Regulatory T cells use programmed death 1 ligands to directly suppress autoreactive B cells in vivo Janine Gotota, Catherine Gottschalka, Sonny Leopolda, Percy A. Knollea, Hideo Yagitab, Christian Kurtsa,1,2, and Isis Ludwig-Portugalla,1,2 aInstitutes of Molecular Medicine and Experimental Immunology (IMMEI), Rheinische Friedrich-Wilhelms-Universität, 53105 Bonn, Germany; and bDepartment of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan Edited by David Tarlinton, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3050, Australia, and accepted by the Editorial Board May 4, 2012 (received for review January 20, 2012) The mechanisms by which regulatory T cells (Tregs) suppress auto- Programmed death-1 (PD-1, CD279) is an activation-induced antibody production are unclear. Here we have addressed this member of the extended CD28/CTLA-4 family that suppresses T question using transgenic mice expressing model antigens in the cells (18–21). It has been associated with exhausted memory kidney. We report that Tregs were essential and sufficient to sup- T cells in chronic viral infection (22, 23) and with cytotoxic T-cell press autoreactive B cells in an antigen-specific manner and to cross-tolerance (24). PD-1 has two known ligands, PDL-1 (B7-H1, prevent them from producing autoantibodies. Most of this sup- CD274) and PDL-2 (B7-DC, CD273) (25, 26), and both of them pression was mediated through the inhibitory cell-surface-mole- are sufficient to mediate T-cell suppression (27, 28). Tregs have cule programmed death-1 (PD-1). Suppression required PD-1 been shown to express PDL-1, but such expression was dispens- expression on autoreactive B cells and expression of the two PD- able for T-cell suppression in vitro (29). Also B cells specific for 1 ligands on Tregs. PD-1 ligation inhibited activation of autoreactive foreign antigens express PD-1 ligands and interact with follicular B cells, suppressed their proliferation, and induced their apoptosis. helper T cells known to express high levels of PD-1, resulting in Intermediate PD-1+ cells, such as T helper cells, were dispensable increased germinal center B-cell survival and plasma cell differ- fi for suppression. These ndings demonstrate in vivo that Tregs use entiation (30). On the other hand, PD-1 knockout mice develop PD-1 ligands to directly suppress autoreactive B cells, and they high levels of auto-Ab (31), which is hard to reconcile with identify a previously undescribed peripheral B-cell tolerance mech- a positive effect on antibody production. Thus, the role of PD-1 on IMMUNOLOGY anism against tissue autoantigens. B cells is unclear. To address these open questions, we have used mice expressing peripheral tolerance | autoimmunity | autoantibodies | inhibitory receptors OVA and HEL in kidney glomerular podocytes (32), which allow detailed studies in an organ where auto-Ab–mediated diseases are utoantibodies (auto-Ab) cause various autoimmune dis- prevalent (33). We demonstrate direct suppression of B cells by Aeases, such as systemic lupus erythematosus and certain Tregs and identify PD-1 signaling as the underlying mechanism. forms of glomerulonephritis. Depletion of autoreactive B cells ameliorates many, but not all, autoimmune diseases. However, Results fi this approach causes severe immunosuppression due to the Tregs Speci cally Suppress Auto-Ab Production Against Glomerular fi general loss of B cells. Specific control of autoreactive B cells is Auto-Ag. To study how Tregs speci cally suppress auto-Ab pro- required for improved therapies. duction against peripheral tissue antigens, we used transgenic mice Regulatory T cells (T ) are powerful suppressors of auto- expressing a membrane-bound fusion protein of OVA and HEL regs under the control of the nephrin promoter in kidney podocytes reactive T cells with high therapeutic potential (1–3). Tregs also suppress auto-Ab production (4, 5). We recently showed in vivo (NOH mice) (32). We used a vaccination scheme of applying OVA in aluminium hydroxide (Alum) three times (experimental scheme that they do so in an antigen-specific (Ag-specific) manner (6, 7). in Fig. 1A), which induced robust anti-OVA titers after 3 wk in These studies used rat insulin promoter HEL/OVA (ROH) mice nontransgenic wild-type (WT) control mice (6, 7). When we vac- expressing ovalbumin (OVA) and hen egg lysozyme (HEL) in cinated NOH mice with this scheme, OVA-specific IgG antibody pancreatic islet β-cells. Autoreactive OVA- and HEL-specificB B fi titers were sevenfold lower compared with WT mice (Fig. 1 ), cells, but not B cells speci c for a foreign antigen, failed to indicating immune tolerance. Consistent with our previous studies proliferate in response to in vivo autoantigen (auto-Ag) challenge on pancreatic auto-Ag (6, 7), treatment with the antibody PC61 1 d and instead underwent apoptosis in a strictly T -dependent + reg before vaccination depleted about 90% of the FoxP3 Tregs (Fig. fashion. Tregs can affect B cells indirectly by suppressing the S1) and restored anti-OVA antibody production in NOH mice to T-helper (Th) cells required for antibody production (8, 9). This 85% of that in WT controls (Fig. 1B). Antibody production against did not rule out that Tregs might also suppress B cells directly. the foreign antigen β-galactosidase (β-Gal) was unchanged in + Cell culture systems have revealed that CD25 Tregs can kill NOH and WT mice, and PC61 treatment had no effect either (Fig. – cocultured B cells (10 12). A recent in vivo study showed that 1C), demonstrating auto-Ag–specific suppression by Tregs. These Tregs enter germinal centers and suppress B cells in this site (13, 14). The question whether this occurred directly or indirectly remained open (15). This question is difficult to address in vivo Author contributions: J.G., C.K., and I.L.-P. designed research; J.G., C.G., S.L., and I.L.-P. performed research; P.A.K. and H.Y. contributed new reagents/analytic tools; J.G., C.G., because it requires an experimental system where Tregs can sup- P.A.K.,H.Y.,C.K.,andI.L.-P.analyzeddata;andJ.G.,C.K.,andI.L.-P.wrotethepaper. press B cells but not Th cells. fl Another open question concerns the molecular mechanisms by The authors declare no con ict of interest. This article is a PNAS Direct Submission. D.T. is a guest editor invited by the which Tregs suppress. In principle, Tregs may suppress other T cells Editorial Board. by (i) secreting inhibitory mediators; (ii) deprivation of survival 1C.K. and I.L.P. contributed equally to this study. factors; (iii) killing target cells by granzyme/perforin; and (iv) 2To whom correspondence may be addressed. E-mail: [email protected] or isis.ludwig- modulation of DCs by ligating inhibitory T-cell receptors (16, 17). [email protected]. The exact contribution of these mechanisms in relevant in vivo This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. situations and the mechanisms by which Tregs suppress are unclear. 1073/pnas.1201131109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201131109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Blocking PD-1 restores Ag-specific auto- Ab titers in vivo. (A) Experimental scheme for B and C:miceweredepletedofTregs with PC61 Ab on days −4, −1, 6, and 13 (white arrows) and immunized with 10 μgOVAand10μg β-Gal in Alum on days 0, 7, and 14 (black arrows). (B and C) IgG titers against OVA (B)andβ-Gal (C)in NOH (black bars) or nontransgenic wild-type (WT) control mice (white bars) after depletion of Tregs on day 21. (D) Experimental scheme for E and F: mice were depleted of Tregs with PC61 Ab on days −4and−1 and immunized with OVA/Alum on day 0. (E and F)Percentage(E) and mean fluorescence intensity (MFI) (F)ofPD- 1+ OVA-specific B cells on day 3. (G) Experi- mental scheme for H and I: mice were immu- nizedondays0,7,and14andinjectedwithPD- 1–blocking RMP1-14 or isotype control Abs on thesamedays.(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 Bonfer- roni). Data are representative of two experi- ments using three to four mice in each group. findings indicate that Tregs prevent autoreactive B cells from by central tolerance mechanisms. To address this possibility, we producing anti-glomerular auto-Ab. immunized these mice with OVA-nitrophenol (NP) or BSA-NP as a control and determined the response of NP-specific B cells. PD-1 Mediates Peripheral B-Cell Tolerance Against Glomerular Auto- Such B cells were not autoreactive yet still were controlled by Ag. To identify candidate molecules for B-cell suppression, we OVA-specific Th cells or Tregs. NP-specific antibody serum titers isolated OVA-specific B cells (representative FACS plot in Fig. in NOH mice were 2.8-fold lower than in WT control mice after S2) from immunized NOH or WT mice and determined ex- immunization with OVA-NP, but were unchanged when mice pression of molecules previously implicated in peripheral im- were immunized with NP-BSA (Fig. 1I). When PD-1 was mune tolerance, such as PD-1 or Fas (11, 18, 34). OVA blocked, anti-NP titers were restored to 67% of those in WT vaccination increased PD-1 mRNA expression in OVA-specific controls, which was not significantly different (Fig.