To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.
basic leucine zipper transcription factor, ATF-like
Cytotoxic T-Lymphocyte Antigen 4
enzyme-linked immunosorbent assay
fluorescence activated cell sorting
Immune Tolerance Network
mean survival time
paired box protein Pax-5
suppressor of cytokine signaling 1
Signal Transducer and Activator of Transcription 1
tumor necrosis factor alpha.
With the recent development of new therapeutic strategies tolerance induction in transplant patients might be achievable . Due to new improved and humanized antibodies even targeting of co-receptors such as CD4 in a nondeletional manner has experienced a renaissance (www.biotest.de). The translation of those therapeutic approaches into the clinic has been proven rather difficult [2, 3]. So far only induction of mixed chimerism by infusion of donor hematopoetic stem cells combined with recipient conditioning has resulted in immunosuppression-free graft survival in transplant patients [4-8]. Many factors interfere with tolerance induction in the clinic. Patients have a higher pool of memory particularly cross-reactive memory T cells [9, 10]. Tolerance induction protocols are therefore accompanied at least initially with IS treatment for safety reasons. CNI seem to be most effective in preventing reactivation of preformed memory T cells [3, 11]. Indeed, although clinical studies are focusing on CNI-sparing protocols, mainly because of their nephrotoxicity, some patients have to be (re)-converted to CNI because of rejection . Consequently, CNI remain the standard immunosuppression at least for high-responders [13-15].
Although CNI are clinically very effective, conflicting findings have been reported as to whether their concomitant use with tolerance induction protocols has beneficial or adverse effects on promoting long-term graft acceptance . Effect of concomitant application of CsA on allograft survival has been investigated in tolerance induction protocols based on bone marrow-mediated induction of chimerism [17-19], costimulatory blockade [16, 20, 21] but also nondeletional targeting of co-receptors such as CD4 and CD8 [22, 23]. When reviewing these studies it appears that regardless of the protocol used concomitant CsA treatment acts synergistic or has no effect when the tolerance protocol used on its own does not or to a rather low extent induce permanent acceptance [19, 22, 24, 25]. In contrast, when using other tolerance induction protocols with a high percentage of permanent graft acceptances, an additional CsA treatment can but does not always lead to tolerance abrogation [17, 20, 23, 26, 27]. In addition, the tolerance-abrogating effect of CsA is dependent on dose, time of application and the transplantation model studied [16, 28, 29]. However, especially from the recent results of clinical trials utilizing the mixed chimerism approach to achieve immunosuppression-free graft survival, we have learned that CNI do not appear to completely antagonize tolerance induction in all settings [4-8]. Furthermore, many of the operational tolerant liver and kidney transplant patients had received CNI previously [30-35].
A simultaneous CsA application can prevent the development of allo-specific regulatory T cells (Tregs) [28, 36, 37]. CsA can also inhibit activation induced apoptosis of allo-reactive effector T cells, a mechanism that plays an important role for peripheral tolerance induction .
Whether CNIs also influence other processes leading to impaired long-term graft function has not been studied. To ensure safe and successful application of CNI regarding their control of memory T cell re-activation in combination with tolerance induction protocols the following questions have to be addressed: Which mechanisms are involved in CNI-mediated tolerance abrogation? Which intervening treatments can be applied to antagonize CNI-mediated interference with tolerance induction?
In accordance to some previous published observations in experimental models we observed an abrogation of anti-CD4mAb-mediated tolerance induction by simultaneous transient CsA treatment. Simultaneous CsA application resulted in intragraft up-regulation of genes associated with B cell migration and activation such as CXCL13, CCL19 and BATF leading to B cell activation, alloantibody production and complement–mediated destruction of glomeruli. Administration of a depleting polyclonal anti-rat B cell IgG serum or splenocytes from tolerant recipients 3 weeks after transplantation could arrest B cell accumulation activation in vivo and thereby diminish complement mediated destruction.
Materials and Methods
Male inbred Lewis (LEW, RT1l) and Dark Agouti (DA, RT1av1) rats, aged 8–12 weeks, were purchased from Harlan-Winkelmann GmbH (Borchen, Germany). The mount and feeding of the animals was carried out in strict accordance to the guidelines of the FELASA (Federation of European Laboratory Animal Science Associations).
Kidney transplantation and immunosuppressive treatment
Orthotopic kidney transplantation was performed in bilaterally nephrectomized recipients using standard method . For the induction of permanent graft acceptance rats were treated with the nondepleting anti-CD4mAb (RIB5/2) at a dose of 10mg/kg b.w./day intraperitoneally on Days −1 until +3 as previously described [39-42]. Some recipients additionally received 10 mg/kg b.w./day CsA subcutaneously on Days 0–9. CsA (Sandimmun®) was purchased from Novartis Pharma GmbH, Nürnberg, Germany.
Histology and immunohistology
Kidney grafts were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 1–2 µm sections and deparaffinized. Those sections were used for hematoxylin and eosin (H&E), PAS (periodic-acid-schiff-reaction), trichrome fibrin, methenamin silver staining and mounted in gelatine (Merck, Darmstadt, Germany) or for staining of C3D (DAKO Glostrup, Denmark), PAX5 (eBioscience, San Diego, CA) and anti-rat IgG (AbD Serotec, Raleigh, NC). Cryo-preserved tissue was cut into 4 µm sections, air-dried overnight and in case of TCR-α/β (clone R73, kindly provided by T. Hünig, Würzburg) and CXCL13 (antibodies online, Aachen, Germany) staining, fixed in ice-cold acetone. For detailed information see Supplementary Material and Methods.
DNA-microarray and quantitative RT-PCR
The relative gene expression of immune related genes was examined with a customized PIQOR™ cDNA microarray (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Gene expression analysis of replicates from allografts from anti-CD4mAb or anti-CD4mAb + CsA-treated recipients on Days 3, 25 and 150 after transplantation were studied. Allografts from untreated recipients recovered on Day 3 after transplantation served as a reference. Total RNA was isolated using the Strataprep-Total-RNA-Miniprep-Kit™ (Stratagene, Heidelberg, Germany). One microgram of each total RNA was used for linear amplification. aRNA samples from allografts of anti-CD4mAb or anti-CD4mAb + CsA-treated recipients were labeled with Cy5, the reference aRNA sample was labeled with Cy3. Hybridization, scanning and data analysis were performed as described in detail elsewhere . Only genes displaying a net signal intensity twofold higher than the mean background were used for further analysis (see also Supplementary Material and Methods for detailed information). All microarray experiments were performed according to the MIAME guidelines. Microarray data have been submitted to Geo (www.ncbi.nlm.nih.gov/geo; GSE40454).
For qPCR analysis total RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). mRNA expression was analyzed using the 7500 Real-Time PCR System (Applied Biosystems, Darmstadt, Germany). QPCR primer/probes for β-actin were purchased from Eurogentec (Seraing, Belgium). For primer/probe sequences refer to Sawitzki et al. . The rat qPCR panels for CXCL13, IgG, CD79b and PNOC were purchased from Applied Biosystems. The cycle number at which the reporter fluorescence reaches the threshold (Ct value) was used for quantitative measurement. Values are given as . The gene of interest expression level is given in relation to the housekeeping gene expression (β-actin) .
Flow cytometry and ELISA
1 × 106 DA thymocytes were incubated with indicated dilutions of recipient's sera for 45 min at 4°C. Samples were incubated with the respective FITC-labeled antibody (goat-anti-rat-IgG-Fab2-FITC, Serotec Ltd., Oxford, UK) for 30 min at 4°C. As a positive control serum from a LEW rat immunized with DA splenocytes was used and the obtained signal set to 100%. After washing, cells were fixed with 2% paraformaldehyde and analyzed on a FACSCalibur (Becton Dickinson, Germany). Peripheral B and T cell frequencies of B cell-depleted and control recipients were determined by staining peripheral blood leukocytes after water lysis for 20 min at 4°C with anti-CD45RA-FITC and anti-CD4-APC antibodies (Serotec Ltd., Oxford, UK). The amount of in vitro secreted rat IgM and IgG was quantified using the ELISA Starter Accessory Kit (E101) (Bethyl, Montgomery, TX).
Generation, purification and functional characterization of a polyclonal anti-rat B cell serum
The polyclonal anti-rat B cell serum was generated by immunizing two rabbits with 2 × 108 and 5 × 107 purified Lewis B cells (CD43 microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) in a 14 day interval (for detailed information see Supplementary material and methods). For in vivo B cell depletion anti-CD4mAb + CsA-treated recipients received 4.75 mg purified anti-rat B cell serum on Days 25–27 after transplantation.
Ex vivo restimulation of B cells
Bone marrow, spleens, mesenteric lymph nodes and kidney grafts from anti-CD4mAb + CsA-treated recipients were recovered on Day 150 after transplantation. Leukocytes of bone marrow and spleens were enriched by hypotonic water lysis. Graft infiltrating leukocytes (GICs) were isolated from kidney grafts by pressing each diced organ through a sieve followed by enzymatic digestion with 0.02% collagenase IV and 0.002% DNase I (Sigma, Hamburg, Germany) in PBS for 30 min at 37°C. Lymphoid cells were separated from the tissue suspension using a Ficoll gradient (Amersham, Uppsala, Sweden). Isolated leukocytes were restimulated with Staphylococcus aureus Cowan I (SAC) inactivated particles (1:25 000, Calbiochem, Germany) for 24 h.
Biopsies of 23 kidney transplant recipients were analyzed by qPCR. All patients gave informed written consent before participating in the study, which was approved by the local institutional review board. CNI free patients were treated with Rapamycin, mycophenolate mofetil and steroids. Biopsies were indicated by cause or by protocol. CNI R (rejection) patients were treated with cyclosporine A or tacrolimus along with mycophenolate mofetil and steroids, biopsies were indicated by cause or by protocol. All biopsies revealed positive C4d staining, additionally performed cross-match proved the presence of donor-specific antibodies. Thus the diagnosis of chronic antibody-rejection was proven. In two patients acute T cell–mediated rejection was present along with AMR. CNI normal patients were treated with cyclosporine A or tacrolimus along with mycophenolate mofetil and steroids, biopsies were indicated by protocol and revealed normal histology without C4d. Patient characteristics are shown in Supplementary Table 1.
Values are reported as mean ± standard deviation (SD). Group comparisons were performed using the parameter-free Mann–Whitney U-test and Wilcoxon's-test using SPSS software. Survival of rats was analyzed by the method of Kaplan–Meier. Differences were considered significant at p ≤ 0.05.
Co-administration of CsA to anti-CD4mAb therapy abrogates tolerance induction
DA (RTlavl) kidneys were transplanted to Lewis (RTll) recipients as previously described [39, 44]. Untreated recipients acutely rejected their kidney grafts (MST = 6.2 ± 0.4). For induction of permanent graft acceptance rats were treated with the nondeletional modulating anti-CD4mAb (RIB5/2). The treatment results in the induction of donor-specific tolerance, which can be transferred into naïve syngeneic recipients by adoptive transfer of splenocytes or graft infiltrating cells [39, 40, 42, 45-47]. Additional application of 10 × 10 mg/kg b.w./day. CsA from Day 0–9 resulted in chronic rejection of the allografts (Figure 1A; 104.1 ± 56). CsA-therapy given alone only prolonged graft survival (MST = 73.8 ± 49.5).
Early after transplantation (Day 3 and 5 post-Tx) a significantly decreased intragraft infiltration of α/β-TCR+-T cells was observed in recipients receiving anti-CD4mAb + CsA (Figure 1B). After withdrawal of CsA a significantly higher infiltration of α/β-TCR+- T cells was detected suggesting a rebound effect. The additional application of CsA also resulted in a significantly higher percentage of glomerulosclerosis and proteinuria (Figure 1C and D). Histological examinations revealed characteristic findings of acute to chronic rejection with mononuclear interstitial infiltrates, mesangial glomerulosclerosis, fibrosis, tubular atrophy and vascular changes in grafts from recipients receiving anti-CD4mAb + CsA (Figure 2A–E and Supplementary Figure 1). Those alterations were quantified applying the Banff nomenclature as shown in Figure 2F.
High intragraft expression of the B cell chemokine CXCL13 in recipients receiving additionally CsA
Microarray analysis was performed to elucidate the molecular mechanisms of CsA-mediated tolerance abrogation. The most highly and significantly up-regulated gene in grafts from anti-CD4mAb + CsA-treated recipients was CXCL13 (Supplementary Figures 2–4). The increased expression was already detectable on Day 3 after transplantation, a time point at which no T cell infiltration was detectable. Expression of genes characteristic for inflammatory processes, for example, STAT1, SOCS1 and T cell infiltration (CD3D), was inhibited or not induced during CsA treatment; however it was enhanced after CsA withdrawal. On Day 25 after transplantation other genes regulating B cell migration and activation such as CCL19, LTB and BATF showed higher expression in grafts from anti-CD4mAb + CsA-treated recipients. Figure 3A illustrates the hybridization signal intensity of the above-mentioned mRNAs in comparison to RNA recovered from untreated allografts on Day 3 after transplantation.
The selectively high expression of CXCL13 upon additional CsA therapy was verified by qPCR (Figure 3B). Immune histology of grafts from anti-CD4mAb + CsA-treated recipients collected on Day 60 posttransplant revealed a strong CXCL13 protein expression within glomeruli (Figure 3C).
Co-administration of CsA leads to intragraft accumulation of IgG+ cells and alloantibody production
Additional application of CsA resulted in significantly higher levels of circulating alloantibodies (Figure 4A). In order to determine whether the alloantibodies were produced locally within the graft, mRNA expression of rat IgG was analyzed. A constant increase in IgG expression was detectable in samples from recipients receiving CsA (Figure 4B). We also detected an accumulation of PAX5+ B cells and IgG+ cells in specimens of anti-CD4-mAb + CsA-treated recipients (Figure 4C and D). Those cells were arranged in lymphocyte foci (Figure 4D), of which we detected increased numbers in grafts from anti-CD4mAb + CsA-treated recipients (Figure 4C).
As illustrated in Figure 4E we detected a strong C3d deposition in peritubular capillaries of grafts recovered from recipients receiving anti-CD4mAb + CsA. C3d deposits were hardly detectable following anti-CD4mAb monotherapy. We also tested whether graft infiltrating B cells are able to induce allograft rejection. 2 × 106 FACS-purified graft infiltrating B cells collected from anti-CD4mAb + CsA-treated recipients 40 days posttransplant were transferred to recipients receiving anti-CD4mAb monotherapy 7 days after transplantation. Although the transferred B cells did not induce acute kidney graft deterioration, long-term kidney graft function was impaired as highlighted by an increase in serum creatinine (anti-CD4mAb + B cells: 225.3 ± 76.9, anti-CD4mAb monotherapy: 52.6 ± 8.8).
Enhanced CXCL13 transcription in biopsies of chronically rejecting kidney graft recipients receiving CNI and the cellular source of CNI-induced CXCL13 expression
We studied CXCL13 transcription in biopsies of kidney transplant patients receiving CNI maintenance therapy with or without clinical and histological signs of chronic antibody-mediated rejection and compared the results to biopsies of patients receiving a CNI free maintenance immunosuppression with signs of impaired graft function (Supplementary Figure 5 and Supplementary Table 1). We detected the highest intragraft CXCL13 expression in biopsies of patients receiving CNI-based therapy with clinical and histological signs of chronic antibody-mediated rejection (Figure 5, p = 0.009 vs. CNI free R and p = 0.019 vs. CNI normal).
Next, we wondered in which cells CNIs can induce CXCL13 expression and how this may affect B cell activation. As the immune histology revealed a predominant CXCL13 production within glomeruli, we hypothesised, that mesangial cells as a major regulatory glomerular cell type maybe able to express CXCL13. We have isolated primary mesangial cells from kidneys of DA rats. Treatment of mesangial cells with different CNIs such as CsA, FK506 or NCI3 resulted in a dose-dependent increase in CXCL13 transcription (Supplementary Figure 6A). Addition of TNF-α did further enhance CXCL13 expression by mesangial cells. CsA treatment did also increase the chemotactic activity of supernatants recovered from mesangial cells (Supplementary Figure 6B). As shown in Supplementary Figure 5C, CsA- or CsA + TNF-α-treated mesangial cells did induce higher IgM but also IgG production by co-cultured B cells.
Differential expression of B cell-related genes in tolerance versus chronic rejection
Recently increased peripheral expression of B cell-related genes such as CD79b and PNOC (Prepronociceptin) have been observed in “operational” tolerance . To determine the relevance of these genes for the CsA-mediated tolerance abrogation, intragraft expression of CD79b and PNOC was analyzed. As shown in Figure 6A expression of PNOC was higher in tolerance developing anti-CD4mAb-treated recipients whereas recipients with characteristic findings of chronic rejection (anti-CD4mAb + CsA) showed significant higher expression levels of CD79b (Figure 6B).
IgG fraction of a polyclonal anti-rat B cell serum specifically depletes B cells
To study the relevance of intragraft B cell accumulation for the development of chronic rejection, we tested, whether early B cell depletion could rescue long-term graft function. We generated a polyclonal rabbit serum by immunizing rabbits with purified B cells from Lewis rats. The purified IgG fraction specifically competed for binding to CD45RA+ B cells but not to T cells (Figure 7A). The serum induced complement-mediated lysis of B cells from Lewis rats (Figure 7B). When we administered the purified IgG fraction in vivo into anti-CD4mAb + CsA-treated recipients we detected a significant reduction in peripheral blood B but not T cell frequencies lasting at least 15 days (Figure 7C). The early depletion of B cells could diminish intragraft CXCL13 and IgG expression in anti-CD4mAb + CsA-treated recipients collected on Day 100 after transplantation (Figure 7D). In contrast, B cell depletion had no effect on intragraft CD3 transcription.
Antibody-mediated B cell depletion prevents alloantibody production and chronic alterations
The early transient depletion of B cells 3 weeks posttransplant prevented intragraft B cell accumulation on Day 100 after transplantation (Figure 8A). Peripheral B cell frequencies returned to normal levels at that time point (data not shown). The B cell depletion also inhibited alloantibody production (Figure 8C) and affected the generation of memory B cells as ex vivo stimulation of leukocytes isolated from bone marrow, mesenteric lymph nodes, spleens and kidney grafts from recipients who received the anti-B cell serum with SAC resulted in significant less IgG secretion (Figure 8B). Most importantly, the transient B cell depletion could ameliorate the histomorphological alterations for example degree of glomerulosclerosis in grafts from anti-CD4mAb + CsA-treated recipients (Figure 8D).
Transfer of splenocytes from tolerant recipients can rescue tolerance in anti-CD4mAb + CsA-treated recipients
We recently showed that splenocytes collected from anti-CD4mAb-treated kidney allograft recipients contain regulatory cells, which can transfer tolerance onto naïve syngeneic recipients [39, 44]. 1 × 108 splenocytes from anti-CD4mAb-treated tolerant recipients were injected i.v. into anti-CD4mAb + CsA-treated recipients on Day 20 after transplantation. This time point was chosen because significant differences in alloantibody production were detectable already between anti-CD4mAb- and anti-CD4mAb + CsA-treated recipients and to ensure that there is no CsA remaining in the circulation of the recipients. Alloantibody production was dramatically abolished on Day 60 post-Tx after transfer of splenocytes (Figure 9A). PAS and EL-DO staining of histology slides from kidney grafts on Day 150 after transplantation revealed a reduced degree, although not significant, of glomerulosclerosis upon adoptive transfer (Figure 9B). Transfer of splenocytes from tolerant recipients diminished intragraft accumulation of PAX5+ B cells (Figure 9C).
Here we describe abrogation of an anti-CD4mAb-induced tolerance by an additional transient application of CsA. The additional CsA application induced local selective expression of B cell chemokine CXCL13 resulting in B cell infiltration, B cell activation and subsequently in alloantibody production and complement-mediated destruction of the glomeruli. However, transient B cell depletion or transfer of splenocytes from tolerant recipients, at a time point when the B cell activation is already detectable, can reverse this process.
As pointed out earlier our findings on CNI-mediated tolerance abrogation are in accordance with some previously published results from other experimental transplant models utilizing costimulatory blockade or co-receptor targeting [16, 20, 23]. But it has to be pointed out again that in some reports no detrimental effect of an additional CNI treatment could be reported [22, 24, 48]. Also for the induction of microchimerism, the only so far successful approach to achieve tolerance in patients, conflicting data on synergy/compatibility or antagonism of CNIs have been published in experimental models [17, 48].
The microarray analysis of graft samples from anti-CD4mAb + CsA-treated recipients revealed an up-regulation of genes associated with B cell attraction. The selective gene expression was already detectable during the CsA treatment. CXCL13 is a B cell chemokine regulating the migration of B cells into secondary lymphoid tissues [49, 50]. CXCL13 and its receptor CXCR5 are required for normal development of secondary lymphoid organs but have also been detected in inflammatory lesions with extranodal lymphoid neogenesis [51, 52]. Steinmetz et al.  described high intragraft CXCL13 expression in B cell clusters of kidney transplants undergoing rejection. CXCL13 expression and B cell recruitment have been also detected during renal interstitial injury (interstitial nephritis and chronic IgA nephropathy) [53, 54]. However, the cellular source of increased CXCL13 expression could not been determined. Generally, follicular dendritic cells are believed to be the main CXCL13 producers in lymphoid and inflamed tissue . Interestingly, there were no follicular dendritic cells detectable in grafts from anti-CD4mAb + CsA-treated recipients (data not shown). In our experiments, CXCL13 was exclusively produced within the glomeruli. In accordance with its function, increased CXCL13 production resulted in massive intragraft B cell infiltration. Furthermore, local B cell infiltration including IgG+ cells was associated with IgG alloantibody production and complement deposition. These infiltrating B cells play an active role for the pathogenesis of CNI-mediated tolerance abrogation as transfer of graft infiltrating B cells into recipients receiving anti-CD4mAb monotherapy caused kidney graft deterioration. B cells may contribute to pathogenic alterations in two ways. Alloantibodies produced by B cells cause complement activation and deposition as presented in our manuscript. B cells may also function as local antigen presenting cells and amplify the activation of infiltrating T cells after withdrawal of CNI. Indeed we observed an increase in T cell numbers and cytokine production at later time points after transplantation (Figure 1B and data not shown). However, transfer of splenocytes from tolerant recipients on Day 20 after transplantation could prevent further production of IgG alloantibodies and diminished glomerulosclerosis.
We also showed that a CNI-based immunosuppressive maintenance therapy is associated with an up to 100-fold increase in local CXCL13 transcription. Patients on a CNI-based immunosuppressive maintenance therapy with an alloantibody-mediated rejection and C4d deposition show enhanced intragraft CXCL13 expression (Supplementary Figure 5). However, whether here similar regulatory mechanisms are operating as in our experimental transplant model needs to be further investigated. Such an investigation was clearly beyond the scope of our work.
We also determined a cellular source of CNI-induced CXCL13 expression. Primary mesangial cells isolated from glomeruli responded with an increase in CXCl13 transcription when stimulated with different CNIs (Supplementary Figure 6). Interestingly, addition of TNF-α as one major local inflammatory mediator following transplantation could further increase CXCL13 expression in mesangial cells, resulting in a perfect milieu for B cell attraction/infiltration and activation (Supplementary Figure 6). When the initial CNI concentration is weaned off freshly activated T cells can deliver help to infiltrated B cells resulting in B cell activation and antibody production.
We also tested whether a transient B cell depletion at a time point, where signs of B cell activation such as alloantibody production were already detectable, could rescue tolerance induction. Indeed, depletion of B cells could reduce intragraft B cell accumulation, inhibit differentiation of alloantibody-producing cells and ameliorate intragraft alterations.
B cells have a well-established role in allograft rejection, both in hyperacute and antibody-mediated acute rejection  as well as in promoting cellular immunity [56, 57]. Novel data suggest that B cells may also play an important role in allograft survival and the development of operational transplant tolerance [35, 58]. Whereas enhanced frequencies of naïve and transitional B cells have been associated with long-term allograft survival, memory B cells have been linked with decreased allograft survival [58, 59]. Newell et al.  described that increased level of IL-10 producing regulatory B cells are associated with positive outcome in renal transplantation patients.
Furthermore, recent results from EU- and ITN-sponsored network projects have reported a B cell signature associated with operational tolerance, suggesting that B cells may contribute to the tolerant state [35, 59]. Top-ranked significant B cell-related genes included CD79b and PNOC . CD79b is a membrane protein that forms a heterodimer with CD79a and is expressed by all B cell subsets including plasma cells [61, 62]. PNOC is a secreted protein that binds the opioid receptor-like receptor (OPRL-1). Arjomand et al.  found that human peripheral blood lymphocytes and especially CD19+ B cells express PNOC. Our own preliminary results revealed enhanced PNOC expression in naïve and transitional B cells (data not shown). Anton et al.  showed that nociceptin modulates adaptive immune responses such as antibody production. Waits et al.  found that N/OFQ could up-regulate CTLA-4 on T cells, which is known to have immunosuppressive properties.
Our results revealed higher expression of intragraft PNOC in grafts from tolerance developing anti-CD4mAb-treated recipients on Day 60 posttransplant whereas CD79b was increased in grafts from recipients receiving CsA. These data may hint to a CNI-mediated disturbance of intragraft B cell subpopulations with less PNOC expressing transitional and naïve B cells to activated memory B cells and plasma cells also expressing CD79b.
Our data represent unexpected findings of CNI, which could be relevant when designing new treatment, especially drug weaning, protocols for transplant patients.
In case of an antagonizing effect of simultaneously applied CNIs we need mechanisms, which counterbalance local induction of CXCL13 and drive the B cell compartment toward a “tolerant” phenotype supporting establishment of transplantation tolerance.
We provide evidence that early depletion of B cells or transfer of splenocytes from tolerant recipients are two treatment options that can prevent CNI-mediated B cell activation and chronic graft rejection if applied early after transplantation.
We thank Simone Spieckermann for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft SFB650 to B. S. and H.-D. V. and from the Grant Agency of the Czech Republic P301/11/1568 to O. V.
The authors of this manuscript have no conflicts of interest to disclosure as described by the American Journal of Transplantation.