Costimulating aberrant T cell responses by B7-H1 autoantibodies in rheumatoid arthritis (original) (raw)
Costimulatory B7-H1 autoantibodies in RA serum. In an attempt to evaluate the potential role of autoantibodies in prolonged activation of T cells in RA, purified IgG from the sera of RA patients were evaluated for their ability to regulate the proliferation of T cells in vitro. In the presence of suboptimal doses (30–50 ng/ml) of anti-CD3 mAb to mimic TCR signaling, purified IgG from the sera of two RA patients, but not control IgG, significantly enhanced the proliferation of purified CD4+ T cells in vitro in a dose-dependent fashion (Figure 1a). In the absence of anti-CD3 mAb, purified IgG from RA patients had no effect (data not shown). The soluble form of these autoantibodies did not have any activity in the same assay (Figure 1b). The costimulatory activity of the autoantibodies was completely blocked by inclusion of soluble B7-H1Ig fusion protein containing human B7-H1 extracellular portion and mouse IgG2a Fc (14), but not by PD-1Ig or control IgG (mouse IgG2a) (Figure 1c). Our results thus suggest that the costimulation activity for CD4+ T cell was mediated by autoantibodies against B7-H1 in the sera of RA, rather than by the soluble form of B7-H1 per se or autoantibodies to PD-1.
Detection, costimulatory function, and disease association of B7-H1 autoantibodies in RA patients. CD4+ T cells were cultured with immobilized (a) or soluble (b) sera IgG at the initiated dose of 20 μg/ml in the presence of suboptimal anti-CD3 mAb. The titers of RA 1 and RA 2 patients are 0.32 and 0.25 (OD450), respectively. (c) For blockade, the control (ctl) Ig, PD-1Ig, or B7-H1Ig at 3 μg/ml were added before the addition of CD4+ T cells. The growth of T cells was detected after 72 hours of culture. (d) To examine specificity of RA sera binding to B7-H1, diluted sera were preincubated with PBS, 2 μg/ml of soluble B7-H1Ig or control Ig (mIgG2a). (e) Diluted sera of 63 patients with RA and 54 healthy donors were tested for binding to B7-H1Ig by ELISA. Samples with OD450 values greater than 0.123 were considered positive. P value for differences between cohorts is shown (t test). (f) The sera at 1:1,000 dilution were examined for binding to B7-1Ig–coated vs. B7-H1Ig–coated ELISA plates. (g) Mock/624mel or B7-H1/624mel cells were stained with diluted (1:5) sera from 16 ELISA-positive RA patients. The inset bar shows the average percentage of positive staining. A typical histogram of FACS assay is shown on the left. (h) Sixty-three RA patients were sorted according to the active status of their disease, and the presence of B7-H1 autoantibodies. Statistical analysis of the correlation was performed as P = 0.0179 in a Fisher exact test.
To directly demonstrate the existence of autoantibodies against B7-H1 in the sera of RA patients, sera from 63 patients with RA were examined by a specific sandwich ELISA using plates coated with purified B7-H1Ig. Autoantibodies binding to B7-H1 were detected by anti-human IgG mAb. Our ELISA is highly specific for human B7-H1 because binding of patients’ sera could be selectively blocked by preincubation of sera with soluble B7-H1Ig, but not control mIgG2a (Figure 1d). We used 0.123 OD450 as a cut-off based on the mean (0.057) + 2 SD (0.033) of the values with sera from 54 healthy donors at 1:1,000 dilutions. As shown in Figure 1e, sera from 18 of 63 sera from patients with RA (29%) had elevated autoantibodies to B7-H1, while only 4% of 54 healthy donors were marginally positive for the presence of autoantibodies to B7-H1 (P = 0.0002). Similar to the findings of Matsui et al. (13), we did not detect any autoantibodies to B7-1, even in the sera that were positive for B7-H1 Ab’s (Figure 1f). The presence of B7-H1 autoantibodies was also tested by binding of 624 melanoma line (624mel) that was transfected to express human B7-H1 (B7-H1/624mel) (16). A significant portion of sera from 16 patients, which were positive in ELISA assay, also stained for B7-H1/624mel but not Mock/624mel. The specificity of the binding was also confirmed by complete blockade with B7-H1Ig, which was preincubated with the diluted sera (data not shown). In addition, all the samples that were negative in ELISA for the presence of autoantibodies to B7-H1 did not bind B7-H1/624mel (data not shown). Our results indicate that a significant population of patients suffering from RA has elevated autoantibodies to B7-H1.
Correlation of B7-H1 autoantibodies with RA activity. As a preliminary functional determinant, we examined the possible relationship between the presence of B7-H1 autoantibodies and disease activity. Active disease was defined as the presence of at least nine tender joints, six swollen joints, and one or both of the following: 1 hour of morning stiffness or elevated Westergren sedimentation rate (19). We found a significant correlation between active disease and the presence of autoantibodies in 63 patients with RA. Eighty-nine percent of RA patients in the B7-H1 autoantibody-positive group had active disease, while only 56% of RA patients in B7-H1 autoantibody-negative group demonstrated disease activity (P = 0.017) (Figure 1h). These results suggest that autoantibodies to B7-H1 may be involved in disease progression by a direct effect on T cells.
Reverse costimulation of CD4+ T cells by B7-H1 mAb. Autoantibodies from sera are polyvalent with multiple different specificities, limiting the potential for functional analysis. To facilitate further study of the effect of autoantibodies to B7-H1 on T cell response, we generated hybridomas that secrete mAb against human B7-H1. We identified two mAbs, 2H1 and 5H1, which specifically bound to B7-H1 on 293 cells transfected with human B7-H1 plasmid (B7-H1/293), but not mock-transfected 293 cells (Mock/293) by FACS analysis (Figure 2a). In these and later studies, 5H1 and 2H1 showed identical features and were used interchangeably.
Preferential expression of B7-H1 mAb on activated CD4+ T cells. (a) On the left, 293 cells were transfected with mock (pcDNA3 vector) or human B7-H1 plasmid (pcDNA3-B7-H1cDNA) for 48 hours. B7-H1/293 cells were pretreated with 20 μg of control Ig (mIgG1) or 5H1 before staining with PD-1Ig (5 μg). On the right, activated M99 CTL cells were pretreated with 10 μg of mIgG1 or 5H1 before staining with B7-H1Ig (10 μg). B7-H1 mAb, PD-1Ig, or B7-H1Ig were used to stain the transfected 293 cells and activated M99 CTL cells. Representative fluorescence histograms of isotype control reagents (open lines) and B7-H1 mAb or fusion proteins (filled lines) are shown. (b) Induction of B7-H1 expression on human T cell subsets. Human PBMCs were activated with PHA for indicated times and subjected to FACS analysis with B7-H1 mAb and mAb to CD4, CD8, or CD45RO. The numbers indicate the percentage of B7-H1 and CD4, CD8, or CD45RO double-positive cells in total populations, and the percentage in parentheses indicates the percentage of B7-H1–positive cells in each CD4+, CD8+, or CD45RO+ subsets.
To determine whether or not our mAb can block interaction between B7-H1 and PD-1, we examine the ability of 5H1 mAb to block the binding of PD-1Ig to B7-H1/293 cells. As shown in Figure 2a, PD-1Ig bound B7-H1/293 cells but not mock/293 cells. Inclusion of 5H1 mAb up to 20 μg/ml during staining did not interfere with the binding of PD-1Ig. Inclusion of 5H1 mAb, however, could inhibit the binding of B7-H1Ig to M99 T cell clone from 31% to 8% (Figure 2a), suggesting that 5H1 could partially block the binding of B7-H1 to a non–PD-1 receptor (16). Similar blocking function was also found using 2H1 mAb (data not shown).
FACS analysis using B7-H1 mAb showed that B7-H1 is not detectable on freshly isolated PBMC subsets with CD4, CD8, or CD45RO markers. However, stimulation by phytohemagglutinin (PHA), a T cell mitogen, rapidly upregulated expression of B7-H1 on 64.2% of CD4+ cells at 24 hours and 78.2% at 48 hours. Meanwhile, only 44.7% and 32.1% of CD8+ T cells expressed B7-H1 at 24 hours and 48 hours after PHA stimulation, respectively. High levels of B7-H1 (84.2% at 24 hours and 62.3% at 48 hours) were also detected on CD45RO+ memory T cells (Figure 2b). We have also found that stimulation of CD4+ T cells with immobilized CD3 mAb in optimal doses rapidly upregulate the expression of B7-H1 within 24 hours, while suboptimal doses of CD3 mAb requires more than 48 hours to induce B7-H1 expression (data not shown). We conclude that B7-H1 is inducible on human T cells and is preferentially expressed on activated CD4+T cells and CD45RO+ memory T cells.
To evaluate the function of CD4+ T cell–associated B7-H1, we stimulated purified human CD4+ T cells with suboptimal concentrations (30 ng/ml) of anti-CD3 mAb in combination with B7-H1 mAb. While anti-CD3 mAb alone induced an absent or weak T cell response, significant increases in T cell proliferation were observed by inclusion of B7-H1 mAb. This effect, however, was less potent than that of anti-CD28 mAb (Figure 3a). Costimulatory activity was also observed using immobilized human PD-1Ig fusion protein, suggesting an agonistic effect of B7-H1 mAb. The effect of the B7-H1 mAbs was dose dependent in a range of 2.5 to 10 μg/ml (Figure 3b). Immobilization of B7-H1 mAb was critical for the effect since B7-H1 mAb in soluble form in doses up to 20 μg/ml were ineffective (Figure 3c). TCR signaling was also required for proliferation, because B7-H1 mAb did not stimulate T cell proliferation in the absence of anti-CD3 mAb (Figure 3a). Inclusion of soluble B7-H1Ig, which competitively inhibits the interaction between T cell–associated B7-H1 and B7-H1 mAb, significantly reduced the costimulatory effect of B7-H1 mAb on T cells. In contrast, soluble B7-1Ig or control Ig had no inhibitory effect (Figure 3d), confirming the specificity of the response.
B7-H1 mAb costimulates human CD4+ T cell proliferation. (a) Purified human CD4+ T cells were cultured with immobilized 10 μg/ml of control (ctl) Ab, B7-H1 mAb (2H1), PD-1Ig, and 2 μg/ml of CD28 mAb in the presence of precoated different dose of CD3 mAb. Cultures were pulsed with 3H-TdR for a final 16 hours, and the cells were harvested at 72 hours. (b) Dose-dependent costimulation of immobilized anti–B7-H1 mAb in the presence of suboptimal dose (30 ng/ml) of CD3 mAb. Titration of mAb or fusion protein was started at 20 μg/ml of control Ab, B7-H1 mAb, PD-1Ig, and 4 μg/ml of CD28 mAb. (c) Human CD4+ T cells were costimulated with 20 μg/ml of soluble control (ctl) Ab, B7-H1 mAb (2H1), PD-1Ig, and 2 μg/ml of CD28 mAb as the same condition in a. (d) Blocking of B7-H1 mAb–mediated costimulation by soluble B7-H1Ig. Soluble control Ig, B7-1Ig, or B7-H1Ig (10 μg/ml) was preincubated with immobilized control (ctl) Ab or B7-H1 mAb (2H1) for 30 minutes before the addition of T cells. B7-H1 costimulation was assayed in the presence of suboptimal dose of CD3 mAb for 72 hours of culture. (e) FACS analysis of IL-2 receptor (CD25) expression in CD4+ T cells after B7-H1 mAb costimulation. (f) IL-10 secretion from CD4+ T cells in the presence of immobilized CD3 mAb (500 ng/ml), and B7-H1 mAb (10 μg/ml), or CD28 mAb (2 μg/ml).
The costimulatory functions of B7-H1 mAb are very similar to the autoantibodies isolated from RA patients, which induced both phenotypic T cell changes and distinct patterns of cytokine secretion. Specifically, B7-H1 mAb induced high-levels of CD25 expression on CD4+ T cells, an effect similar to that using anti-CD3/CD28 mAb (Figure 3e). Additionally, anti-CD3/B7-H1 mAb costimulation led to increased secretion of IL-10 (Figure 3f). A small increase of IFN-γ secretion was also observed in culture supernatant, while IL-2 and IL-4 were not detected (data not shown). Taken together, our results suggest a reverse-signaling function of B7-H1 for CD4+ T cell costimulation.
Promoting apoptosis of activated CD4+ T cells by B7-H1 mAb. We have shown that one role of B7-H1 mAb is to promote proliferation of CD4+ T cells. This suggests that B7-H1 autoantibodies in RA patients may contribute to the persistent activation of newly recruited T cells when they encounter self antigens. In RA patients, however, many CD4+ T cells are already mature/activated. The effect of B7-H1 triggering by Ab’s on activated T cells may be different from those observed on primary T cells. To examine the effect of B7-H1 mAb on activated CD4+ T cells, we employed an in vitro culture system in which optimal doses of anti-CD3 mAb can drive T cell proliferation without additional costimulation. In this setting, B7-H1 mAb significantly increased apoptosis of CD4+ T cells, as determined by double staining of PI and AV (Figure 4a). The cell death induced by B7-H1 mAb was completely abrogated by preincubation of the mAb with B7-H1Ig, but not with control Ig (Figure 4b). Similarly, immobilized PD-1Ig also increased apoptosis of activated T cells. Neither soluble nor immobilized mAb to B7-H1 or PD-1Ig alone had an effect (data not shown). In a different setting, CD4+ T cells were preactivated by optimal doses of anti-CD3 mAb for 48 hours, in which all CD4+ T cells express high levels of B7-H1, and further stimulated with B7-H1 mAb. Similarly, preactivated T cells also had increased apoptosis after exposure to a coligation of immobilized anti-CD3/B7-H1 mAb, but not to ligation of B7-H1 mAb alone (data not shown). Our results suggest that B7-H1 mAb in the presence of strong TCR signaling promotes apoptosis of activated CD4+ T cells.
B7-H1 mAb promotes programmed cell death of activated CD4+ T cells. (a) Human CD4+ T cells were cultured with 10 μg/ml of immobilized control (ctl) Ab, B7-H1 mAb, PD-1Ig in the presence of precoated optimal dose (500 ng/ml) of CD3 mAb. The cells were analyzed by FACS to determine apoptotic cells (positive in AV and negative in PI staining). (b) Blocking of B7-H1 mAb-induced apoptosis by soluble B7-H1Ig. Control Ig or B7-H1Ig at 10 μg/ml was preincubated with immobilized control Ab or B7-H1 mAb for 30 minutes before the addition of T cells. Percentages of apoptotic CD4+ T cells were shown at 72 hours of culture. (c) Expression of apoptotic genes by B7-H1 mAb costimulation. Purified human CD4+ T cells (5 × 106) were cultured with immobilized B7-H1 mAb or control mAb at 10 μg/ml in the presence of optimal dose of CD3 mAb. The mRNA levels of each gene from B7-H1 mAb-stimulated T cells were presented as the fold induction, relevant to that from control mAb-treated T cells. (d) FACS analysis of expression of TRAIL protein on CD4+ T cells 3 days after anti-CD3/B7-H1 mAb costimulation. (e) Purified human CD4+ T cells were stimulated in the presence of indicated mAb or fusion protein in immobilized form as indicated in the legend of a and the activated form of caspase-3 at the indicated time point was stained by FAM-DEVD-FMK and subjected to FACS analysis. (f) Blocking of B7-H1 mAb-induced apoptosis of activated CD4+ T cells by anti–IL-10 neutralizing mAb. Purified human CD4+ T cells were stimulated with immobilized CD3 mAb and B7-H1 mAb for 72 hours, and the apoptotic cells were analyzed by AV and PI staining. Control Ab and mAbs to Fas ligand, IL-2, or IL-10 at 10 μg/ml was included at the beginning of the culture.
To define the mechanism of the apoptotic effect, we used DNA arrays for expression analysis of apoptosis-related genes stimulated by B7-H1 mAb. Up to 72 hours after ligation with anti-CD3/B7-H1 mAb, mRNA from CD4+ T cells was extracted and hybridized to a DNA array membrane. In three separate experiments, transcription of caspase-10 and caspase-3 genes was reproducibly increased. TRAIL was also upregulated (Figure 4c). Enhanced gene expression was also confirmed by protein analysis. Specifically, elevated TRAIL was found in anti-CD3/B7-H1 mAb–stimulated T cells by staining with anti-TRAIL Ab in FACS analysis (Figure 4d). Significant increases of active caspase-3 were also detected at 48 and 72 hours after stimulation (Figure 4e). Neither B7-H1 mAb nor anti-CD3 (at suboptimal doses) alone stimulated these changes. Anti-CD3 at high dose (1 μg/ml) could induce the activation of caspase 3 but not the expression of TRAIL in activated CD4+ T cells (data not shown). These observations emphasize dependence of a TCR signal in the effect of B7-H1 mAb. Our results suggest that B7-H1 mAb upregulates caspases and TRAIL on T cells, which may facilitate activation-induced death of CD4+ T cells.
IL-10 is a potent immunosuppressive cytokine that stimulates Th2 CD4+ T cell responses and enhances the apoptosis of activated human T cells (20–23). B7-H1 mAb stimulated secretion of IL-10 from activated CD4+ T cells (Figure 3f), providing presumptive evidence that IL-10 might play a role in the increased apoptosis induced by B7-H1 mAb. To test this hypothesis, we examined whether neutralization of IL-10 in anti-CD3/B7-H1 mAb inhibited apoptosis. As shown in Figure 4f, inclusion of anti–IL-10 mAb significantly reduced the amount of apoptosis induced by anti-CD3/B7-H1 mAb at 72 hours. In contrast, neutralizing Ab’s against Fas ligand and IL-2 had no effect. Our results suggest that IL-10 facilitates, at least in part, the induction of apoptosis by B7-H1 mAb ligation.