Chloroquine Suppresses Effector B-Cell Functions and Has Differential Impact on Regulatory B-Cell Subsets

Abstract

Objectives: Chloroquine (CQ) is approved for treatment of B-cell mediated diseases such as rheumatoid arthritis and systemic lupus erythematosus. However, the exact mode of action in these diseases has not been studied and it remains unclear which effect CQ has on B-cells. Thus, it was the aim of this study to investigate to which extent CQ affects functionality of effector and regulatory B-cell.

Methods: For this purpose, B-cells were isolated from peripheral blood of healthy controls and renal transplant patients. B-cells were stimulated in presence or absence of CQ and Interleukin-10 (IL-10) and Granzyme B (GrB) secretion were assessed. In addition, effector functions such as plasma cell formation, and Immunoglobulin G (IgG) secretion were studied.

Results: CQ suppressed Toll-Like-Receptor (TLR)-9 induced B-cell proliferation in a dose-dependent manner. IL-10^pos regulatory B-cells were suppressed by CQ already at low concentrations whereas anti-IgG/IgM-induced GrB secreting regulatory B-cells were less susceptible. Plasma blast formation and IgG secretion was potently suppressed by CQ. Moreover, purified B-cells from renal transplant patients were also susceptible to CQ-induced suppression of effector B-cell functions as observed by diminished IgG secretion.

Conclusion: In conclusion, CQ had a suppressive effect on IL-10 regulatory B-cells whereas GrB secreting regulatory B-cells were less affected. Effector functions of B-cells such as plasma blast formation and IgG secretion were also inhibited by CQ. Effector B-cells derived from renal transplant patients already under immunosuppression could be suppressed by CQ. These findings may partly explain the clinical efficacy of CQ in B-cell mediated autoimmune diseases. The application of CQ in other disease contexts where suppression of effector B-cells could offer a benefit, such as renal transplantation, may hypothetically be advantageous.

Keywords: B-cells; chloroquine; effector B-cells; regulatory B (Breg) cells; renal transplantation.

Figure 1

Dose-dependent suppression of TLR9-induced B-cell proliferation by CQ. PBMCs or purified B-cells were labeled with CFSE and stimulated with CpG in presence of different concentrations of CQ. After 72 hours, CD19⁺ B-cell proliferation was determined by CFSE-dilution. (A) Impact of CQ on B cell proliferation with CpG stimulation in PBMCs. (B) Impact of CQ on B cell proliferation with CpG stimulation in purified B cells. (C) Impact of CQ on vitality of B-cells after stimulation with CpG in PBMCs. (D) Impact of CQ on vitality of B-cells after stimulation with CpG in purified B-cells, P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. *p < 0.05, **p < 0.01, ***p < 0.0001 (against CpG).

Figure 2

CQ affects B-cell proliferation depending on the stimulus. PBMCs were isolated and labeled with CFSE followed by stimulation with different stimuli in presence of CQ at four concentrations (0.05 µM, 0.5 µM, 10 µM, 25 µM). Rapamycin and tacrolimus were also used as controls. After 72 hours, CD19⁺ B-cell proliferation was determined by CFSE-dilution. (A) Impact of CQ on B cell proliferation upon stimulation with CpG. (B) Impact of CQ on B cell proliferation upon IgM/IgG stimulation. (C) Impact of CQ on B cell proliferation upon Poly-S stimulation. P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. **p < 0.01, ***p < 0.0001 (against CpG, IgG+IgM or Poly-S as control conditions).

Figure 3

Effect of CQ on IL-10^pos regulatory B-cells. PBMCs were cultured in presence of different stimuli and CQ at four concentrations (0.05 µM, 0.5 µM, 10 µM, 25 µM) for 72 hours. Rapamycin and tacrolimus were also used as controls. After 72 hours, the production of IL-10 by CD19⁺ B-cells was determined by flow cytometry. (A) Impact of CQ on IL-10⁺ CD19⁺ B-cells upon CpG stimulation in PBMC. (B) Impact of CQ on IL-10⁺ CD19⁺ B-cells upon IgG+IgM stimulation in PBMCs. (C) Impact of CQ on IL-10⁺ CD19⁺ B-cells upon Poly-S stimulation in PBMCs. P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. *p < 0.05, **p < 0.001, ***p < 0.0001 (against CpG, IgG+IgM or Poly-S as control conditions).

Figure 4

Effect of CQ on GrB^pos regulatory B-cells. Purified B cells were isolated and stimulated via TLR9 or BCR plus IL-21, in presence or absence of CQ, rapamycin or tacrolimus for 72 hours. After 72 hours, GrB⁺ CD19⁺ B-cells were determined by flow cytometry. (A) Impact of CQ on GrB⁺ CD19⁺ B-cells upon CpG plus IL-21 stimulation. (B) Impact of CQ on GrB⁺ CD19⁺ B-cells upon IgG/IgM plus IL-21 stimulation. P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. *p < 0.05 (against CpG+IL-21 or IgG+IgM+IL-21 as control conditions).

Figure 5

Effect of CQ on plasmablast formation depending on the stimulus. Purified B cells were stimulated for 6 days and were monitored for differentiation into plasma cells by flow cytometry. (A) Impact of CQ on plasma cells upon CpG plus IL-21 and IL-2 stimulation. (B) Impact of CQ on plasma cells upon IgG+IgM plus IL-21 and IL-2 stimulation. P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. *p < 0.05, **p < 0.001 (against CpG+IL-2+IL-21 or Poly-S+IL-2+IL-21 as control conditions).

Figure 6

Chloroquine suppresses antibody-synthesis by plasma cells. To assess if CQ has impact on antibody-secretion by B-cells, a human IgG-specific ELISpot analysis was performed. Isolated CD19⁺B cells were initially seeded at 5×10⁴ cells/well under Poly-S stimulation in presence of CQ at different concentrations for four days. Then, cells were harvested and transferred to ELISpot plates at a density of 500 cells/well or 1000 cells/well to be cultured for another 24 hours. (A, B) Impact of CQ on IgG-secretion of plasma cells using purified B cells. P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. *p < 0.05, **p < 0.001, ***p < 0.0001 (against Poly-S as control condition).

Figure 7

Effect of CQ on IgG-secretion of plasma cells derived from renal transplant patients. To further investigate the effect of CQ on B-cells from renal transplant patients, 22 patients were enrolled. Isolated CD19 ⁺ B cells derived from renal transplant patients were seeded at a concentration of 5 × 10⁴ cells/well under Poly-S stimulation in presence of CQ for 4 days. B cells were then harvested and transferred to ELISpot plates at a density of 500 or 1000 cells/well for further 24 hours. (A) Impact of CQ on IgG-secretion of plasma cells at a density of 500 cells/well. (B) Impact of CQ on IgG-secretion of plasma cells at a density of 1000 cells/well. P-values were calculated by repeated-measures ANOVA and correction for multiple comparisons were done by Dunnett’s test. **p < 0.001, ***p < 0.0001 (against Poly-S as control condition).