Suppression of expression and function of negative immune regulator PD-1 by certain pattern recognition and cytokine receptor signals associated with immune system danger

Xuemei Zhong1,3, Chunyan Bai3, Wenda Gao4, Terry B. Strom4 and Thomas L. Rothstein1,2,3

Departments of 1 Medicine and 2 Microbiology, Boston University School of Medicine and 3 Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston, MA 02118, USA
4 Division of Immunology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

Correspondence to: T.L. Rothstein; E-mail: trothstein{at}bu.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Stimulation of certain cytokine and pattern recognition receptors enhances adaptive immune responses, and in chronic situations, may play a role in the loss of self-tolerance. We hypothesized that in addition to upregulating positive immune receptors (i.e. co-stimulatory molecules), certain cytokine and pattern recognition signals might downregulate negative immune receptors, removing a potential barrier to lymphocyte responsiveness. The newly identified CD28 family member Programmed Death-1 (PD-1) is an inhibitory receptor involved in peripheral tolerance, as evidenced by the frank autoimmunity and autoantibody formation found in PD-1-deficient mice. Here we report that antigen-receptor induced PD-1 expression on murine B cells is markedly reduced by certain signals associated with immune system danger, including LPS, CpG oligodeoxynucleotides and several pro-inflammatory cytokines, through distinct signaling pathways. We further report for the first time that engagement of PD-1 inhibits cell cycle progression in primary B cells and that modulation of PD-1 expression by CpG or IL-4 significantly reverses such inhibition. Our data suggest a novel mechanism for enhancement of normal immune responses and disruption of normal tolerance mechanisms.

Keywords: B lymphocytes, CpG, danger signals, inflammatory cytokines, lipopolysaccharide, PD-1


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Programmed death 1 (PD-1) is a 50–55 kDa type I transmembrane protein originally identified in apoptotic T cells (1). PD-1 is a member of the immunoglobulin superfamily and shares homologous sequence with CD28 and CTLA4. PD-1 expression on naive lymphocytes is low, but rises following activation, particularly following antigen receptor engagement (2). Despite its name, PD-1 appears to have little to do with cell death. Instead, like CTLA4, PD-1 is thought to negatively influence lymphocyte activation. B and T cell stimulation is enhanced in the absence of PD-1, and murine anti-viral responses against adenovirus are more effective in PD-1-deficient, as compared to normal, animals (3,4). These and other reports suggest that PD-1 might act to moderate lymphocyte responses, and that, conversely, loss of PD-1 might enhance or accelerate adaptive immunity. However, physiological downregulation of PD-1 expression has not been reported.

The danger model postulates that immune responses are determined as much or more by the presence of signals indicating that an antigen is associated with danger to the organism as by the foreign nature of an antigen (5). Danger signals that enhance immune responses can be either exogenous or endogenous (6). Exogenous danger signals are typically those elaborated by microbial pathogens and include lipopolysaccharide (LPS) and unmethylated CpG sequences that trigger pattern recognition (Toll-like) receptors. Endogenous danger signals include molecules released from stressed cells and encompass pro-inflammatory cytokines such as IL-12 and IL-18 (7). Considering that danger signals foster beneficial immune responses and that PD-1 engagement may limit B cell activity, we hypothesized that danger signals act to downregulate expression of PD-1 in order to remove a potential barrier to B cell responsiveness. To investigate this possibility, we examined PD-1 expression on primary B cells stimulated in various ways to determine whether PD-1 expression can be inhibited through specific receptor engagement, and we evaluated the function of PD-1 in primary B cells to determine whether alterations in PD-1 expression are accompanied by modulation of PD-1 activity.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Male BALB/cJ, C57BL/6, C3H/HeJ, C3H/HeOuJ, C.129S2-Stat6tm1Gru, C57BL/10ScN and C57BL/10ScSn mice of 6–8 weeks age were obtained from The Jackson Laboratory (Bar Harbor, ME). MyD88-deficient mice (8) were a kind gift from Dr Ann Marshak-Rothstein (Boston University School of Medicine, Boston, MA).

Primary B cell culture
Spleen cells were depleted of RBC and non-viable cells by sedimentation on Lympholyte M (Cedarlane Laboratories Ltd, Hornby, Ontario, Canada). B cells were isolated by negative depletion using the magnetic-activated cell sorting (MACS) purification system (Miltenyi Biotec, Aubum, CA). All cells were cultured in RPMI 1640 medium (Bio Whittaker, Walkersville, MD) supplemented with 10% FBS (Sigma-Aldrich, St Louis, MO), 10 mM HEPES (Calbiochem Novabiochem, San Diego, CA), 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.

Flow cytometric analysis
Cells were washed, blocked with rat anti-mouse CD16/CD32 antibody (clone 2.4G2), stained with immunofluorescent antibodies, and then analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA) with appropriate gating. Images were constructed with FlowJo 4.3.2 software (Tree Star, San Carios, CA). The following antibodies were obtained from BD Pharmingen (San Diego, CA): FITC-conjugated rat anti-mouse CD45R/B220 (clone RA3-6B2), R-PE-conjugated hamster anti-mouse PD-1 (clone J43), R-PE-conjugated hamster IgG2 isotype standard (clone B81-3), FITC-conjugated rat anti-mouse CD25 (clone 7D4), rat anti-mouse CD16/CD32 (Fc{gamma}III/II receptor) (clone 2.4G2).

Proliferation assay
B cells were cultured for 60 h in round bottomed 96-well microtiter plates in quadruplicate. Tritium incorporation was assessed after exposure to 0.5 µCi [3H]thymidine (20 Ci/mmol; New England Nuclear, Boston, MA) during the last 12 h of culture.

Reagents
Affinity purified F(ab')2 fragments of polyclonal goat anti-mouse IgM antibody (anti-IgM) and whole anti-IgM antibody were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). LPS was obtained from Sigma-Aldrich. Stimulatory CpG oligodeoxynucleotide (ODN) (1826) (9) and inhibitory CpG ODN (2088) (10) were kind gifts from Dr Ann Marshak-Rothstein. Recombinant mouse IL-1{alpha}, IL-1ß, IL-6, IL-12, IL-18, GM-CSF, IFN{gamma} and TNF{alpha} were obtained from R&D systems (Minneapolis, MN) or eBioscience (San Diego, CA). Recombinant mouse IL-2 and recombinant human TGF-ß1 were obtained from Biosource International (Camarillo, CA). Recombinant mouse IL-4 and IL-10 were purchased from BD Pharmingen (San Diego, CA) or eBioscience (San Diego, CA). Soluble recombinant CD40 ligand fusion protein was obtained from transfected J558L cells (11) and crosslinked with anti-CD8 antibody (53-6-72) as previously described (12). Tosylactivated Dynalbeads (Dynal Inc., Lake Success, NY) were coated with either goat anti-mouse IgM (anti-IgM), goat anti-mouse IgM plus PD-L1-Fc (human IgG4 Fc) (13), or goat anti-mouse IgM plus irrelevant human IgG (Sigma-Aldrich, St Louis, MO). Beads (107) were coated with 5 µg of each protein. The integrity and chemical bonding of these proteins to the bead surface was confirmed by flow cytometry (data not shown).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
PD-1 expression is induced by BCR engagement
To confirm activation-induced upregulated expression of PD-1, MACS-purified splenic B cells were evaluated before (day 0) and after stimulation for 1–4 days with mitogenic F(ab')2 fragments of goat anti-mouse IgM (anti-IgM) (Fig. 1A). Anti-IgM produced a dramatic increase in PD-1 expression within 24 h, as previously reported (2). We now show that continued anti-IgM treatment produced an even larger increase in PD-1 expression at 48–72 h that continued through 96 h and was of greater magnitude than that which has been noted in the past (14). We observed similarly increased and prolonged PD-1 expression when B cell stimulation with anti-IgM was limited to 24 h and was then followed by culture in medium for various periods of time (Fig. 1B). Thus, the persistent B cell receptor (BCR)-triggered, upregulated expression of PD-1 does not require continued anti-IgM, at least during the subsequent 3 days. Note that the duration of antigen receptor-induced PD-1 upregulation shown here in B cells mirrors that reported for T cells (14). In view of these characteristics, we then used anti-IgM-induced upregulation of PD-1 as a benchmark against which to test the possibility that danger signals modulate PD-1 expression.



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Fig. 1. PD-1 expression is induced by BCR engagement. (A) Anti-Ig induces upregulation of PD-1 expression within 24 h. MACS-purified splenic B cells from BALB/c mice were harvested immediately (Day 0) or cultured at 2 x 106/ml in the presence of F(ab')2 fragments of goat anti-mouse IgM at 5 µg/ml (anti-IgM) for 1–4 days as indicated. B cells were stained with anti-PD-1 (shaded) and isotype control (dotted) fluorescent antibodies, followed by flow cytometric analysis. One of four comparable experiments is shown. (B) Anti-Ig-induced PD-1 expression is stable. MACS-purified splenic B cells from BALB/c mice were incubated with F(ab')2 fragments of goat anti-mouse IgM at 5 µg/ml (anti-IgM) for 24 h and then washed three times after which B cells were cultured in medium alone for an additional 24, 48 and 72 h (harvested at 48, 72 and 96 h, as indicated). B cells were stained with anti-PD-1 and isotype control fluorescent antibodies. One of two comparable experiments is shown.

 
LPS substantially diminishes BCR-induced upregulation of PD-1 through TLR4
To determine whether PD-1 expression is influenced by LPS, splenocytes and purified splenic B cells were stimulated with anti-IgM in combination with LPS. LPS produced a substantial decline in anti-IgM-stimulated, upregulated PD-1 expression in purified BALB/c B cells (Fig. 2A). As might be expected, the loss of PD-1 produced by LPS was much less pronounced when spleen cells were obtained from TLR4 mutant C3H/HeJ mice, in comparison to spleen cells obtained from normal control C3H/HeOuJ mice (Fig. 2B). To further evaluate the role of TLR4, additional experiments were carried out using B cells obtained from C57BL/10ScN mice in which the Tlr4 gene is naturally deleted, in comparison to B cells obtained from wild-type control C57BL/10ScSn mice. At several doses, particularly 5 to 25 µg/ml, LPS produced substantial loss of anti-IgM-induced, upregulated PD-1 expression in control C57BL/10ScSn B cells but little change in upregulated PD-1 expression in mutant Tlr4 null mice (Fig. 2C). Thus, LPS acts through TLR4 to partially reverse the elevated PD-1 expression produced by BCR engagement.



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Fig. 2. LPS substantially diminishes BCR-induced upregulation of PD-1 through TLR4. (A) LPS reverses anti-Ig-induced upregulation of PD-1 expression. MACS-purified splenic B cells from BALB/c mice were cultured for 48 h with anti-IgM at 5 µg/ml, LPS at 25 µg/ml, or both reagents together. B cells were harvested and stained with anti-PD-1 and isotype control fluorescent antibodies, followed by flow cytometric analysis. One of two comparable experiments is shown. (B) TLR4 mutation blocks the effect of LPS on PD-1 expression. Total splenocytes were isolated from C3H/HeJ and control C3H/HeOuJ mice. Cells were cultured for 72 h in the presence of anti-IgM at 4.5 µg/ml, with or without LPS at 25 µg/ml. Cells were harvested, stained, and B220+ cells analyzed for PD-1 expression in comparison to unstained control B cells. One of two comparable experiments is shown. (C) TLR4 deletion blocks the effect of LPS on PD-1 expression. MACS-purified splenic B cells from C57BL/10ScSn (ScSn) and C57BL/10ScN (ScN) mice were cultured for 68 h in the presence of anti-IgM at 4.5 µg/ml, with or without LPS at the indicated concentrations. Cells were harvested, stained, and analyzed for PD-1 expression in comparison to unstained control B cells. One of two comparable experiments is shown.

 
A stimulatory CpG ODN substantially diminishes BCR-induced upregulation of PD-1 through MyD88
The influence of LPS/TLR4 in reducing PD-1 expression suggested that engagement of other pattern recognition receptors might produce a similar effect. To determine whether TLR9 mediates downregulation of PD-1, MACS-purified B cells were stimulated with anti-IgM in combination with either stimulatory CpG-containing ODN 1826 or inhibitory ODN 2088 at 1–2 µg/ml (9,10). Note that appropriate ODN doses were determined separately and varied from batch to batch. ODN 1826 produced almost complete loss of the induced level of PD-1 expression that typifies anti-IgM-stimulated BALB/c B cells (Fig. 3A). In contrast, ODN 2088 had little or no effect on PD-1 expression. Further, the inhibitory ODN 2088 was dominant, in that combining ODN 2088 with ODN 1826 abolished ODN 1826-mediated modulation of anti-IgM-induced PD-1. To confirm the roll of Toll-like receptor signaling, B cells from MyD88-deficient mice were tested in comparison to purified control C57BL/6 B cells. ODN 1826 produced virtually complete loss of anti-IgM-induced PD-1 expression in C57BL/6 B cells. In marked contrast to these results, ODN 1826 failed to alter the elevated PD-1 expression produced by anti-IgM in B cells obtained from MyD88-deficient animals, so that in these B cells ODN 1826 was as ineffective as ODN 2088 (Fig. 3B). Thus, CpG ODN acts through MyD88 to reverse the elevated PD-1 expression produced by BCR engagement.



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Fig. 3. Stimulatory CpG oligodeoxynucleotide substantially diminishes BCR-induced upregulation of PD-1 through MyD88. (A) CpG ODN 1826, but not CpG ODN 2088, diminishes anti-Ig-induced upregulation of PD-1. MACS-purified B cells from BALB/c mice were cultured for 65 h with anti-IgM at 5 µg/ml, stimulatory CpG ODN 1826 at 1 µg/ml and inhibitory CpG ODN 2088 at 2 µg/ml, as indicated. Cells were harvested and stained with anti-PD-1 and isotype control fluorescent antibodies, followed by flow cytometric analysis. One of seven comparable experiments is shown. (B) MACS-purified B cells from MyD88-deficient (KO) and control C57BL/6 (WT) mice were cultured for 72 h with anti-IgM at 4.5 µg/ml, ODN 1826 at 1 µg/ml, or ODN 2088 at 1 µg/ml, as indicated. Cells were harvested, stained, and analyzed for PD-1 expression in comparison to unstained control B cells. One of two comparable experiments is shown.

 
Inflammatory cytokines diminish BCR-induced upregulation of PD-1
To extend the analysis of danger signals in regulating PD-1 expression, B cells were treated with various pro-inflammatory cytokines in combination with anti-IgM. Cytokines were used at both 25 ng/ml and 50 ng/ml; both doses produced similar results, which are shown in Fig. 4 for 50 ng/ml. IL-12 and IL-18 produced a decrease in anti-IgM-induced PD-1 expression, although less than that produced by LPS or CpG, whereas several other cytokines, including IL-1{alpha}, IL-1ß, IL-10, TNF{alpha}, TGFß and GM-CSF had little effect (Fig. 4A). The combination of pro-inflammatory cytokines IL-12 and IL-18 produced a more substantial decline in anti-IgM-induced, upregulated PD-1 expression than either cytokine alone (Fig. 4B), consistent with synergy between IL-12 and IL-18 observed in other settings (15,16). Both Th1 cytokine IFN{gamma} and Th2 cytokine IL-4 were found to markedly diminish the upregulated level of PD-1 produced by anti-IgM, resulting in reductions of, on average, 50–70% or more in PD-1 expression, respectively, similar to the level of inhibition produced by CpG, LPS and the combination of IL-12 plus IL-18. Thus, IL-4 and IFN{gamma} markedly reduce elevated PD-1 expression produced by BCR engagement, whereas some classical pro-inflammatory cytokines, such as IL-12 and IL-18, have a lesser, but noticeable effect, which is augmented in combination.



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Fig. 4. Inflammatory cytokines diminish BCR-induced upregulation of PD-1. (A) IL-4, IL-12, IL-18 and IFN{gamma} diminish anti-Ig-induced upregulation of PD-1 expression. MACS-purified splenic B cells from BALB/c mice were cultured for 72 h in the presence of anti-IgM at 5 µg/ml, with or without various recombinant cytokines at 50 ng/ml, as indicated. B cells were harvested and stained with anti-PD-1 and isotype control fluorescent antibodies, followed by flow cytometric analysis. In each panel, Control 1 denotes B cells treated with anti-IgM alone and stained with isotype control antibody, and Control 2 denotes B cells treated with anti-IgM plus cytokine and stained with isotype control antibody. One of 10 comparable experiments is shown. (B) IL-12 and IL-18 act in synergy to reduce PD-1 expression. MACS-purified splenic B cells from BALB/c mice were cultured for 72 h in the presence of anti-IgM at 5 µg/ml, with or without IL-12 and IL-18 at 50 ng/ml, as indicated. Cells were harvested and stained with anti-PD-1 and isotype control fluorescent antibodies, followed by flow cytometric analysis. In each panel, Control 1 denotes B cells treated with anti-IgM alone and stained with isotype control antibody, and Control 2 denotes B cells treated with anti-IgM plus cytokine and stained with isotype control antibody. One of six comparable experiments is shown. (C) IL-4 acts through STAT6 to alter PD-1 expression. MACS-purified splenic B cells were obtained from STAT6-deficient mice (KO) and from control BALB/c mice (WT) and were cultured for 68 h in the presence of anti-IgM at 4.5 µg/ml with or without IL-4 at 25 ng/ml, as indicated. B cells were harvested, stained, and analyzed for PD-1 expression in comparison to unstained control B cells. One of three comparable experiments is shown. (D) IL-4 acts coincident with, or after, anti-IgM to reduce PD-1 expression. MACS-purified B cells from BALB/c mice were cultured for 72 h in the presence of anti-IgM at 4.5 µg/ml and IL-4 at 25 ng/ml. B cells were treated with IL-4 for 12 or 24 h, washed, and treated with anti-IgM for 60 or 48 h (–12 h and –24 hr, respectively), or B cells were treated with anti-IgM and IL-4 concurrently for 72 h (0 h), or B cells were treated with anti-IgM for 24 h, washed, and treated with IL-4 for 48 h (+24 hr) or B cells were treated with anti-IgM alone for 72 h (solid line). B cells were then stained and analyzed for PD-1 and CD25 expression in comparison to unstained control B cells. One of six comparable experiments is shown.

 
IL-4 acts through STAT6 to alter PD-1 expression
To delineate the role of STAT6 in mediating IL-4-induced reduction of PD-1 expression, MACS-purified B cells from STAT6-deficient mice were tested in comparison to control BALB/c B cells. IL-4 produced marked loss of anti-IgM-induced PD-1 expression in control B cells as expected. In direct contrast, IL-4 produced little change in PD-1 expression of B cells from STAT6-deficient animals (Fig. 4C). Although the effect of IL-4 is mediated by STAT6, it is not longlasting. B cells treated with IL-4, and washed before exposure to anti-IgM, showed undiminished upregulation of PD-1 expression. However, IL-4 added after anti-IgM diminished PD-1 expression to the same extent as IL-4 added concurrently (Fig. 4D). Notably, IL-4 had little or no effect on expression of another activation antigen, CD25, demonstrating the specificity of PD-1 modulation.

PD-1 engagement inhibits B cell stimulation, and diminished PD-1 expression correlates with inhibition of PD-1 function
PD-1 function has not previously been documented in primary B cells, nor in B cells expressing physiological levels of this inducible receptor. In previous work with T cells and PD-1-transfected B cell lines, PD-1 activity was elicited by co-crosslinking PD-1 and antigen receptors (17,18). To test the role of PD-1, we triggered primary B cell receptor signaling alone or in combination with PD-1 engagement using Dynalbeads coated with either anti-IgM, anti-IgM plus recombinant PD-L1-Fc, or anti-IgM plus control Ig. Cell cycle progression was assessed by measuring thymidine incorporation during the last 12 h of 60 h cultures. Because the level of thymidine incorporation produced by anti-IgM coated beads was modest in comparison to the level stimulated by soluble F(ab')2 fragments of anti-IgM, a well-recognized phenomenon that accompanies the use of immobilized BCR ligands, we enhanced the level of S phase progression by stimulating B cells in the presence of soluble CD40L. The presence of CD40L did not alter downregulation of PD-1 induced by IL-4 or CpG (Fig. 5A). Anti-IgM-coated beads produced substantial B cell proliferation in the context of CD40L, increasing thymidine incorporation 1.5–2.0-fold from the level produced by CD40L alone (~4 x 104 c.p.m.). PD-L1-Fc produced a 33.6 ± 6.1% (mean ± SEM) (P < 0.05) decline in thymidine incorporation by anti-IgM-stimulated B cells, demonstrating the relevance of PD-1 as a modulator of primary B cell activity. Importantly, in the presence of IL-4, engagement of PD-1 by PD-L1 had no effect on B cell proliferation in response to anti-IgM coated beads. Moreover, in the presence of CpG as well, PD-1 engagement had no effect on B cell proliferation (and control oligonucleotide was inactive; data not shown) (Fig. 5B). Thus, two very different agents, IL-4 and CpG, that each diminish B cell PD-1 expression, eliminated the functional effect of PD-L1 in terms of inhibiting B cell proliferation.



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Fig. 5. PD-1 engagement inhibits B cell stimulation and diminished PD-1 expression correlates with inhibition of PD-1 function. (A) CD40L does not alter downregulation of PD-1 by IL-4 and CpG. MACS-purified splenic B cells from BALB/c mice were cultured for 72 h with CD40L plus anti-IgM at 5 µg/ml in the presence of IL-4 at 25 ng/ml or CpG oligonucleotide 1826 at 1 µg/ml, and then stained and analyzed for PD-1 expression in comparison to unstained and unstimulated control B cells. One of two comparable experiments is shown. (B) Diminished PD-1 expression is accompanied by loss of PD-1-induced inhibition. MACS-purified splenic B cells from BALB/c mice were cultured with Dynalbeads for 48 h at a cell:bead ratio of 1:2. Dynalbeads were coated with either whole anti-IgM, whole anti-IgM plus PD-L1-Fc, or whole anti-IgM plus control (ctrl) Ig, as indicated. All cultures contained soluble CD40L; some cultures contained IL-4 at 25 ng/ml or stimulatory CpG oligonucleotide 1826 at 1 µg/ml, as indicated. [3H]Thymidine was added for an additional 12 h of culture and the level of incorporated radioisotope expressed as a percent of the level present after stimulation with anti-IgM-coated beads alone. Mean values and standard errors of the means of three independent experiments are shown. Mean c.p.m. values for incorporated [3H]thymidine were: (i) for untreated samples, anti-IgM, 87714; anti-IgM plus PD-L1-Ig, 58227; anti-IgM plus control Ig, 93680; (ii) for IL-4 treated samples, anti-IgM, 189418; anti-IgM plus PD-L1, 178467; anti-IgM plus control Ig, 171405; (iii) for CpG treated samples, anti-IgM, 157659; anti-IgM plus PD-L1, 154537; anti-IgM plus control Ig, 178394.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The work presented in this study demonstrates that anti-IgM-induced PD-1 expression on B cells is diminished by treatment with LPS, CpG and cytokines such as IL-4, IL-12, IL-18 and IFN{gamma}. Further, this loss of PD-1 expression is accompanied by loss of PD-L1-induced inhibition of BCR-stimulated proliferation. Thus, we have shown for the first time that PD-L1 inhibits the behavior of primary B cells expressing physiological levels of PD-1, that induction of PD-1 expression is counteracted by specific receptor signaling, and that diminished PD-1 expression is accompanied by modulated PD-1 function.

This is the first report in primary B cells that co-stimulation of both BCR and PD-1 actually inhibits B cell proliferation, although the extent of inhibition (30–40%) was relatively modest. This may be partially explained by the confounding factor that PD-1 ligand is also expressed on activated B cells (14) and may already be affecting B cell proliferation even in the absence of ligand provided by Dynal beads. Exogenous PD-L1 may thus replace, in part, endogenous PD-1 engagement, leading to measured inhibition that appears to be less than the true level. In this view, the use of B cells from PD-L1 knock out mice would be expected to produce greater inhibition. Despite this potential complication, the level of inhibition we found in primary B cells is similar to that of a previous report using ectopic expression of PD-1 in a B cell line (17).

The stimuli that we found diminished PD-1 expression and function can, for the most part, be grouped together as ‘danger’ signals. However, the degree of concurrence delineated in this study between stimuli that diminish PD-1 expression and danger stimuli is not perfect. Typical danger cytokines of IL-1{alpha}, IL-1ß and TNF{alpha} (6) had no effect on PD-1 expression, at least by themselves. Conversely, IL-4 produced dramatic reduction in PD-1 expression and is not always thought of as a ‘danger’ signal, although it shares important ‘danger’ characteristics (19), including a role in inflammation (20,21).

Danger signal-triggered reduction of PD-1 expression fits with a speculative model for the function of PD-1 in normal and aberrant immune responses. According to this model, in the event that autoreactive B cell receptors interact with self-antigen in the periphery, expression of PD-1 would be upregulated and B cell activity would be suppressed through PD-L binding to PD-1. This is supported by the observation that PD-L is widely distributed on many peripheral tissues (22,23). Conversely, B cell responses would be enhanced by loss of PD-1 expression, which, as we show here, could occur through the influence of bacterial or macrophage products that are associated with danger signaling. In this light, reduction of PD-1 expression and function by CpG and IL-4 might be viewed as an appropriate facilitation of anti-bacterial and T-dependent responses, respectively. However, the autoimmune phenotype of PD-1-deficient animals (3,24) indicates that there is a real risk to diminished PD-1 expression and function, in the form of dysregulation of B cells in general, and of autoreactive B cells in particular, the consequences of which may be quite substantial. Thus our results may explain the autoantibody formation that accompanies IL-4 overexpression (25,26) and the resistance to autoimmunity (24) associated with deficiencies of IL-12 (27,28). However, the overall extent to which normal immune and autoimmune responses are influenced by loss of PD-1, here shown to result from specific receptor signaling and to be accompanied by functional consequences in primary B cells, remains to be determined.


    Acknowledgements
 
X.Z. is supported by an American Heart Association fellowship. This work was supported by US Public Health Service grants AI29690 and AI40181 awarded by the National Institutes of Health.


    Abbreviations
 
BCR   B cell receptor
LPS   lipopolysaccharide
MACS   magnetic-activated cell sorting
ODN   oligodeoxynucleotide
PD-1   programmed death-1
PD-L   programmed death-1 ligand
TLR   toll-like receptor

    Notes
 
Transmitting editor: S. Akira

Received 30 January 2004, accepted 28 May 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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