A single dose of the anti-CD20 monoclonal antibody rituximab induces a nearly complete B cell depletion in peripheral blood, but not in secondary lymphoid organs. Modulation of this remaining B cell population due to rituximab treatment may contribute to the therapeutic effects of rituximab. To assess the in vivo effects of rituximab we used lymph nodes (LNs) collected during renal transplant surgery in patients who had received rituximab 4 weeks earlier in preparation for an ABO-incompatible transplantation. Rituximab treatment resulted in a lower percentage of naïve (IgD+CD27−) and a higher percentage of switched memory (IgD−CD27+) B cells. Remarkably, transitional (CD24++CD38++) B cells were virtually lacking in the LNs of rituximab-treated patients. Moreover, LN-derived B cells from rituximab-treated patients produced different amounts of various Ig-subclasses after anti-CD40/IL-21 stimulation ex vivo. Finally, after stimulation of allogeneic T cells with LN-derived B cells from rituximab-treated patients, the proliferated T cells showed a decreased production of IL-17. In conclusion, after treatment with rituximab there remains a B cell population with different functional capacities. Consequently, the effect of rituximab on the immune response will not only be determined by the extent of B cell depletion, but also by the functional properties of the remaining B cells.
activation-induced cytidine deaminase
carboxyfluorescein succinimidyl ester
peripheral blood mononuclear cells
After transplantation of a solid organ graft, B cells can play a major role in graft rejection via the production of alloantibodies, but they can also induce an immune response by acting as professional antigen presenting cells, or by the production of various cytokines . It has recently been shown that a subset of human B cells can display regulatory function (Breg). This subset, which is predominantly found within the CD24++CD38++ B cell population and produces IL-10, is very small in healthy individuals but was found to be increased in patients with autoimmune diseases [2, 3] and in tolerant transplant patients who had stable graft function despite receiving no immunosuppression for at least 1 year . Since B cells can play such a variety of roles in the immune response, the effects of anti-B cell therapy have to be analyzed carefully.
The chimeric anti-CD20 monoclonal antibody rituximab (RTX) triggers B cell lysis through antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity or apoptosis induction [5, 6]. RTX is used to reduce autoantibody levels in various autoimmune disorders . However, RTX can also ameliorate chronic inflammatory diseases mediated by T and B cells, such as rheumatoid arthritis  and multiple sclerosis , suggesting that the therapeutic effect of RTX not solely depends on inhibition of antibody production. In renal transplantation, RTX is used successfully in ABO-incompatible (ABOi) transplantation , in desensitization protocols , and for treatment of antibody-mediated rejection .
Although a single dose of RTX induces a nearly complete B cell depletion in peripheral blood (PB), there often remains a residual B cell population in secondary lymphoid organs [13-15]. We have previously shown that exposure of human PB B cells to RTX in an in vitro nondepleting stimulation model affects B cell phenotype and function, resulting in an altered outcome of B cell–T cell interaction . This triggered us to study the functional properties of the nondepleted but potentially modulated B cells that remain present in secondary lymphoid organs after treatment with RTX. To this end, we collected lymph nodes (LNs) during renal transplant surgery in patients who had received RTX 4 weeks earlier in preparation for an ABO-incompatible renal transplantation . The phenotypic and functional properties of B cells isolated from these LNs were compared with B cells isolated from LNs collected from renal transplant patients not treated with RTX.
Materials and Methods
Collection and preparation of samples
PB samples were obtained before renal transplantation and buffy coats from healthy donors were purchased from Sanquin Blood Bank (Nijmegen, The Netherlands). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Lucron, Dieren, The Netherlands). Iliac LN were obtained during transplant surgery. Spleen cells (SPL) were obtained from deceased organ donors. LN and SPL samples were first homogenized and subsequently forced through 75-µm netwell filters (Costar, Corning International, Amsterdam, The Netherlands) to obtain single-cell suspensions. Mononuclear LN and SPL cells were isolated by density gradient centrifugation using Lymphoprep (Nycomed Pharma, Roskilde, Denmark). All samples were cryopreserved in liquid nitrogen until analysis.
Patients scheduled for an ABO-incompatible renal transplantation received a single dose of 375 mg/m2 RTX (Mabthera, Roche Pharma AG, Grenzach-Wyhlen, Germany) intravenously 4 weeks before transplantation. Prior to RTX administration, 25 mg prednisolone was given intravenously. Two weeks before transplantation, treatment with tacrolimus (0.2 mg/kg/day), mycophenolate mofetil (2 g/day) and prednisolone (10 mg/day) was started. Intravenous immunoglobulin (IVIG, Nanogam, Sanquin, Amsterdam, The Netherlands; 0.5 g/kg) was administered the day before surgery. Patients scheduled for a regular, ABO-compatible living donor kidney transplantation were used as controls. Table 1 summarizes the characteristics of all patients. None of the patients had a systemic auto-immune disease as cause of renal insufficiency and none of them received any immunosuppressive drug during the last 3 months prior to transplantation (or the administration of RTX in the ABO-incompatible group). The study was performed in accordance with the regulations set by the Medical Ethics Committees of the participating hospitals. Informed consent was obtained from all participants.
|Group||ID||Sex||Age (years)||Cause of renal failure||Type of dialysis||Re-Tx||Leukocyte count at Tx (109/L)||Lymphocyte count at Tx (109/L)|
|Control (Tx RTX−)||1||M||68||Hypertension||HD||No||4.8||1.7|
|ABOi (Tx RTX+)||1||M||68||Hypertension||PD||No||16.2||1.6|
|4||M||67||Vascular and hypertension||HD||No||8||NA|
B cells were purified from SPL and LN cells by negative selection using monoclonal antibodies directed against CD3(UCHT1), CD8(RPA-T8), CD14(M5E2), CD16(3G8), CD33(P67.6), CD56(B159) and CD235a (GA-R2 (HIR2)) (BD Biosciences, Erembodegem, Belgium) combined with sheep anti-mouse Ig-coated magnetic beads (Dynal, Denmark). This resulted in a CD19+ B cell enrichment of more than 90%. CD3+ T cells were positively selected from PBMCs of healthy donors using anti-CD3 magnetic microbeads (Miltenyi Biotec, Utrecht, The Netherlands) resulting in a purity of more than 95%.
For functional studies, 5 × 104 cryopreserved LN and SPL cells were cultured in RPMI-1640 medium supplemented with pyruvate (0.02 mM), glutamax (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) (all from Gibco, Paisley, United Kingdom), and 10% heat-inactivated pooled human serum (HPS) in 96-well round bottom plates (Greiner, Frickenhausen, Germany) in a 37°C, 95% humidity, 5% CO2 incubator. In selected conditions, 5 µg/mL RTX was added to the culture medium.
Flow cytometry and CFSE labeling
For cell surface staining, the following fluorochrome-conjugated mAbs were used: CD19(SJ25C1)-PeCy7, CD20(2H7)-PeCy7, CD25(M-A251)-PE (BD Biosciences), CD3(UCHT1)-ECD, CD4(13B8.2)-PeCy5, CD5(BL1a)-APC Alexa Fluor 700, CD8(SFCI21Thy2D3)-ECD, CD19(J3-119)-ECD, CD19(J3-119)-APC Alexa Fluor 750, CD24(ALB9)-APC, CD27(1A4-CD27)-PeCy5.5, CD38(LS198-4-3)-PeCy7, CD45(J.33)-FITC, CD45(J.33)-Krome Orange, CD45RO(UCHL1)-ECD, CD56(N901)-PeCy5, IgD(IADB6)-FITC, IgM(SA-DA4)-PE (Beckman-Coulter, Mijdrecht, The Netherlands), CD127(EBIORDR5)-PeCy7 (eBioscience, Uithoorn, The Netherlands), CD8(DK25)-PE and CD27(M-T271)-PE (Dako, Glostrup, Denmark). Isotype-matched antibodies were used to define marker settings. Intracellular analysis of IL-2(MQ1-17H12)-PE (BD Biosciences), IL-4(8D4-8)-PeCy7, IL-17(EBIO64CAP17)-PE and IFNγ(4S.B3)-PeCy7 (eBioscience) was performed after fixation and permeabilization, using Fix and Perm reagent (eBioscience). Before intracellular cytokine measurement, the cells were stimulated for 4 h with PMA (12.5 ng/mL), ionomycin (500 ng/mL) and Brefeldin A (5 µg/mL; Sigma–Aldrich, Zwijndrecht, The Netherlands).
To study cell division by flow cytometry, 8 × 106 cells were labeled with 0.5 µM CFDA-SE (Molecular Probes, Leiden, The Netherlands) prior to stimulation. The cell phenotype was analyzed by five-color flow cytometry (FC500) or 10-color flow cytometry (Navios™), and data were analyzed using CXP or Kaluza® software respectively (all from Beckman-Coulter).
Immunoglobulin isotyping assay
CD19+ LN B cells (5 × 104 cells) were cultured in the presence of anti-CD40 monoclonal antibody (αCD40 mAb, 1 µg/mL, Bioceros, Utrecht, The Netherlands) and recombinant human IL-21 (100 ng/mL, ZymoGenetics, Seattle, WA) for 8 days in culture medium supplemented with 10% fetal bovine serum (FBS). Supernatants were stored at −20°C until analysis. Human IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE were determined using a human Bio-Plex Pro™ immunoglobulin isotyping assay (BioRad, Veenendaal, The Netherlands) according to the manufacturer's instructions.
Real-time quantitative PCR of activation-induced cytidine deaminase (AID)
CD19+ SPL B cells (5 × 104 cells) were cultured in the presence or absence of 5 µg/mL RTX with or without addition of 1 µg/mL αCD40 mAb and 50 ng/mL IL-21. After 4 days of culture, total RNA was extracted, cDNA was synthesized and transcripts were quantified as described previously . Probes with the following identification number were used: AID, Hs00757808_m1 (Applied Biosystems, Foster City, CA). Results were normalized using the human HPRT1 Endogenous Control (4333768T; Applied Biosystems) and expressed as the relative fold change compared to the control condition.
In vitro T cell proliferation assay
CD19+ LN B cells were added to 5 × 104 CFSE-labeled allogeneic CD3+ T cells in a 1:1 ratio. Intracellular cytokine production by proliferating (CFSElow) CD3+CD4+ and CD3+CD8+ T cells was analyzed by flow cytometry at Day 7.
Statistical analysis was performed using GraphPad Prism 5.03. Paired t-tests were used to compare results obtained with SPL cells cultured in the presence or absence of RTX. To test differences between both patient groups, a Mann–Whitney's U-test or an unpaired t-test was performed. p-Values <0.05 were considered statistically significant and are indicated with asterisks.
A single dose of RTX induces a nearly complete B cell depletion in PB, but not in LNs of renal transplant recipients
RTX treatment resulted in a nearly complete depletion of B cells from the peripheral lymphocyte population (CD19+; 0.12 ± 0.05% vs. 4.9 ± 1.1%; p = 0.002) while T cell and NK cell percentages were not affected by RTX (Figure 1A). Importantly, the percentage of CD19+ B cells in LNs of RTX-treated patients did not differ from that of the untreated patients (35.1 ± 8.5% vs. 40.3 ± 11.0%; p = 0.61; Figure 1A). Likewise, RTX treatment had no effect on the percentages of other lymphocyte populations in the LNs.
To exclude that LN B cells were not depleted by RTX treatment because they lack CD20 expression we first demonstrated that CD19+ LN B cells of untreated patients express CD20 (Figure 1B, C). Remarkably, LN B cells of RTX-treated patients were CD19+ but CD20− suggesting that RTX influenced detection of CD20 on LN B cells. However, we were unable to detect RTX on the surface of the LN B cells of RTX-treated patients using an anti-idiotype antibody  (Supplementary Figure S1).
B cells remaining in the LNs after RTX treatment are predominantly of an IgD−CD27+ switched memory phenotype associated with an altered Ig-isotype production
Since RTX treatment failed to deplete CD19+ B cells in the LNs, we examined the remaining CD19+ LN B cells in more detail. RTX treatment resulted in significant reduction of the percentages of naïve B cells (IgD+CD27−, 1.9 ± 1.0% vs. 31.0 ± 14.7%; p = 0.01), transitional B cells (CD24++CD38++, 0.1 ± 0.05% vs. 2.3 ± 1.4%; p = 0.01) and CD24+CD38+ (mature) B cells (8.4 ± 4.4% vs. 28.6 ± 16.5%; p = 0.02), and in an increase in the percentages of switched memory B cells (IgD−CD27+, 86.1 ± 8.2% vs. 53.0 ± 17.0%; p = 0.02) percentages and CD24++CD38− (memory) B cells (85.5 ± 6.2% vs. 67.5 ± 17.2%; p = 0.07). Accordingly, the percentage of B cells positive for the naïve B cell markers CD5 and IgM was also reduced after RTX treatment (1.3 ± 0.5% vs. 9.1 ± 4.5%; p = 0.01 and 13.5 ± 5.9% vs. 70.3 ± 12.4%; p = 0.01 respectively; Figure 2A, B). Plasma cells (CD19low CD20− CD38high) were virtually lacking in the LNs, irrespective of treatment with RTX (data not shown). Similar results were obtained after 3 days exposure of human SPL B cells to RTX in vitro (Supplementary Figure S2).
To define whether the RTX-induced shift of LN B cells from a naïve to a switched memory phenotype influenced Ig-isotype production, LN B cells were stimulated ex vivo with αCD40 mAb and IL-21 to mimic the in vivo help of follicular helper CD4+ T cells . After 8 days Ig-isotypes were measured in the culture supernatant. LN B cells from RTX-treated patients produced lower amounts of IgM (0.6 ± 0.2 µg/mL vs. 14.1 ± 7.8 µg/mL; p = 0.01) and IgG2 (0.9 ± 0.3 µg/mL vs. 2.3 ± 1.5 µg/mL; p = 0.02), with a trend toward a higher IgG1 production compared to LN B cells from untreated patients (77.8 ± 33.0 µg/mL vs. 44.3 ± 15.7 µg/mL; p = 0.17; Figure 3A). The production of IgA, IgE, IgG3 and IgG4 was not affected by RTX treatment.
In vitro exposure to RTX enhances the mRNA expression of activation-induced cytidine deaminase in stimulated human splenic B cells
To determine whether the RTX-induced population shift from a naïve to a switched memory phenotype was accompanied by class-switch recombination (CSR), we studied expression of AID which plays a key role in CSR . CD19+ SPL B cells were cultured in the presence or absence of RTX with or without addition of αCD40 and IL-21. After 4 days of culture AID mRNA expression was determined by quantitative PCR (Figure 3B). AID mRNA expression levels tended to be higher when SPL B cells were stimulated with αCD40/IL-21 and exposed to RTX compared to the control condition (p = 0.07).
T cell stimulation with LN B cells from RTX-Treated patients resulted in a weaker Th17 response
Based on our previous finding that in vitro exposure of human PB B cells to RTX altered the B cell–T cell interaction , we analyzed the intracellular cytokine production by T cells after stimulation with LN B cells. LN B cells from RTX-treated or untreated patients were added to allogeneic CFSE-labeled CD3+ T cells. After 7 days of culture, we measured the intracellular cytokine production of IL-2, IL-4, IL-17 and IFNγ by proliferating (CFSElow) T cells. Interestingly, the percentage of CFSElow CD4+ T cells that produced IL-17 was lower upon stimulation with LN B cells from RTX-treated patients as compared to LN B cells from untreated patients (2.6 ± 0.6% vs. 4.6 ± 1.4%; p = 0.003; Figure 4).
In this study, we showed that at 4 weeks after administration of a single dose of RTX there is a nearly complete B cell depletion in PB, but not in LNs of renal transplant recipients. Exposure of human B cells to RTX resulted in a lower percentage of naïve (IgD+CD27−) and a higher percentage of switched memory (IgD−CD27+) B cells. Concomitantly, there was a change in the production of Ig-subclasses after ex vivo stimulation with αCD40 mAb and IL-21. Finally, CD4+ T cells showed lower IL-17 production upon stimulation with LN B cells from RTX-treated patients as compared to LN B cells from untreated patients in an in vitro stimulation assay.
The observed persistence of B cells in lymphoid tissues after RTX treatment, has been reported by others before [8, 13, 15, 22-26]. However, in contrast to our finding of unaffected numbers of LN B cells, these studies described at least some degree of B cell reduction in synovial tissue, spleen or LNs. Renal transplant recipients treated with RTX for antibody-mediated rejection, showed a complete depletion of circulating B cells with a 50% reduction of B cells in tertiary lymphoid organs . RTX therapy of patients with autoimmune thrombocytopenia resulted in complete B cell depletion in PB and in a reduction in SPL B cells . In patients with rheumatoid arthritis, the number of synovial and bone marrow B cells were decreased after RTX treatment, but there was no complete depletion [8, 22, 23]. Genberg et al.  reported an average of 50% reduction of the percentage of CD19+ B cells in LNs after treatment with RTX as induction therapy in renal transplant patients. The variation in B cell depletion might be explained by differences in RTX treatment regimen, choice of immunosuppressive agents and heterogeneity of the patient populations. The majority of the patients were treated with at least two doses of 375 mg/m2, or 1000 mg for RA patients, with different time intervals before obtaining secondary lymphoid tissue [8, 22-24], while our patients only received a single dose of 375 mg/m2 4 weeks before collecting the LNs. Notably, there is a trend to use less intensive dosing regimens of RTX for auto-immune diseases, and like in our study, a single dose of RTX is currently applied in several conditions [27, 28].
Why there is such a wide discrepancy between depletion of B cells in PB and LN after RTX treatment is an intriguing question. A possible explanation could be an inability of RTX to reach the B cells in the LNs. However, we clearly demonstrate that LN B cells that remain after RTX treatment were CD19+CD20−, probably due to modulation of CD20 by the binding of RTX [29-32], which indicates that the LN B cells had been exposed to RTX. Exhaustion of the complement system is another possible explanation, which could not be further addressed in the current study . Finally, a high concentration of the B cell activation factor (BAFF) in LNs, might favor the survival of B cells .
Although treatment with RTX had minimal effect on the proportion of LN B cells, it induced a striking population shift from a naïve to a switched memory phenotype, which can be the result of two nonmutually exclusive processes. First, RTX might selectively deplete and/or inhibit naïve but not memory B cells leading to a relative increase of memory B cells. Second, binding of RTX to naïve B cells might induce differentiation into memory B cells. Using an in vitro nondepleting B cell stimulation model, we have previously shown that RTX inhibited the proliferation of CD27− naïve, but not of CD27+ memory B cells . In agreement with these findings, it has been reported that RTX administered in vivo as part of a desensitization protocol in kidney transplantation decreased the number of splenic naïve B cells, but had no effect on the number of CD27+ memory B cells . In general, as compared to naïve cells, memory B cells are characterized by a high expression of activation and prosurvival molecules, which allows them to respond quickly during an immune response and to persist for long time . These properties may also result in a relative resistance of memory B cells to depletion by RTX. In summary, these observations suggest that selective inhibition of naïve, but not of memory B cells can indeed contribute to the observed shift from a naïve to a switched memory B cell phenotype. Next to its effect on the phenotype of B cells, RTX also affected the Ig-isotype production with a decrease in IgM production. Combined with a trend towards increased AID mRNA expression, this suggests that the population shift might be accompanied by CSR. Taken together, the LN B cell population shift from a naïve to a switched memory phenotype after treatment with RTX might be due to both a selective depletion of naïve B cells and a direct effect on signaling cascades resulting in CSR and transition from a naïve to a switched memory phenotype.
Treatment with RTX can be effective in chronic inflammatory diseases, such as rheumatoid arthritis [8, 35] and multiple sclerosis . The improvement of these conditions by RTX has been associated with a reduced Th17 response . In the current study, we found that LN B cells obtained from RTX treated patients resulted in a weaker Th17 response when used as stimulators in an allogeneic mixed lymphocyte reaction. Likewise, it has been observed that RTX reduces the IL-17 production by PBMCs after stimulation with Candida albicans in vitro . Moreover, the Th17-cell frequency has been shown to correlate with the frequency of both switched memory B cells and serum BAFF levels . These findings suggest a close relationship between Th17-cell homeostasis and B cell maturation which can be affected by RTX. In a previous study, we observed that B cells that were treated with RTX in vitro, were able to induce a Th2-like shift in proliferating T cells. However, it should be noted that in the present study we investigated LN-derived B cells, whereas in the previous study PB B cells were used. The B cell subset distribution between the two is clearly different, which might explain a different T cell response regardless of any further treatment (e.g. RTX), as was observed by comparing CD27− and CD27+ B cells to stimulate allogeneic CD4+ T cells .
We acknowledge that the changes in the B cell repertoire that we observed at 4 weeks after RTX treatment, could theoretically also have been caused by steroids that were given at the time of RTX administration, or by the treatment with immunosuppressive drugs (tacrolimus, mycophenolate mofetil and prednisone from 2 weeks before transplantation), or IVIG (1 day before transplantation). However, none of these drugs directly targets B cells. It has been shown that IVIG does not affect B cell responses in vitro , and treatment with IVIG as part of desensitization protocols had no effect on B cells in the spleen . In SLE patients treated with mycophenolate mofetil, the number and phenotype of B cells were similar to that in controls without immunosuppressive therapy . Tacrolimus had minimal effect on B cell proliferation and survival after stimulation in vitro . Finally, treatment of healthy volunteers with a single dose of prednisolone (30 mg) resulted in a decrease of the absolute counts of total lymphocytes, B cells, T cells and NK cells, but the counts returned to baseline levels within 13–26 h after administration . Moreover, the only differences between RTX-treated and untreated patients were those found in the B cell compartment. The fact that LN B cells from treated patients were negative for CD20, strongly indicates that RTX was involved in the observed changes in B cell phenotype and function.
In summary, we have demonstrated that a single dose of RTX depletes B cells in PB, but not in LNs at 4 weeks after administration. Exposure of human B cells to RTX results in a relative increase of B cells with a switched memory phenotype. Consequently, the effect of rituximab on the immune response will not only be determined by the extent of B cell depletion, but also by the functional properties of the remaining B cells.
The authors thank L. Boon (Bioceros, Utrecht) for kindly providing the αCD40 mAb. Technical support was kindly provided by F.W. Preijers and H. Tijssen (Radboud University Medical Centre). We also thank M.C. Warlé (Radboud University Medical Center), M.M. Idu and E.B. Remmerswaal (Academic Medical Centre, Amsterdam) for harvesting the LNs.
Funding source: This study was supported by research funding from the Dutch Kidney Foundation (nr C09-2301).
The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. L.H. has received research funds from Roche, the manufacturer of rituximab. Roche had no role in study design, data collection, preparation of the manuscript and decision to publish. The other authors of this manuscript have no conflicts of interest to disclose.