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Transplant Immunology 31 (2014) 207–209

Contents lists available at ScienceDirect

Transplant Immunology
journal homepage: www.elsevier.com/locate/trim

Anti-B cell therapy with rituximab as induction therapy in


renal transplantation
I. Joosten a, M.C. Baas b, E.G. Kamburova a, M.W.F. van den Hoogen b, H.J.P.M. Koenen a, L.B. Hilbrands b,*
a
Dept. of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands
b
Dept. of Nephrology, Radboud University Medical Center, Nijmegen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Available online 28 September 2014 Traditionally, antirejection therapy in organ transplantation has mainly been directed at T cells. During recent
years, the role of B cells in acute rejection has attracted more attention. In the Radboud University Medical Center
Keywords: (Nijmegen, The Netherlands) we performed a randomized, placebo controlled study to assess the efficacy and
Kidney transplantation safety of rituximab as induction therapy after renal transplantation. In parallel we investigated the effects of
Immunosuppression rituximab on the numbers and function of B and T cells. An overview of the results, which have largely been
B cells
published in peer reviewed papers, is presented below.
Rituximab
© 2014 Elsevier B.V. All rights reserved.

1. Introduction 1.1. The effect of rituximab induction therapy on acute rejection after
renal transplantation
Acute rejection is an important predictor of chronic transplant glo-
merulopathy after renal transplantation. A tubulo-interstitial infiltrate We performed a single center, randomized, double-blind, placebo-
of activated T cells with tubulitis and/or endovasculitis is the histologi- controlled study from December 2007 to June 2012 [2]. Patients (n =
cal hallmark of acute cellular rejection. Therefore, induction therapy 280) were randomized for a single intra-operative dose of rituximab
with an IL-2 receptor antagonist or depleting anti-T cell antibodies is (375 mg/m2) or placebo, added to a standard immunosuppressive reg-
commonly used to lower the incidence of acute rejection [1]. During re- imen consisting of tacrolimus, mycophenolate mofetil (MMF), and ste-
cent years the role of B cells and antibodies in acute rejection has gained roids. IL-2 receptor antagonists or anti-T-cell antibodies were not used
attention, based on the negative prognostic impact of donor-specific as induction therapy in this trial. Patients were stratified according to
anti-HLA antibodies, the presence of B cell clusters in biopsies of pa- panel-reactive antibody (PRA) value and rank number of transplanta-
tients with severe rejection, and the frequent finding of capillary depo- tion. The incidence of the primary endpoint, the incidence of biopsy
sition of C4d, indicating complement activation after antibody proven acute rejection (BPAR) within six months after transplantation,
engagement, in patients with acute rejection. Therefore, depletion of B was comparable between rituximab-treated (23/138, 16.7%) and
cells in renal transplant recipients might help to prevent allograft rejec- placebo-treated patients (30/142, 21.2%, P = 0.25). Immunologically
tion. On the other hand, B cells with regulatory functions have been de- high-risk patients (PRA N 6% or re-transplant) not receiving rituximab
scribed in tolerant transplant patients who had stable allograft function had a significantly higher incidence of rejection (13/34, 38.2%) com-
without the use of immunosuppressive drugs. Since B cells can play pared to other treatment groups (rituximab-treated immunologically
multiple roles in the immune response, the effects of anti-B cell therapy high-risk patients, and rituximab- or placebo-treated immunologically
have to be analyzed carefully. This prompted us to investigate the effec- low risk (PRA ≤ 6% or first transplant) patients) (17.9%, 16.4%, and
tiveness and safety of the anti-B cell monoclonal antibody rituximab as 15.7%, P = 0.004). When the group of immunologically high-risk pa-
induction therapy after renal transplantation. In parallel, we investigat- tients was analyzed separately, there was a clear trend toward a lower
ed the effects of rituximab on the composition of the peripheral B and T incidence of BPAR with rituximab treatment as compared to placebo
cell repertoires and assessed how rituximab affects functional aspects of (17.9% vs. 38.2% during the first six months, P = 0.06 by log-rank
these cells. test). Although most rejections were T cell mediated, rituximab-
treated patients tended to have less antibody mediated rejections, com-
pared to placebo-treated patients (4/138, 2.9% vs. 11/142, 7.7% P = 0.11
by Fisher's exact test). Patient and graft survival (at six months, and
after a median duration of follow-up of 4.0 years, range 1.9–6.4 years)
as well as graft function and proteinuria (at six months and at
⁎ Corresponding author. 24 months) were comparable between the rituximab and placebo
E-mail address: luuk.hilbrands@radboudumc.nl (L.B. Hilbrands). group. Neutropenia (b 1.5 × 109/L) occurred more frequently in

http://dx.doi.org/10.1016/j.trim.2014.09.011
0966-3274/© 2014 Elsevier B.V. All rights reserved.
208 I. Joosten et al. / Transplant Immunology 31 (2014) 207–209

rituximab-treated patients (24.3% vs. 2.2%, P b 0.001). After 24 months, using human splenic mononuclear cells (SMNC; obtained from organ
the cumulative incidence of infections and malignancies was compara- donors). The SMNC population was treated with rituximab in the pres-
ble. We concluded that a single dose of rituximab as induction therapy ence or absence of sub-optimal concentrations of (rabbit) complement
did not reduce the overall incidence of BPAR, but might be beneficial and/or CpG as stimulatory agent. First, we showed that in a CpG free en-
in immunologically high-risk patients. Currently, we are analyzing the vironment, the B cells that survived the exposure to rituximab and com-
effect of rituximab on the course of donor-specific anti-HLA antibodies plement showed a higher expression of CD38, indicating activation,
(DSA) already present at the time of transplantation as well as on the than the B cells remaining after culture without rituximab and comple-
development of de novo DSA after transplantation. ment. However, in the presence of CpG, the B cells remaining after expo-
sure to rituximab and complement revealed a significantly less naïve
1.2. The effect of rituximab on the composition of the peripheral B and T cell (IgD+), more memory (CD24+ and CD27+), less activated (CD38+)
population in renal transplant patients and less regulatory (IL-10+) phenotype than the B cells treated with
CpG only. Next, we showed that the splenocyte population that
The design of the randomized trial enabled us to study the effects of remained after partial B cell depletion with rituximab and complement
standard immunosuppression (tacrolimus, MMF and steroids), with or in a CpG free environment tended to have a stronger capacity to induce
without the addition of rituximab induction therapy on the phenotype T cell proliferation than the splenocytes that were not exposed to ritux-
and function of T and B cells over time. To avoid bias by other immuno- imab and complement. The expression of CD25, CD27 and CD62L of the
logical events as much as possible, we investigated only cytomegalovi- divided allogeneic T cells did not vary with the pre-treatment of the
rus (CMV) seronegative patients who received a kidney from a CMV stimulator splenocyte population (unpublished observations).
seronegative donor (and thus did not develop CMV infection), did not Taken together, exposure of B cells to rituximab, either obtained
experience a rejection episode, and were not treated with other immu- from peripheral blood or from the spleen, appears to increase the poten-
nosuppressive drugs during the follow-up period. 14 patients were in- tial of these cells to induce T cell proliferation.
cluded in the group treated with tacrolimus, MMF and steroids, and
12 patients in the group additionally treated with RTX [3]. 1.4. Ex vivo phenotype and function of lymph node B and T cells upon
We found that in patients treated with tacrolimus/MMF/steroids the rituximab treatment
proportion of central memory CD4+ and CD8+ T cells was higher at
3 months post-transplant compared to pre-transplant levels. In addi- To assess the effects of rituximab on B and T cells in secondary lym-
tion, the ratio between the percentage of central memory CD4+ and phoid organs, we used lymph nodes collected during renal transplant
CD4+ regulatory T cells was significantly higher up to 24 months surgery in patients who had received rituximab four weeks earlier in
post-transplant compared to pre-transplant levels. Interestingly, treat- preparation for an ABO-incompatible transplantation [5]. Rituximab
ment with tacrolimus/MMF/steroids resulted in a shift toward a more treatment resulted in a lower percentage of naïve (IgD+CD27−) and a
memory-like B-cell phenotype post-transplant. However, addition of a higher percentage of switched memory (IgD−CD27+) B cells. Remark-
single dose of rituximab, leading to long-lasting B-cell depletion, result- ably, transitional (CD24++CD38++) B cells were virtually lacking in
ed in a high percentage of transitional B cells in the small fraction of the lymph nodes of rituximab-treated patients. Moreover, lymph
repopulated B cells at 12 months. Notably, induction therapy with ri- node-derived B cells from rituximab-treated patients produced differ-
tuximab had no effect on the T-cell phenotype and function up to ent amounts of various Ig-subclasses after anti-CD40 mAb/IL-21 stimu-
24 months after transplantation. lation ex vivo as compared to B cells not exposed to rituximab. As
expected, rituximab treatment did not alter the phenotype of T cells in
1.3. The effect of rituximab on T cell activation and differentiation upon the lymph nodes.
stimulation with B cells in vitro When lymph node-derived B cells from rituximab-treated patients
were used for stimulation of allogeneic T cells, the T cells that proliferat-
It should be appreciated that although in vivo administration of a ed showed a decreased production of IL-17 as compared to T cells
single dose of rituximab results in full B cell depletion in peripheral exposed to B cells from control patients. In conclusion, after treatment
blood, there remains a residual B cell population in secondary lymphoid with rituximab there remains a B-cell population with altered function-
organs. These non-depleted B cells might be altered by exposure to ri- al capacities. Consequently, the effect of rituximab on the immune re-
tuximab, with subsequent immunomodulatory effects. To mimic this sponse will not only be determined by the extent of B-cell depletion,
in vivo situation, we cultured B cells that were isolated from buffy but also by the functional properties of the remaining B cells.
coats obtained from healthy blood donors, in the presence of rituximab,
while the absence of Fc-receptor bearing cells, such as NK cells, and 1.5. Cytokine release after treatment with rituximab in renal transplant
complement prohibited the lysis of B cells. So, we were able to examine patients
the effect of (non-depleting) rituximab treatment on B cells with regard
to their phenotype and function as antigen presenting cells. We ana- Administration of rituximab can be followed by acute infusion
lyzed proliferation, activation, and differentiation of CD19+ B cells upon reactions due to the release of cytokines, especially in patients with
rituximab treatment by means of CFSE-based multiparameter flow high B-cell counts like lymphoma patients. A rise in the level of (proin-
cytometry [4]. Rituximab inhibited the proliferation of CD27− naïve, but flammatory) cytokines could lead to activation of the immune system
not of CD27+ memory B cells. Interestingly, upon stimulation with the and therefore be of clinical relevance.
co-stimulatory anti-CD40 monoclonal antibody (mAb) and IL-21 in the Rituximab can deplete B cells via antibody-dependent cellular cyto-
presence of rituximab there was an enrichment of B cells that underwent toxicity (ADCC) by natural killer (NK) cells, complement-dependent cy-
only one or two cell divisions, and displayed an activated naïve totoxicity (CDC), and apoptosis. The observation that polymorphisms in
phenotype (CD27−IgD+CD38−/+). The potency of prestimulated B cells the FcγRIIIa gene affect the effectiveness of rituximab, indicates that
to induce T cell proliferation was increased by exposure of the B cells to ADCC plays an important role. NK cells express the Fc-receptor FcγRIIIa
rituximab. Of note, after stimulation with rituximab-treated B cells, prolif- (CD16), which has a high affinity for binding to IgG1, the isotype of ri-
erated T cells displayed a more Th2-like phenotype. Overall, these results tuximab. FcγRIIIa appears in two allelic forms that differ by the amino
demonstrate that rituximab can affect human B cell phenotype and func- acid on position 158. FcγRIIIa homozygous for valine (VV) has a higher
tion, resulting in an altered outcome of B–T cell interaction. affinity for IgG1 than FcγRIIIa with phenylalanine at that position (VF or
To mimic the in vivo situation in the secondary lymphoid organs FF). Patients with the high affinity receptor show a better clinical re-
even more closely, we set-up an in vitro partial–depleting system sponse to rituximab.
I. Joosten et al. / Transplant Immunology 31 (2014) 207–209 209

We studied whether cytokine release also occurs after rituximab or comparison with other induction agents, like basiliximab or anti-
treatment in renal transplant patients with normal B-cell numbers. In thymocyte globulin. Moreover, for better interpretation of our findings
addition, we studied the mechanism of this cytokine release in vitro, es- it is important to analyze the effect of rituximab on the presence and
pecially with respect to activation of FcR bearing innate immune cells. concentration of DSA.
Twenty renal transplant recipients (10 rituximab-treated, 10 placebo- Our studies have also indicated that rituximab has several effects be-
treated) were recruited from the previously mentioned randomized clin- yond pure B cell depletion. The repopulating B-cell compartment after
ical trial. Rituximab or placebo was infused during surgery and blood B-cell depletion contained a relatively high number of transitional B
samples were taken before, and 2, 4, and 24 h after administration, and cells including regulatory B cells which might promote tolerance. In
analyzed for IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, TGFβ, TNFα, IFNγ, and vitro exposure of B cells to rituximab appeared to increase the potential
MIP-1β. In vitro, healthy donor peripheral blood mononuclear cells, puri- of these B cells to induce T cell proliferation, whereas the resulting T-cell
fied B cells, monocytes, NK cells, or combinations thereof were incubated population showed a more Th2-like phenotype. After in vivo treatment
with rituximab, rituximab-F(ab′)2, or medium, and MIP-1β, IL-10, IFNγ, with one dose of rituximab, we found that a B cell population remains in
and TNFα levels were measured in the culture supernatant [6]. lymph nodes, which mainly consists of switched memory (IgD-CD27+)
We showed that rituximab-treated patients had higher serum levels B cells that are less able to induce a Th17 response. Finally, we found
of IL-10 (101 ± 35 pg/ml versus 41 ± 9 pg/ml; P b 0.01) and MIP-1β that rituximab infusion in patients with normal B cell numbers can
(950 ± 418 pg/ml versus 125 ± 32 pg/ml; P b 0.001) compared to lead to cytokine release, especially IL-10 and MIP-1β. The cumulative
placebo-treated patients at 2 h after start of infusion. There was no in vivo consequences of these findings have not yet been clarified.
difference in the level of other cytokines. In vitro, significant levels of This means that careful evaluation of the cellular and humoral immune
MIP-1β were only detected when rituximab was added to a co-culture responses in rituximab treated patients is indicated and will remain the
of B and NK cells. Incubation with rituximab-F(ab′)2 did not result in in- subject of further research.
creased MIP-1β levels. Levels of MIP-1β were higher in patients with a
high affinity Fc-receptor compared to those with a lower affinity
Acknowledgments
FcγRIIIa (1356 ± 184 pg/ml versus 679 ± 273 pg/ml; P b 0.01).
In conclusion, next to B-cell depletion, rituximab can modulate the
The research described in this paper was financially supported by a
immune response by inducing cytokine secretion, especially IL-10 and
grant of the Dutch Kidney Foundation (C09.2301) and by the pharma-
MIP-1β. NK cells appear to be responsible for the MIP-1β secretion in
ceutical companies Roche Nederland BV and Astellas Pharma.
a B-cell and Fc-receptor dependent manner.

2. Conclusion References
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