PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis

Introduction

Immune thrombocytopenia (ITP) is an autoimmune disease in which autoreactive T and B cells are activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. The binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases.

Methods

Thirty-four patients with primary active ITP who were diagnosed and/or followed up and 26 healthy controls were enrolled in this study. Platelet counts in all ITP patients were less than 30×10⁹ /L at sampling. They had not been treated with any immunosuppressive agents for at least one week prior to sampling for this study.

To determine the role of the PD-1/PD-L signalling pathway in ITP, we detected PD-1 expression on T cells and PD-L expression on dendritic cells (DCs) in both ITP patients with active disease and healthy controls by flow cytometry. To investigate the effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells, PBMCs from ITP patients and healthy controls with autologous platelets were cultured with soluble anti-CD3 monoclonal antibody (mAb) and anti-CD28 mAb in the presence (PD-L1-Fc⁺ group) or absence (PD-L1-Fc^- group) of the PD-L1-Fc (0.5 μg/mL) at 37 ˚C with 5% CO₂ for 4 days. The cells were harvested and stained for flow cytometry to detect the apoptosis, activation and proliferation of T cells. IL-2 and IFN-γ levels in the co-culture supernatant were assayed by ELISA.

Results

Enhanced PD-1 expression on T cells and decreased PD-L1 expression on mDCs in ITP patients

We found that PD-1 mean fluorescence intensities (MFIs) increased in CD4⁺ cells (P < 0.01) and in CD8⁺ cells (P < 0.01) from ITP patients compared with those from healthy controls. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls (P < 0.01, Figure 1).

PD-L1-Fc promoted T cell apoptosis

Annexin V-FITC and propidium iodide (PI) were used to detect T cell apoptosis. The percentage of apoptotic cells and dead cells were analysed to determine T cell apoptosis levels. In ITP patients, the percentage of apoptotic cells and dead cells were higher in PD-L1-Fc⁺ group than in the PD-L1-Fc^- group (P < 0.01 for both CD4⁺ and CD8⁺ T cells). However, we found no significant difference in apoptosis between PD-L1-Fc^- and PD-L1-Fc⁺ groups in healthy controls (Figure 2).

PD-L1-Fc inhibited T cell activation and proliferation

CD25 MFIs were analysed to determine the activation level of cocultured T lymphocytes. Compared with healthy controls, CD25 expression on CD4⁺ and CD8⁺ T cells was significantly increased in ITP patients (P < 0.05 for both CD4⁺ and CD8⁺ T cells). These results suggest that ITP patients had more activated CD4⁺ and CD8⁺ T cells than in healthy controls. PD-L1-Fc significantly inhibited T cell activation in ITP patients (P < 0.01, n=34) but not in healthy controls (P = 0.0834 in CD4⁺ T cells, P = 0.6834 in CD8⁺ T cells, n=26, Figure 3).

To analyse proliferation, the frequency of divided cells was calculated according to the loss of CFSE fluorescence intensity. PD-L1-Fc inhibited proliferation of T cells from ITP patients (P < 0.01 in both CD4⁺ T and CD8⁺ T cells, n=34, Figure 4). We found no significant difference in proliferation in T cells from healthy controls (CD4⁺ T cells P = 0.9758, CD8⁺ T cells P = 0.5658, n=26).

PD-L1-Fc inhibited IL-2 and IFN-γ production

IL-2 secretion was higher in ITP than in healthy controls in the absence of PD-L1-Fc (P = 0.0308, Figure 5). PD-L1-Fc significantly inhibited IL-2 and IFN-γ production in ITP patients. IL-2 and IFN-γ levels were lower in the PD-L1-Fc⁺ group than in the PD-L1-Fc^- group in ITP (P < 0.01 for both IL-2 and IFN-γ, n=34). However, we did not detect a significant difference in secretion of IL-2 (P = 0.2016, n=26) or IFN-γ (P = 0.2989, n=26) in healthy controls.

Conclusions

In summary, our study suggests that the aberrant PD-1/PD-L negative costimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP by promoting T cell apoptosis, inhibiting T cell activation and proliferation, and reducing secretion of inflammatory factors.

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