Buffy coats from healthy blood donors serving as healthy controls (HC) were provided by the Institute for Transfusion Medicine and were used for the experiments. In addition, 26 patients were enrolled after renal transplantation. Details on the patient demographics are given in Table 1. This study was approved by the local ethics committee, and all patients provided informed consent.

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell, Cologne, Germany). B-cells were isolated from PBMCs using a bead-/column-based magnetic separation method (Miltenyi Biotec, Bergisch Gladbach, Germany). B-cells were purified by negative selection, and purity was typically above 90%. When B-cells were isolated from renal transplant patients, a direct isolation method from whole blood was used. This method is a magnetic bead-based negative selection system (MACSExpress, Miltenyi Biotec), and purity was typically above 90%. To track proliferation, cells were labeled with 2 µM carboxyfluorescein-succinimidyl-ester (CFSE, Sigma–Aldrich, Taufkirchen, Germany).

B-cell cultures were performed as described previously (13). Isolated B-cells were cultured in RPMI1640 GlutaMAX (Thermo Fisher Scientific, Darmstadt, Germany) at a concentration of 0.5 × 10⁶ cells/ml supplemented with 100 U/ml penicillin (Sigma–Aldrich), 100 µg/ml streptomycin (Sigma–Aldrich), 10% fetal calf serum (Greiner-Bio one, Frickenhausen, Germany), nonessential amino acids (NEAA; Sigma–Aldrich) and sodium pyruvate (Sigma–Aldrich) in 96-well U-bottom plates (Sigma–Aldrich). For the plasmablast formation assay and the phenotyping of costimulatory molecules on B-cells, PBMC were cultured. Depending on the assay, the cells were cultured for three to six days in a 5% CO₂ atmosphere at 37°C. To induce the production of IL-10^pos B-cells, isolated B-cells were stimulated with CpG (ODN2006, 0.1 μM, InvivoGen, Toulouse, France) for 72 h, followed by restimulation with phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) and ionomycin (1 µg/ml, both Sigma–Aldrich) in the presence of brefeldin A (5 µg/ml, BFA, BD Biosciences). For detection of Granzyme B (GrB^pos) B-cells, isolated B-cells were stimulated with CpG (ODN2006, 0.1 μM) in the presence of IL-2 (50 ng/ml, Miltenyi Biotec) or IL-21 (50 ng/ml, Miltenyi Biotech) for 72 h, followed by restimulation with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml, Sigma–Aldrich) and ionomycin (1 µg/ml, Sigma–Aldrich) in the presence of brefeldin A (BFA, BD Biosciences). For the plasmablast formation assay, unseparated B-cells were stimulated for six days with either CpG (ODN2006, 0.1 μM, Invitrogen) in the presence of IL-2 (50 ng/ml, Miltenyi Biotech) or IL-21 (50 ng/ml, Miltenyi Biotech). To detect IgG secretion, isolated B-cells were stimulated with Poly-S (resiquimod plus IL-2, 1:1000 final dilution, CTL Europe GmbH) for four days and then transferred to a prepared ELISpot plate for incubation for an additional 24 h. The two FTI lonafarnib (Selleckchem, TX, USA) and tipifarnib (Selleckchem, TX, USA) were used at different concentrations for the assays. Tacrolimus (1.25 ng/ml, Sigma–Aldrich) and rapamycin (12.5 ng/ml, Sigma–Aldrich) were used in selected experiments.

For the detection of IgG-secreting human cells, a commercially available ELISpot kit was used (Human IgG Single-Color ELISpot, CTL Europe GmbH) according to the manufacturer's instructions. For the final incubation step, fixed numbers of purified B-cells were used (500 or 1,000 cells per well), and spots were counted using an ELISpot Reader (AID, Straßberg, Germany).

For GrB and IL-10 detection, purified B-cells were harvested at the end of the culture period and stained with an anti-CD19 Pacific Blue antibody (clone J3-119, Beckman Coulter, Krefeld, Germany) and 7AAD (BioLegend, Eching, Germany) followed by fixation/permeabilization (Cytofix/Cytoperm Kit, BD Biosciences, Heidelberg, Germany). B-cells were then intracellularly stained for GrB (anti-human GrB antibody, clone GB11, PE, eBioscience) or IL-10 (anti-human IL-10 antibody, APC, clone JES3-9D7, Biolegend). For the plasmablast formation assay, PBMC were harvested after culture and stained with anti-human CD19 (clone J3-119, FITC, Beckman Coulter), anti-human CD38 (clone HIT-2, PE, Biolegend) and anti-human CD27 (clone O323, APC-H7, Biolegend). Appropriate isotype controls were used to confirm the specificity of staining. Flow cytometric measurement was performed the same day by fluorescence activated cell sorting (FACS) with a NAVIOS^TM flow cytometer (Beckman Coulter). The software Kaluza, version 2.1 (Beckman Coulter) was used to analyze the FACS data. The gating strategy is depicted in Supplementary Figure 1. After gating on lymphocytes, viable B-cells were discriminated by 7AAD and CD19 staining. Viable cells were defined as CD19⁺ B-cells being 7AAD^neg. Subsequently, doublets were excluded. Proliferation data was analyzed by gating on the cells that have undergone at least one round of division and is given as “Proliferated fraction” (Figure 1). This parameter is most commonly used for the analysis of CFSE-derived data (14). The term “proliferated fraction” is synonymous with “Fraction diluted” and “Proliferative fraction”.

All values are expressed as the mean ± standard deviation (SD). Unless otherwise stated, statistical significance was calculated by repeated-measures ANOVA and corrections for multiple comparisons were done by Dunnett's test. A p-value below 0.05 was considered as statistically significant.

Purified B-cells from HC were used to investigate the effect of FTI on differentiation of B-cells. First, it was determined whether FTI have an impact on the proliferation of stimulated B-cells. For this purpose, CFSE-labeled B-cells were stimulated with CpG plus IL-2 in the presence or absence of FTI. Lonafarnib and tipifarnib suppressed B-cell proliferation in a dose-dependent manner (Figure 1). Viability was assessed after 72 h of culture and was assessed in two different ways: viable 7AAD^negCD19^pos B-cells as percentage of total cells and viable 7AAD^neg CD19^pos B-cells within the CD19^pos B-cell population. In absence of lonafarnib and tipifarnib, 69 ± 5% of total cells were CD19^pos7AAD^neg whereas 91 ± 3% within the CD19^pos B-cell population were negative for 7AAD. At the lowest and intermediate concentrations of FTI, the overall fraction of CD19^pos7AAD^neg cells was slightly decreased as compared to stimulation without FTI. Viability within the B-cell population was comparable (total cells: %CD19^pos7AAD^neg and CD19^posB-cells: %7AAD^neg; lona 34.2 ng/ml: 63 ± 4% and 91 ± 0.3%; tipi 24.5 ng/ml: 57 ± 2% and 89 ± 0.7%; lona 342 ng/ml: 50 ± 4% and 88 ± 1%; tipi 245 ng/ml: 51 ± 5% and 86 ± 2). At the highest concentration of FTI, the fraction of viable cells was greatly reduced (total cells: %CD19^pos7AAD^neg and CD19^pos B-cells: %7AAD^neg; lona 1710 ng/ml: 7 ± 2% and 41 ± 11%; tipi 1,225 ng/ml: 1.65 ± 0.2% and 9.21 ± 4%). Thus, high dosages of FTI induce cell death leading to decreased proliferation. Next, it was studied whether the suppressive effect of both agents is reversible. For this purpose, B-cells were exposed either for 72 h to FTI during stimulation with CPG and IL-2 or exposure to FTI during stimulation was limited to 24 h followed by FTI-free stimulation for another 48 h (Figure 1B). Suppression of B-cell proliferation was reduced during short term exposure vs. long term exposure. In a separate set of experiments, B-cells were exposed for 72 h to FTI while being stimulated followed by FTI-free stimulation for another period of 72 h. At the end of the second culture period, FTI-exposed B-cells showed reduced proliferation as compared to FTI-free conditions; however, proliferation of FTI-exposed B-cells was clearly enhanced when proliferation at the end of the second culture period was compared to the first culture period of 72 h (Supplementary Figure 2). Thus, the suppressive effects of FTI are potentially reversible.

To investigate whether FTI impact the costimulatory capacity of B-cells, the expression of PDL-1 and CD27 was studied. PDL-1 is a co-inhibitor, binds to PD-1 on T-cells and may mediate inhibitory signals to T-cells. CD27 serves on B-cells not only as a memory marker but also has a key role in promoting plasma cell differentiation as costimulator (15, 16). As expected, stimulation with CpG enhanced expression of PDL-1, CD27 and CD25. FTI treatment did not reduce the expression of PDL-1 on B-cells and was comparable to the control condition with CpG only; interestingly, rapamycin also had no influence on PDL-1 expression (Figure 2A). On the contrary, FTI and rapamycin diminished the fraction of CD27⁺ B-cells significantly (Figures 2B, 2D). There was no effect on CD25 expression (Figure 2C). However, the fraction of CD25 expressing B-cells was slightly reduced after treatment with Lonafarnib (Supplementary Figure 3).

It was then tested whether FTI inhibit the maturation of IL-10 and GrB producing B-cells. For this purpose, IL-10 and GrB expressing B-cells were induced during cell culture. IL-10 as well as GrB production were assessed by flow cytometry. As expected, CpG + IL-2 was a potent inducer of IL-10^posand GrB^pos B-cells (Figure 3). Lonafarnib lowered the fraction of IL-10^pos B-cells at a higher concentration (1710 ng/ml) significantly whereas tipifarnib already diminished the fraction of IL-10^pos B-cells at a lower concentration (245 ng/ml) significantly (Figure 3). The same observation was made for GrB⁺ B-cells (Figure 3). However, only at the highest concentrations, IL-10 and GrB production was almost completely suppressed with either tipifarnib or lonafarnib.

Furthermore, the extent to which FTI impact effector B-cell function was investigated. Plasmablast formation and IgG secretion were studied. Plasmablast formation was induced by stimulation with CpG in the presence of IL-21 and IL-2 for six days. The fraction of plasmablasts was then determined by flow cytometry. Lonafarnib and tipifarnib efficiently inhibited plasmablast formation upon CpG stimulation being as potent as rapamycin (Figure 4). Subsequently, it was determined whether IgG secretion could be suppressed by FTI. Purified B-cells were stimulated for four days with Poly-S in the presence of the FTI, rapamycin or tacrolimus. Then, B-cells were transferred to ELISpot plates, and the number of spot-forming units (SFU) was determined after 24 h. Lonafarnib and tipifarnib significantly lowered the number of SFU and were equally potent to rapamycin (Figure 5A). Tacrolimus did not influence IgG secretion (Figure 5A). To assess whether FTI also show significant effects under disease conditions, B-cells derived from patients after renal transplantation were assayed. FTI inhibited IgG secretion to an extent similar to that seen in HC (Figures 5B,C).