinct from those in other autoimmune diseases following RTX. For example, autoreactive B cell clones in patients with RA may be expanded more easily due to the potentially promiscuous nature of B cells with specificity for Fc of IgG or citrullinated proteins 28,43,44. Information regarding the precursor frequency and activation requirements of naive or of memory B cells specific for ADAMTS13 are lacking in TTP patients.

In normal individuals, BAFF-R is expressed on the majority (>98%) of circulating B cells, and binds only BAFF 45. We found here that BAFF-R expression [both the percentage B cells and relative expression (MFI)] was reduced significantly at B cell return, as we have also found previously in patients with RA 46. Despite that the percentage of B cells across all subpopulations had regained levels within the normal range during remission, the expression (MFI) of BAFF-R was reduced markedly for many months after therapy across all B cell subpopulations. This is similar to findings in ontogeny, where naive (often CD5+) B cells express less BAFF-R compared with adult B cells 47. In patients with BAFF-R deficiency, who form a subset of patients with late-onset common variable immunodeficiency (CVID), there are severely reduced numbers of all mature B cell subsets 17, reflecting the need for naive B cells to signal through BAFF/BAFF-R for survival 48. A partial reduction in BAFF-R expression on naive B cells after RTX may therefore decrease the level of expression of anti-apoptotic markers maintained through this receptor, thus reducing the proportion of cells able to mature further.

Serum BAFF levels were raised in most patients at B cell return and decreased with time after treatment during remission. Our analyses showed that, after BCDT, there was a strong negative association with the concentration of serum BAFF with BAFF-R (MFI) expression. Levels of BAFF above those present in normal serum have been shown previously to modulate BAFF-R expression in the absence of productive NF-κB signalling in vitro 49. In our studies, we found that even modest levels of serum BAFF, within normal limits, appeared to be significantly inversely associated with BAFF-R expression, but not with the number of CD19+ B cells per se, in patients with TTP during remission.

The presentation of ADAMTS13 protein to T cells is thought to be via binding to an endogenous ligand (mannose receptor) on dendritic cells 50. Internalization and presentation of peptides to CD4+ T cells will then rely primarily upon microenvironmental factors, such as the supply of autoantigen (ADAMTS13). The local production of cytokines (perhaps induced by complexed autoantigen) will then impact the maturation state of the dendritic cells and the processing and presentation of antigenic moieties. A strong link between the development of idiopathic TTP with human leucocyte antigen (HLA)-DRB1*11 may also favour the presentation of specific peptides to appropriate CD4+ T cells 51,52. The selection of autoreactive B cells specific for conformational epitopes on the native protein will therefore depend upon the selection and affinity maturation of B cells with appropriate B cell receptors (BCR) and prosurvival signals delivered through receptors for BAFF. The autoantibodies to ADAMTS13 in TTP patients are class-switched and somatically mutated 4 and thus have the signature of probably being generated in a T cell-dependent reaction. The answer as to why the regeneration of a pathogenic autoimmune response to ADAMTS13 is so delayed after RTX may reflect the stoichiometry of the selection of ‘new’ clones of B cells with appropriate BCR, BAFF-binding and signalling and the availability of help from (presumably) long-lived T cells. We describe here another possibly contributory mechanism for the delay in disease return. It appears that as a consequence of RTX removing a large proportion of the mature B cell pool, the naive B cells generated thereafter show a marked down-regulation of BAFF-R expression, related inversely to serum BAFF levels.

In conclusion, the continuation of disturbances of B cell homeostasis after RTX in TTP patients may be due initially to the reduction in the ‘bulk’ of B cells by RTX and the resultant raised serum BAFF levels. Expansion of repopulating naive B cells with low numbers of BAFF-R/per cell into such a BAFF-rich environment seems, conversely, to have a negative effect on BAFF-R expression, as shown here. Although BAFF-R expression gradually increased with time after RTX, it failed to reach levels comparable with HC, even after more than 5 years in some patients. The process of B cell repopulation after RTX in patients with TTP follows a similar pattern to ontogeny. The fact that naive B cells (IgD+CD27–) predominate for such long periods after RTX, together with down-regulation of receptors for BAFF, may therefore contribute indirectly to the delay in disease reactivation in patients with TTP. Reduced capacity to generate memory B cells may result as a consequence of the disruption of germinal centre architecture following RTX, as described in patients with systemic lupus erythematosus (SLE) 40, or due possibly to a higher threshold for B cell survival. Signalling through both BAFF-R and the B cell receptor are important separately and simultaneously for mature naive B cell survival, sharing anti-apoptotic signalling through NF-κB; therefore a reduction in expression of BAFF-R may affect this prosurvival pathway 53. This may therefore affect the resumption of autoimmunity by slowing the rate of selection and expansion of B cell clones, perhaps even those with appropriate ADAMTS13 specificity. Relative proportions of different B cell subpopulations and BAFF-R expression following RTX may therefore provide a cellular monitor for factors associated with prolonging remission in patients with TTP. The use of sCD23 as a biomarker of B cell differentiation to CD27+ status may also provide a useful correlate of B cell maturation following B cell-depleting therapies.

Acknowledgments

E. B. and I. d. l. T. were supported by a grant from the Alfonso Martin Escudero Foundation, Madrid, Spain.

Disclosure

E. B., E. O. H., M. A. S., I. d. l. T., J.-P. W. and G. C. have no conflicts of interest to declare.

M. L. has received consultancy and speaker fees from Roche, Chugai, BMS and Lilly, sponsorship to attend medical meetings by Roche and Chugai and research funding from Roche and GSK.

Author contributions

E. B. performed the B cell phenotype studies and performed the analyses of FACS data with M. J. L.; M. A. S. collected and collated clinical and laboratory data on the TTP patients with J.-P. W.; E. O. H. collected and stored clinical samples from patients. G. C. and M. J. L. designed the experiments and G. C. performed BAFF and sCD23 ELISAs. All authors contributed to and approved the final manuscript.

Supporting Information

Additional Supportingporting information may be found in the online version of this article at the publisher's web-site:

Supplementary Data 1. Correlation between decrease in naive B cells and increase in post-switch memory B cells (gated on CD19+ population) with serum levels of sCD23 in patients with thrombotic thrombocytopenic purpura (TTP) in long-term remission [10–68 months after plasma-exchange/rituximab (PEX/RTX)]. Normal range for sCD23 is from 1235–5024 pg/ml.

cei0179-0414-sd1.tif (1.5MB, tif)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data 1. Correlation between decrease in naive B cells and increase in post-switch memory B cells (gated on CD19+ population) with serum levels of sCD23 in patients with thrombotic thrombocytopenic purpura (TTP) in long-term remission [10–68 months after plasma-exchange/rituximab (PEX/RTX)]. Normal range for sCD23 is from 1235–5024 pg/ml.

cei0179-0414-sd1.tif (1.5MB, tif)

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