Regulatory T cells use programmed death 1 ligands to directly suppress autoreactive B cells in vivo

Abstract

The mechanisms by which regulatory T cells (T(regs)) suppress autoantibody production are unclear. Here we have addressed this question using transgenic mice expressing model antigens in the kidney. We report that T(regs) were essential and sufficient to suppress autoreactive B cells in an antigen-specific manner and to prevent them from producing autoantibodies. Most of this suppression was mediated through the inhibitory cell-surface-molecule programmed death-1 (PD-1). Suppression required PD-1 expression on autoreactive B cells and expression of the two PD-1 ligands on T(regs). PD-1 ligation inhibited activation of autoreactive B cells, suppressed their proliferation, and induced their apoptosis. Intermediate PD-1(+) cells, such as T helper cells, were dispensable for suppression. These findings demonstrate in vivo that T(regs) use PD-1 ligands to directly suppress autoreactive B cells, and they identify a previously undescribed peripheral B-cell tolerance mechanism against tissue autoantigens.

Fig. 1.

Blocking PD-1 restores Ag-specific auto-Ab titers in vivo. (A) Experimental scheme for B and C: mice were depleted of T_regs with PC61 Ab on days −4, −1, 6, and 13 (white arrows) and immunized with 10 μg OVA and 10 μg β-Gal in Alum on days 0, 7, and 14 (black arrows). (B and C) IgG titers against OVA (B) and β-Gal (C) in NOH (black bars) or nontransgenic wild-type (WT) control mice (white bars) after depletion of T_regs on day 21. (D) Experimental scheme for E and F: mice were depleted of T_regs with PC61 Ab on days −4 and −1 and immunized with OVA/Alum on day 0. (E and F) Percentage (E) and mean fluorescence intensity (MFI) (F) of PD-1⁺ OVA-specific B cells on day 3. (G) Experimental scheme for H and I: mice were immunized on days 0, 7, and 14 and injected with PD-1–blocking RMP1-14 or isotype control Abs on the same days. (H) Anti-OVA serum IgG titers on day 21 in WT mice (white bar), NOH mice (black bar), NOH mice treated with RMP1-14 (light gray bar), or with isotype control (dark gray bar). (I) Anti-NP titers on day 14 in NOH (black bars) or WT (white bars) mice immunized with either OVA-NP or BSA-NP in Alum. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA and Bonferroni). Data are representative of two experiments using three to four mice in each group.

Fig. 2.

B cells need to express PD-1 to be suppressed by T_regs in NOH mice. (A) Experimental scheme: 5 × 10⁶ IgHEL were transferred into untreated or T_reg-depleted WT (white bars in B) or NOH mice (black bars in B), which were immunized with HEL in Alum on the next day. (B) Absolute numbers of surviving B220⁺ IgMa⁺ HEL⁺ IgHEL cells 3 d after adoptive transfer. (C) Experimental scheme for D–F: Either 5 × 10⁶ IgHEL or IgHEL×PD-1^−/− cells were transferred into WT (white bars in D–F) or NOH mice (black bars in D–F), which were immunized with HEL in Alum on the next day. (D) Absolute numbers of surviving B220⁺ IgMa⁺ HEL⁺ IgHEL cells 3 d after adoptive transfer. (E) Proportion of Ki67⁺ proliferating B220⁺ IgMa⁺ HEL⁺ IgHEL B cells. (F) Representative bar graph of cleaved caspase 3 expression on IgHEL B cells. *P < 0.05 (ANOVA and Bonferroni). Data are representative of two experiments with three mice in each group.

Fig. 3.

Ag-specific T_regs are sufficient for suppression of PD-1⁺ B cells in vivo. (A) Experimental scheme: 1 × 10⁶ T_regs or Th cells were transferred into IgHEL (B) or IgHELxPD-1^−/− (C) mice on day −1, which were then immunized on days 0, 7, and 14 with HEL in Alum i.p. (B) HEL-specific IgMa serum Ab titers on day 21. (C) HEL-specific serum IgMa titers in IgHEL or IgHEL×PD-1^−/− mice after adoptive transfer of T_regs or Th cells. The Ab titers of unimmunized control mice are shown as background. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA and Bonferroni). Data are representative of two experiments with four mice in each group.

Fig. 4.

PDL-1/2 expression by T_regs is required for B-cell suppression. (A) Experimental scheme: 1 × 10⁶ T_regs from spleens of NOH or NOH×PDL-1^−/− mice were transferred into IgHEL mice that were immunized with HEL/Alum i.p. and injected with PDL-2–blocking TY25 Ab on days 0, 7, and 14. The Ab titers of unimmunized control mice were shown as background. (B) HEL-specific serum IgMa titers on day 21. (C) Proportion of cells producing anti-HEL IgMa Ab determined by ELISpot. (D) Proportion of apoptotic IgHEL cells among splenocytes on day 21. *P < 0.05; **P < 0.01 (ANOVA and Bonferroni). Data are representative of two experiments in groups of four mice each.

Fig. 5.

T_regs directly suppress B cells. (A) Experimental scheme: Either 5 × 10⁶ IgHEL (white bars in B–D, F, and G) or IgHEL×PD-1^−/− (black bars in B–D, F, and G) B cells were transferred into PD-1–competent NOH or into NOH×PD-1^−/− mice, which were immunized with HEL/Alum on the next day. (B) Absolute numbers of IgHEL cells in the spleen 3 d after immunization. (C) Proportions of proliferating IgHEL cells. (D) Proportion of apoptotic IgHEL cells at 18 h after immunization. (E–G) Same experiment as in A except that mice were immunized twice in a weekly interval. (F) HEL-specific IgMa serum Ab titers on day 14. (G) Numbers of antibody-forming cells in the spleens measured by ELISpot. (H) Numbers of HEL-specific antibody-forming cells in the spleens of T_reg-depleted NOH–PD-1^−/− mice measured by ELISpot. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA and Bonferroni). Data are representative of two experiments in groups of three to five mice.