For many patients with end-stage renal disease, renal transplantation (RTx) is the treatment of choice. However, long-term graft survival remains unsatisfactory with a 10-year graft survival rate of 56%.1 Antibody-mediated rejection (AMR) causes upto 65% of late graft failures.2 AMR is initiated by donor-specific antibodies (DSA), which are themselves associated with poorer graft survival.3 Current standard of care for active AMR includes plasmapheresis or immunoadsorption, steroids, and intravenous immunoglobulins,4 but chronic AMR lacks effective treatment.
The urgent clinical need for better prevention and treatment of AMR has increased interest in the role of B cells in organ transplantation. Beyond their role as precursors to antibody-secreting cells, B cells have other important functions, such as the secretion of cytokines, antigen presentation to T cells, and costimulation. B-cell–activating factor (BAFF) is an important survival and activation factor for B cells during various stages of development and differentiation.5 Elevated BAFF has been associated with AMR,6 pretransplant immunization,7 DSA,7 interstitial fibrosis and tubular atrophy (IFTA),8 and poor allograft function in RTx patients.9 Belimumab, a monoclonal anti-BAFF antibody, has shown beneficial results in the treatment of systemic lupus erythematosus patients10 and has recently been explored as an add-on to induction and maintenance therapy for RTx patients in a small placebo-controlled trial.11 However, the mechanistic effects on specific B-cell subsets in secondary lymphoid organs and allografts and the resulting impact on humoral responses and DSA formation have not been fully established in the context of organ transplantation. We therefore tested a monoclonal anti-BAFF antibody in a rat RTx model and investigated effects on B-cell subsets, functional molecules, germinal center (GC) dynamics, and antibody formation.
MATERIALS AND METHODS
Animals and Experimental Treatments
Animal experiments were approved by local authorities and performed according to animal protection laws. RTx was performed as previously described.12 Male Brown Norway rats served as donors and male Lewis (LEW) rats as recipients (Charles River Laboratories, Sulzfeld, Germany), as depicted in Figure S2 (SDC, http://links.lww.com/TP/B822). Left Brown Norway kidneys were transplanted orthotopically into LEW rats. Right kidneys were removed. To permit chronic rejection (CR) and DSA development, all rats received reduced dose cyclosporine A (5 mg/kg/day on d0–d7 post-RTx, d7 onward on alternating days) as previously described.13 In addition, some groups received a monoclonal anti-BAFF antibody (CR + AB) (hamster anti-mouse BLYS antibody 10F4, GSK, Hamburg) or an IgG isotype (CR + iso) (hamster IgG isotype; Leinco Technologies, Inc., MO) at 10 mg/kg intraperitoneally on days 3, 17, 31, and 45 post-RTx. Rats were euthanized on d28 or d56 post-RTx. The experimental groups were as follows: RTx CR d28 “CR d28” (n = 6); RTx CR + anti-BAFF antibody d28 “CR + AB d28” (n = 6); RTx CR + IgG isotype d28, “CR + iso d28” (n=4); RTx CR d56 “CR d56” (n = 6); RTx CR + anti-BAFF antibody d56 “CR + AB d56” (n = 6); and for DSA assays, untreated LEW rat serum was used as a control (n = 6). Table S1 (SDC, http://links.lww.com/TP/B822) shows the experimental groups. Figure S2 (SDC, http://links.lww.com/TP/B822) shows rat treatments.
Histology and Immunohistochemistry
Paraffin sections were stained with hematoxylin and eosin and periodic acid-Schiff and evaluated by a nephropathologist using Banff criteria.14 Three micrometer formalin-fixed paraffin-embedded spleen sections were stained with anti-rat IgG-AlexaFluor647 (Thermo Fisher A21472, Waltham) for plasmablasts/plasma cells (PB/PC), anti-CD20 (Santa Cruz sc-393894) for B-cell follicles, anti-Ki67 (eBioscience, 11-5698) for proliferating GC cells, anti-CD3 (Abcam, 5690) for T follicular helper (TfH), and anti-foxp3 (Novus Bio, NB100-39002) for T follicular regulatory cells (Tfr), which were counted within Ki67+ GC regions. Staining was performed according to a standard immunohistochemistry protocol.15 Cells were quantified using Histoquest software.
Spleen and kidneys were chopped and passed through 70 and 30 μm filters. After Ficoll gradient centrifugation, cells were blocked using 10% BSA in PBS and stained. Absolute numbers were determined using Accu-Check counting beads (LifeTechnology PCB100, Darmstadt, Germany) and normalized to spleen or allograft weight or volume of blood. Fc-Block (Innovex Biosciences NB30930, Richmond) was used before staining. Labels used were mouse anti-rat CD45R(B220)-PE-Cy7 (eBioscience/Thermo Fisher HIS24 25-0460, Waltham), mouse anti-rat CD11b/c-PE (eBioscience/Thermo Fisher 12011082, Waltham), mouse anti-rat CD3-APC (eBioscience/Thermo Fisher 17003082, Waltham), mouse anti-rat IgM-Fitc (BioLegend 408905, San Diego), mouse anti-rat CD38-PE (BioLegend 250505, San Diego), CD24-Fitc (Santa Cruz sc-33669, Dallas), mouse anti-rat IgD (antibodies online ABIN118428), anti-mouse-biotin (Dianova 715-065-151, Hamburg, Germany), and streptavidin-BV421 (BioLegend 405226, San Diego).
Quantitative polymerase chain reaction (qPCR) was performed as previously described.13 The sequences of primers are listed in Table S3 (SDC, http://links.lww.com/TP/B822). Copy numbers of target genes were normalized to expression of house-keeping gene hypoxanthine-guanine phosphoribosyltransferase and shown as delta cycle threshold values.
Donor splenocytes were incubated with heat-inactivated recipient serum for 30 minutes at 4°C, washed and stained with anti-rat IgG-Alexa Fluor647 antibody (eBioscience/Thermo Fisher, A21472, Waltham), anti-rat IgG1-APC (eBioscience/Thermo Fisher 17-4812), anti-rat IgG2a-PE Cy7 (eBioscience/Thermo Fisher 25-4817), anti-rat IgG2b-PE (eBioscience/Thermo Fisher 12-4815), or anti-rat IgG2c-biotin (eBioscience/Thermo Fisher 17-4812), and streptavidin-BV421 (BioLegend 405226, San Diego). Finally, cells were stained and gated for CD3-FITC (eBioscience/Thermo Fisher 11-0030) to exclude nonspecific binding by fragment crystallizable receptors.
Creatinine and Proteinuria
Serum creatinine was measured enzymatically using the Trinder method (LTsys LT-CR0106 Labor + Technik, Berlin, Germany). Urine was collected in 24-hour metabolic cages. Proteinuria was measured photometrically using the Pierce method with bicinchoninic acid (Sigma B9643, Darmstadt, Germany) and copper(II) sulfate pentahydrate 4% solutions (Sigma C2284).
Data were analyzed using GraphPad Prism (Version 7.0c, San Diego) and is shown as mean ± SEM. Statistical analysis was performed by Mann-Whitney test and Kruskal-Wallis Test for creatinine and proteinuria. P ≤ 0.05 were considered statistically significant.
Anti-BAFF Treatment Significantly Reduced the Frequency of B Cells in All Compartments
Within allografts, the frequency of total B cells (CD45R+CD3−) was significantly reduced by anti-BAFF treatment at d28 (7.8±1.4% versus 3.0±0.7%, P = 0.009) and d56 (5.2±0.7% versus 2.3±0.3%, P = 0.009) (Figure 1A). There was a nonsignificant trend for reduced absolute B-cell numbers in allografts in anti–BAFF-treated rats (9.9 × 104 ± 2.1 × 104 versus 4.2 × 104 ± 0.8 × 104 B cells/g, P = 0.06 at d28; 5.1 × 104 ± 0.9 × 104 versus 3.6 × 104 ± 1.0 × 104 B cells/g, P = 0.4 at d56) (Figure 1A). In spleens, B-cell frequency was also significantly lower in anti–BAFF-treated rats at d28 (32.8 ± 2.2% versus 14.8 ± 1.5%, P = 0.002) and d56 (29.9 ± 3.8% versus 12.0 ± 1.1%, P = 0.015) as were absolute numbers of B cells (2.8 × 107 ± 0.7 × 107 versus 1.0 × 107 ± 0.2 × 107 B cells/g, P = 0.09 at d28; 2.1 × 107 ± 0.5 × 107 versus 0.6 × 107 ± 0.1 × 107 B cells/g, P = 0.01 at d56) (Figure 1A). In peripheral blood, frequency of B cells was lower in anti–BAFF-treated rats at d28 (19.8 ± 1.5% versus 7.3 ± 1.2%, P = 0.0022) and d56 (18.6 ± 2.8% versus 6.6 ± 0.6%, P = 0.041), and absolute B-cell numbers were decreased at d56 (3.6 × 105 ± 0.9 × 105 versus 1.2 × 105 ± 0.4 × 105 B cells/mL, P = 0.026).
Anti-BAFF treatment did not significantly change T-cell (CD3+) frequency or absolute numbers in allografts (Figure 1A). In spleen, T-cell frequency, but not absolute numbers, significantly increased after anti-BAFF at d28 (47.8 ± 3.3% versus 65.4 ± 1.1%, P = 0.002), but not at d56 (Figure 1A). The number and frequency of myeloid cells (CD11b/c+) was not significantly affected at d28 or d56 in any compartment (data not shown).
BAFF Depletion Specifically Affected Naive B Cells, Transitional B Cells, and Plasmablasts/Plasma Cells
We measured naive B cells based on their intermediate IgM and high CD45R expression (CD45R+IgMint) (Figure S4, SDC, http://links.lww.com/TP/B822)16 and transitional B cells based on their high IgM expression (CD45R+IgMhigh) (Figure S4, SDC, http://links.lww.com/TP/B822) and costaining for CD24 and CD38 (Figure S5, SDC, http://links.lww.com/TP/B822). Naive B cells were significantly reduced after anti-BAFF treatment in renal allografts at d28 (P = 0.026) and d56 (P = 0.004) and spleens at d28 (P = 0.015) and d56 (P = 0.015), compared with CR without anti-BAFF (Figure 1B). In peripheral blood, naive B cells were significantly reduced by anti-BAFF treatment at d56 (P = 0.015). We found a transient reduction of transitional B cells in allografts after anti-BAFF treatment compared with CR at d28 (P = 0.026), but not at d56 (Figure 1B). No differences were found in the spleen or peripheral blood. PB/PC were also significantly reduced after anti-BAFF treatment at d28 (86.4 ± 12.3 cells/mm2 versus 36.0 ± 7.9 cells/mm2, P = 0.009) and d56 (146.2 ± 23.3 cells/mm2 versus 64.1 ± 11.5 cells/mm2, P = 0.026) (Figure 1C).
Anti-BAFF Treatment Altered the Expression of Costimulatory Molecules and Interleukin-6
Splenic mRNA expression of costimulatory molecules CD40 and inducible T cell costimulator (ICOS) ligand was significantly reduced after anti-BAFF treatment (CD40: 0.29 ± 0.03 versus 0.15 ± 0.02, P = 0.002 at d28; ICOS ligand: 0.14 ± 0.01 versus 0.09 ± 0.003, P = 0.002 at d28 and 0.13 ± 0.01 versus 0.08 ± 0.009, P = 0.028 at d56) (Figure 2A). Furthermore, we found a substantial reduction of interleukin (IL)-6 mRNA expression in CR + AB at d28 (0.034 ± 0.004 versus 0.020 ± 0.002, P = 0.026) and d56 (0.033 ± 0.010 versus 0.014 ± 0.007, P = 0.009) (Figure 2B).
Anti-BAFF Treatment Limited GC Expansion and DSA Formation
Although the frequency of B-cell follicles and GCs was unaffected by anti-BAFF treatment (data not shown), B-cell follicles were significantly smaller in anti–BAFF-treated rats (d28: 0.044 ± 0.005 versus 0.026 ± 0.002 mm2, P = 0.009 and d56: 0.039 ± 0.005 versus 0.025 ± 0.003 mm2, P = 0.056) (Figure 2C). More importantly, GC expansion was diminished after anti-BAFF treatment (0.018 ± 0.001 versus 0.012 ± 0.001 mm2, P = 0.009 at d28) (Figure 2C). Fittingly, mRNA expression of activation-induced cytidine deaminase (AID), an essential enzyme for GC B cells, was also significantly lower in anti–BAFF-treated rats (0.007 ± 0.002 versus 0.003 ± 0.0004, P = 0.009 at d28) (Figure 2C).
Follicular B and T cells are known to engage in substantial crosstalk.17 We therefore investigated the effects of anti-BAFF treatment on TfH, which induce antibody responses,18 and on Tfr, which suppress antibody responses.19 Although anti-BAFF treatment did not significantly change TfH, their numbers appeared to correlate with GC expansion (Figure 2D). The percentage of Tfr (assessed as Tfr/TfH × 100) was unaffected by anti-BAFF treatment but increased over time in our CR model (1.6 ± 1.3% versus 4.4 ± 1.4%, P = 0.04) (Figure 2D).
We then evaluated the effect on IgG synthesis and DSA development. Splenic IgG mRNA expression was significantly lower in CR + AB at d28 (7.7 ± 3.9 versus 2.6±1.7, P = 0.04) (Figure 2E). We used a flow crossmatch technique, reviewed in Young et al,20 to measure DSA over time and found that, using mean fluorescence intensity (MFI) as a measure, there was a nonsignificant lower level of serum pan-IgG DSA (MFI = 64 ± 13 versus 37 ± 11, P = 0.10 at d56) (Figure 2E) and all IgG subclasses in anti–BAFF-treated rats trended lower, being statistically significant for only IgG subclass IgG2a (MFI = 31 ± 4.4 versus 16 ± 4.3, P = 0.03) and IgG2c (MFI = 18 ± 0.7 versus 16 ± 0.5, P = 0.04) at d56 (Figure 2F).
Effects of Anti-BAFF Treatment on Fibrosis, Allograft Function, and Rejection
The development of IFTA was related to the presence and severity of rejection. At d56, 2 of 6 CR rats showed no IFTA and 4 of 6 showed low-level IFTA (≤25%). In anti–BAFF-treated rats, 4 of 6 had no IFTA and 2 of 6 showed moderate IFTA (>25%–≤50%) at d56; notably, these 2 had concurrent AMR (Table 1). Proteinuria or creatinine did not differ significantly between the groups (Table 1).
As expected, sustained under-immunosuppression led to cellular rejection in all rats at d28, where 4 of 6 CR rats had Banff IA rejection, 2 of 6 had borderline rejection, and 5 of 6 CR + AB rats had Banff IA and 1 of 6 had Banff IIA rejection at d28 (Table 1). T-cell–mediated rejection persisted until d56 in CR, where 1 of 6 rats had Banff IA rejection and 4 of 6 rats had borderline rejection. In anti–BAFF-treated rats, there was no rejection in 4 of 6 rats at d56, but 2 of 6 rats developed mixed rejection with AMR (Table 1).
Analysis of DSA from the 2 rats which developed AMR showed elevated DSA MFIs in all IgG subclasses, but similar MFIs were also observed in rats not treated with anti-BAFF without AMR (shown in Figure S6, SDC, http://links.lww.com/TP/B822). As discussed below, we did not find an explanation for the occurrence of AMR in these rats, other than a poor response to anti-BAFF treatment in DSA MFI compared with the rest of the group (Figure S6, SDC, http://links.lww.com/TP/B822).
Because of the important functions of B cells and implications of BAFF for worse outcomes in RTx, we investigated the effects of an anti-BAFF intervention in a rat RTx model. We found that anti-BAFF treatment interfered with several mechanisms important to humoral responses, namely the survival of naive B cells, the expression of costimulatory molecules and IL-6, and the expansion of GC. This culminated in reduced antibody-secreting PB/PC and lower DSA of certain IgG subclasses. We also demonstrated that intrarenal B-cell subpopulations are sensitive to anti-BAFF treatment.
BAFF is an important B-cell survival and differentiation factor. Depletion of BAFF or receptor blockade has been shown to reduce naive B cells, transitional B cells, marginal zone B cells, and plasma cells.11,21 In line with previous reports, we observed a strong and sustained reduction of naive B cells in all measured compartments. Transitional B cells were transiently reduced in allografts, and PB/PC were significantly reduced in spleens of anti–BAFF-treated rats. These changes in BAFF-sensitive B-cell populations confirm the biological activity of anti-BAFF treatment in our model and demonstrate that an anti-BAFF strategy can affect intragraft B-cell populations.
Costimulation is important for multiple adaptive immune processes, and the costimulatory CD40-CD40L and ICOS-ICOSL axes are particularly important in humoral responses. CD40 ligation is an essential proliferation signal for B cells provided by cognate TfH,18 which depend on ICOS ligation provided by B cells.22 These interactions are vital for the development of a GC response and the formation of high-affinity antibodies.17 Blocking of the CD40-CD40L axis has been extensively studied as an immunosuppressive strategy in organ transplantation showing promising results in preclinical models23 and is being investigated in an ongoing clinical trial in RTx patients (ClinicalTrials.gov; Identifier: NCT03663335). The expression of both CD40 and ICOS ligand was significantly reduced in anti–BAFF-treated rats in our model, which provides an additional mechanism by which anti-BAFF treatment can modify immune responses in organ transplantation.
B cells and plasmablasts are potent producers of IL-6, a pleiotropic cytokine which induces TfH and supports the development of GCs and survival of PB/PC.24 On the basis of these important functions in humoral responses, IL-6 has been explored and shows promise as a novel therapeutic target for the treatment of AMR in RTx patients.25 We found that anti-BAFF treatment potently reduced IL-6 expression in secondary lymphoid organs in our model, which may have interfered with humoral responses in several ways. Follicular B cells can secrete IL-6, which induces TfH and drives GC formation. In turn, plasmablasts, arising from the GC, and being supported by IL-6 as a survival factor, may themselves secrete IL-6 thereby inducing further TfH and GC formation, promoting their own expansion and survival.26 Plasmablast production of IL-6 thus mediates a positive-feedback loop amplification in this system. The anti–BAFF-mediated reduction of IL-6 expression in our model therefore reinforces the rationale for developing an anti-BAFF treatment strategy to prevent DSA formation in solid organ transplantation.
Because peptide alloantigens are T-dependent antigens, humoral alloreactivity originates in the GC.19 Here antigen-specific B cells are activated and selected by specialized TfH to generate high-affinity antibodies. We found that GC expansion and expression of the essential GC B-cell enzyme AID was diminished by anti-BAFF treatment. Although anti-BAFF had no effect on TfH or the percentage of Tfr, TfH seemed to mirror GC expansion and Tfr increased over time in our CR model, supporting their physiological role of limiting GC responses.19 From a mechanistic point of view, anti-BAFF treatment may have limited GC expansion by diminishing the pool of naive B cells, thereby removing the substrate for GC formation, but anti–BAFF-mediated reduction of costimulatory molecules and IL-6 may have also impacted GC dynamics.
DSA are associated with reduced graft survival and can cause AMR. Although the expression of splenic IgG and PB/PC were reduced after anti-BAFF treatment, pan-IgG DSA MFI was not significantly lower. However, overall there was a trend for lower DSA MFI in anti–BAFF-treated rats, which was significant in IgG subclasses IgG2a and IgG2c. In Belimumab-treated RTx patients, de novo non-HLA antibodies were reduced after 24 weeks of treatment,11 and in Belimumab-treated systemic lupus erythematosus patients, autoantibody titers were not lower until 8 weeks of treatment.27 In light of the observations from Belimumab-treated patients, a significant reduction of DSA by anti-BAFF treatment might have occurred in our model if given more time. However, 2 rats treated with anti-BAFF antibody developed AMR, and these had higher DSA MFI in all IgG subclasses compared with the rest of the group, opening the possibility that these animals did not fully respond to anti-BAFF treatment. Notably, however, similar DSA MFIs were observed in rats not treated with anti-BAFF (CR d56 group), but none developed AMR. The number of naive B cells, PB/PC, GCs, TfH and IL-6 mRNA expression was similar in AMR rats to other rats in the same group, but GCs were larger and AID expression was higher in the rats with AMR. ICOS and CD40 expression was higher in 1 rat with AMR. In summary, we did not identify a specific cause for AMR development in these particular 2 rats or definitively find a link to anti-BAFF treatment.
Our study shows that anti-BAFF treatment can interfere with humoral alloresponses by addressing multiple steps of B-cell activation in a model of RTx. The long-term effect of anti-BAFF treatment on humoral alloreactivity and its implications for outcomes in RTx requires further experimental and clinical investigation.
We acknowledge the contribution of Prof Kerstin Amann as part of the collaboration of our SFB 1350 projects C2 and B6. We thank Stefanie Ellmann and Alexandra Mueller for their technical support.
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