Human Leukocyte Antigen-G Restores Immune Tolerance in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder in which loss of immune tolerance leads to platelet destruction. Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class I antigen including four membrane-bound (mHLA-G, HLA-G1 to -G4) and three soluble (sHLA-G, HLA-G5 to -G7) isoforms due to alternative splicing of the HLA-G primary transcript. HLA-G exerts immunosuppressive functions by interacting with its inhibitory receptors, immunoglobulin-like transcripts (ILTs), expressing differentially on natural killer cells, T cells, and antigen presenting cells.

Upon binding to ILTs, HLA-G suppressed lymphocyte proliferation, promoted apoptosis of cytotoxic lymphocytes and natural killer cells, inhibited antibody secretion from activated B cells, and induced expansion of suppressive T cells, tolerogenic dendritic cells and myeloid-derived suppressor cells. HLA-G supports immune escape, and high level of HLA-G is associated with unfavorable outcomes in cancer patients. Meanwhile, HLA-G-induced tolerance can benefit human allotransplantation and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, but the role of HLA-G in the treatment of ITP is still unclear.

Here we observed significantly less soluble HLA-G in plasma from ITP patients positive for anti-glycoprotein (GP) IIb/IIIa and/or anti-GPIb/IX autoantibodies compared with autoantibody-negative patients or healthy controls. However, no significant difference was found in the sHLA-G levels between anti-glycoprotein (GP) IIbIIIa and anti-GPIb-IX autoantibodies groups. Interestingly, sHLA-G is positively correlated with platelet counts in ITP patients both with and without autoantibodies, suggesting that HLA-G can also protect platelets from destruction through other mechanisms. To elucidate this, we then analyzed both expression of membrane HLA-G and ILTs on PBMCs. Our data found less membrane-bound HLA-G and ILTs on CD4⁺T cells and CD14⁺cells in ITP patients. Moreover, recombinant human HLA-G (rhHLA-G) upregulated expression of ILT2 on CD4⁺and ILT4 on CD14⁺cells. The upregulation of ILT by rhHLA-G indicates that sHLA-G might also benefit ITP patients negative for anti-platelet autoantibodies.

To determine whether rhHLA-G affects T cell differentiation, we tested 17 cytokines by ELISA to investigate the immune environment with or without rhHLA-G, and found that rhHLA-G upregulated Interleukin (IL)-4 and IL-10, and downregulated tumor necrosis factor-α, IL-12, and IL-17 secreted by ITP patient peripheral blood mononuclear cells (PBMCs), indicating a promotion of helper T(Th)2 and inhibition of Th1 and Th17 differentiation. However, our results indicated that rhHLA-G did not expand the CD4⁺CD25⁺Foxp3⁺regulatory T cells(Tregs). In addition, rhHLA-G exerted their immunosuppressive effects by upregulating IL-1β, IL-2, Granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). By reprogramming the cytokine profile in ITP patients, HLA-G established an optimal environment for the impaired cell populations, such as dendritic cells (DCs), to regain their tolerogenic function. Our results showed that rhHLA-G inhibited monocyte-derived DCs maturation by downregulating CD80 and CD86 expression. To further assess whether the rhHLA-G modulated-DCs were functionally tolerogenic, PBMCs were then cocultured with rhHLA-G modulated-DCs. Our results showed that rhHLA-G-modulated DCs from ITP patients suppressed CD4⁺T cell proliferation by 38.8% compared to unmodulated cells. Besides, PBMCs exposed to rhHLA-G were less cytotoxic toward plateletsfrom ITP patients or healthy subjects. This study provides a potential strategy to protect exogenous platelets from destruction in ITP patients.

In conclusion, our data demonstrated that impaired expression of HLA-G and/or ILT is involved in the pathogenesis of ITP, and recombinant human HLA-G can restore immune tolerance in ITP patients via upregulation of ILTs, which indicates that HLA-G can be a diagnostic marker and also a therapeutic option for ITP treatment.