Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses

Abstract

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

Fig. 1

Monoclonal autoantibodies activate RF B cells through either BCR/TLR9-, BCR/TLR7- or both BCR/TLR7- and BCR/TLR9-dependent pathways. (A) Splenic B cells from RF WT (black bar), Tlr7^−/−(grey bar), Tlr9^−/− (white bar) and Tlr7^−/−Tlr9^−/− (hatched bar) mice were activated with the indicated ligands or monoclonal autoantibodies for 30 h (left) or 40h (right) and proliferation was measured by ³H-thymidine uptake. Data represent the mean +/− SEM of 3 independent experiments. (B) RF WT B cells were stimulated with BWR4 in the presence (open squares) or absence (filled circles) of IFN-β for the indicated times. (C) RF WT (open symbols) and RF Tlr9^−/− (closed symbols) B cells were stimulated with PA4 (left) or PL2-3 (right) for the indicated times. For (B) and (C), data is representative of 3 independent experiments. Proliferation was assessed by ³H-thymidine incorporation over the last 6 h of time point. Statistical analysis was performed using unpaired Student’s t test (* P ≤ 0.5, ** P ≤ 0.05, *** P ≤ 0.005).

Fig. 2

RNA-containing ICs induce BLyS-independent survival. (A) CFSE-labeled RF WT B cells were stimulated with the indicated ligands in the absence (top) or presence (bottom) of 50 ng/ml BLyS for 72 h. Proliferation was measure by CFSE dilution and cell death by uptake of TO-PRO-3. The quadrants depict the following: top left - dead divided cells (TOP-RO-3⁺ CFSE diluted); top right – dead undivided (TOP-RO-3⁺ CFSE undiluted); bottom right- live undivided (TO-PRO-3⁻ CFSE undiluted); bottom left – live divided (TO-PRO-3⁻ CFSE diluted). Division numbers are indicated by red arrows underneath the flow plots. Flow plots are representative of >10 experiments. (B, C) B cells from RF WT, Tlr7^−/−, Tlr9^−/− and Tlr7^−/−Tlr9^−/− mice were stimulated as in (A). (B) Representative flow plots of B cells from RF WT, Tlr7^−/−, Tlr9^−/− and Tlr7^−/−Tlr9^−/− mice stimulated with PL2-3. (C) B cells from RF WT, Tlr7^−/−, and Tlr9^−/− mice were stimulated as in (A) with PA4, BWR4, and PL2-3 in the presence or absence of BLyS. Data shown represents % of the recovered cells that had divided and remained alive +/− SEM, n=9. Statistical analysis was performed using 2-way ANOVA and Bonferroni post-test (* P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005). (D) RF WT B cells were stimulated with PL2-3 in the presence or absence of BLyS, with or without a TLR9 inhibitor for 72 h. (E) RF WT B cells were stimulated with IgG2a IC that incorporated a defined CG-rich DNA-fragment for 72 h. (D,E) Representative flow plots of 3 independent experiments.

Fig. 3

BCR/TLR7 coengagement promotes plasma cell differentiation. (A) RF WT B cells were stimulated with the indicated ligands or monoclonal autoantibodies and stained for CD22, CD44 and CD138 to determine the frequency of CD22^lo CD44^hi CD138⁺ plasmablasts. n=4. (B) RF WT, Tlr7^−/−, Tlr9^−/− and Tlr7^−/−Tlr9^−/− B cells were activated with PL2-3 for 72 h and stained as in (A), n=4. (C) RF WT B cells were activated with PL2-3 in the presence of a TLR9 specific inhibitor for 72 h and stained as in (A), n=3. (D) Purified RF FcγR2b^−/− B cells were activated with the indicated ICs for 96 h in the presence of 50 ng/ml BLyS. mRNA levels for bcl-6, Pax5 and prdm1 were determined by qPCR. Data is normalized to media control using the ΔΔCT method and analyzed by Student’s t test; * P value of ≤ 0.5, n=4. (E,F) Expression of B cell differentiation-associated genes in RF WT, Tlr7^−/−, and Tlr9^−/− cells after stimulation with PL2-3. Gene expression changes are reported as ratios relative to RF WT 0 h (unstimulated) as measured by gene expression arrays.

Fig. 4

BCR/TLR7-dependent activation leads to the prolonged expression of IRF4 and BCR/TLR9-dependent activation leads to early expression of IRF8. (A) RF WT, Tlr7^−/−, Tlr9^−/− and Tlr7^−/−Tlr9^−/− B cells were stimulated with indicated ligands for either 24 h (dotted line) or 72 h (black line), permeabilized and stained for IRF4. The filled area represents the isotype control. Representative plots of 3 independent experiments are shown. (B) RF WT (black circles), Tlr7^−/− (grey squares) or Tlr9^−/− (open triangles) B cells were stimulated with PL2-3 for the indicated times and gene expression for IRF4 (left) and IRF8 (right) determined by microarray. (C) RF Tlr7^−/− (black line) and Tlr9^−/− (dotted line) B cells were stimulated with PL2-3 for 14 h (left panel) and 72 h (right panel) in the presence of 50 ng/ml BLyS. Expression of IRF8 (top) and IRF4 (bottom) was measured by flow cytometry, n=2.

Fig. 5

RF WT and Tlr9^−/− B cells express comparable levels of TLR7. (A) Unactivated purified WT (grey line), Tlr7^−/− (black line) and Tlr9^−/− (dashed line) RF B cells or B cells activated with the indicated ligands for 24 hr, were permeabilized and stained for TLR7 expression. Unstained cells are indicated by the filled histogram. Representative plots of 3 experiments are shown. Data from the 3 experiments is summarized as MFI of the TLR7 staining intensities over background in the graph on the right. (B) RF WT, Tlr7^−/− and Tlr9^−/− splenic B cells were activated with the indicated concentrations of the synthetic TLR7 ligand CL097 for 30 h and proliferation was measured by ³H-thymidine uptake. Data represent the mean +/− SEM of 8 independent experiments.

Fig. 6

BCR/TLR7 activation promotes AFC differentiation in vivo. (A) BALB/c CD45.1 recipients were injected i.v. with 15 x 10⁶ WT, Tlr7^−/−, Tlr9^−/− and Tlr7^−/−Tlr9^−/− VPD450-labeled RF B cells and 50 μg PL2-3 on day 0 and injected again with PL2-3 on day 3. Spleens were harvested on day 6. B cell engraftment was ascertained by CD45.2 staining and proliferation was measured by dilution of VPD450. Representative plots of 3 independent experiments are shown. (B) The number of cell divisions based on the data in (A) was calculated for each mouse and the mean value +/− SEM are shown, n=3 mice/group. Statistical analysis was performed using Student’s t test, * P ≤ 0.05. (C) BALB/c CD45.1 recipients were injected with AM14 B cells as above but injected with PL2-3 on day 0, 3, 7 and 10. Additional BALB/c CD45.1 mice were only injected with PL2-3, and not RF B cells (none). Spleens were harvested on day 13; the number of clonotype positive IgG+ AFCs was measured by ELISpot, data is compiled from 4 independent experiments, One-way ANOVA including Tukey’s multiple comparison test was used for statistical analysis; ** P ≤ 0.005, *** P ≤ 0.0005.