The programmed death-1 ligand 1:B7-1 pathway restrains diabetogenic effector T cells in vivo

Abstract

Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti-PD-L1 Abs that have different blocking activities. Anti-PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti-PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti-PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4(+) and CD8(+) T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4(+) and CD8(+) T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.

Figures

Figure 1. Anti-PD-L1 mAb 10F.2H11 does not interfere with functional PD-L1:PD-1 signaling, whereas 10F.9G2 does

A, Binding of 10F.9G2, 10F.2H11 and Rat IgG2b isotype control mAb to PD-L1-Ig fusion protein was compared by ELISA. B, Binding of 10F.9G2 or 10F.2H11 to PD-L1-transfected 300.19 cells assessed by flow cytometry. Directly conjugated mAb (left panel) or fluorescently conjugated anti-rat IgG secondary (right panel) were used. C, Functional engagement of PD-1 is inhibited by 10F.9G2, but not 10F.2H11. PD-1-overexpressing transgenic T cells were cultured on plates coated with anti-CD3 and either PD-L1-Ig fusion protein (L1-Ig) or control protein (C-Ig). After coating, plates were incubated with the indicated concentrations of anti-PD-L1 Abs or control Ab, washed and then T cells were added. Proliferation was measured by CFSE dilution of 7-AAD-negative T cells by flow cytometry. Data are representative of at least two independent experiments.

Figure 2. 10F.9G2 and 10F.2H11 recognize distinct epitopes of PD-L1

A, B. Analysis of epitopes recognized by 10F.9G2 and 10F.2H11 mAbs. PD-L1-transfected 300.19 cells were pre-incubated with 20 μg/ml purified antibody and then stained with fluorescently conjugated MIH5, 10F.9G2, 10F.2H11 or isotype control as indicated. C, Schematic depiction of the binding and blocking capabilities of the 10F.9G2 and 10F.2H11 mAbs. Data are representative of at least two independent experiments.

Figure 3. 10F.2H11 precipitates diabetes in NOD females in an age-dependent manner

6-7 week-old (A) or 13 week-old (B) NOD females were treated with 0.5 mg antibody i.p. on day 0, followed by 0.25 mg on days 2, 4, 6, 8 and 10 or until they became diabetic. n=10 for each group; data are representative of three independent experiments. C, 6-7 week old mice were treated on days 0 and 2 and pancreata were harvested on day 3. H&E stains of representative sections are shown. Left panel – 10F.9G2 (insulitis); middle panel – 10F.2H11 (peri-insulitis with mild insulitis); right panel – Rat IgG2b isotype control (no insulitis). Scale bars in bottom right-hand corners represent 200 μm. Pancreata were scored as either normal (white) or as having peri-insulitis (light grey) or insulitis (dark grey).

Figure 4. T cells from diabetic mice are more susceptible than T cells from prediabetic mice to the effects of 10F.2H11 antibody

CD4+ and CD8+ T cells from prediabetic (A) or diabetic (B) NOD females were transferred i.v. into NOD SCID recipients which were then treated with 0.5 mg antibody i.p. on day 0, followed by 0.25 mg on days 2, 4, 6, 8 and 10. *p<0.05 over the first 59 days for 10F.9G2 vs. 10F.2H11 treatment. For transfers from diabetic donors, the effects of 10F.9G2 vs. 10F.2H11 mAb administration were not significantly different over any time frame. Data are pooled from two independent experiments with similar results; n = at least 9 per group. Pancreata for histology were harvested 10 days after transfer of prediabetic (C) or diabetic (D) T cells. Left panels – 10F.9G2 (insulitis in C and D); middle panels – 10F.2H11 (no insulitis in C, insulitis in D); right panels – Rat IgG2b isotype control (no insulitis in C or D).

Figure 5. Both 10F.2H11 and 10F.9G2 antibodies precipitate diabetes transferred by islet antigen-specific transgenic CD4+ or CD8+ effector T cells

2 × 104 CD4+Foxp3-GFP- T cells purified from BDC2.5 Foxp3-GFP NOD TCR transgenic mice (A) or 4.5 × 106 CD8+ T cells purified from diabetic 8.3+ NOD TCR transgenic mice (B) were transferred i.v. to NOD SCID recipients, which were then treated with 0.5 mg antibody i.p. on day 0, followed by 0.25 mg on days 2, 4, 6, 8 and 10. Data are representative of two and four experiments, respectively, each with at least four mice per group. *p<0.05; **p<0.005

Figure 6. 10F.2H11 and 10F.9G2 mAb treatment increases islet infiltration by CD8+ effector T cells

NOD SCID recipients of 8.3+ NOD TCR transgenic T cells were treated with anti-PD-L1 antibodies or isotype control and analyzed on day 11 post transfer. Pancreatic infiltrates were analyzed for the percentage (A) and number (B) of CD3+CD8+ T cells. Pooled data from 3 independent experiments each with at least 3 mice per group are shown. *p<0.05. Pancreas sections showing islets were stained with H&E (C) and scored for insulitis (D). Left panel – 10F.9G2 (insulitis); middle panel – 10F.2H11 (insulitis); right panel – Rat IgG2b isotype control (no insulitis).

Figure 7. 10F.2H11 and 10F.9G2 mAbs increase CD8+ effector T cell expansion, activation and cytokine production

NOD SCID recipients were given 8.3+ NOD TCR transgenic T cells, treated with anti-PD-L1 antibodies or isotype control, and analyzed on the indicated day post transfer. Splenic CD3+CD8+ T cells were evaluated by flow cytometry for numbers (A), activation (B), cytokine production (C) and B7-1 and PD-1 expression (D). For B and C, representative flow cytometry plots (i) and cumulative data (ii) are shown. (A-C) shows data representative of three independent experiments, each with five mice per group; (D) shows representative FACS plots and pooled data from two independent experiments **p<0.005; ***p<0.0005