IgG Anti-platelet Autoantibodies Research Articles

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4\u201328/IGHJ4 gene in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4–28 in ITP patients. One hundred eighty-six distinct IGHV4–28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4–28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4–28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4–28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.

What do we learn from immunomodulation in patients with immune thrombocytopenia?

Current therapeutic strategies for autoimmune diseases primarily rely on immunosuppression, but global immune suppression results in an increased risk for severe infection and malignancy. In contrast, immuomodulation is another therapeutic approach employing intrinsic or environmental regulators that exert modulatory effects by intervening multiple checkpoints of the immune system, leading to correction of dysregulated immune responses. We have learned that immunomodulation by intravenous immunoglobulin is highly efficacious and safe in patients with immune thrombocytopenia (ITP), an autoimmune disease mediated by IgG antiplatelet autoantibodies. Recently, another types of immunomodulatory treatment are also effective for ITP. These include eradication of Helicobacter pylori and thrombopoietic agents, such as thrombopoietin receptor agonists. These treatment modalities are shown to exert immunomodulatory action by suppressing multiple checkpoints of the pathogenic loop of ITP, although only certain subsets of the patients show robust responses. Understanding mechanisms underlying immunomodulation is highly useful in clarifying pathogenesis of immune-mediated diseases and developing novel therapeutic approaches.

Induction of immune tolerance to platelet antigen by short-term thrombopoietin treatment in a mouse model of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder caused by IgG anti-platelet autoantibodies. Thrombopoietin (TPO) receptor agonists are highly effective in inducing the recovery of platelet counts in ITP patients. Although these agents are thought to promote platelet production without affecting the autoimmune pathogenesis of the disease, a small subset of ITP patients exhibits sustained platelet recovery after treatment termination. To investigate mechanisms involved in this sustained recovery, we evaluated the effects of short-term TPO treatment using a mouse ITP model generated by Foxp3(+) T regulatory cell (Treg) depletion. After treatment, platelet recovery was sustained, along with complete suppression of both anti-platelet autoantibody production and T-cell responses to platelet autoantigens. TPO treatment also promoted the peripheral induction of Foxp3(+) Tregs in conjunction with elevated circulating TGF-β levels. In summary, thrombopoietic agents are capable of inducing immune tolerance to platelet autoantigens, thereby suppressing the autoimmune pathogenesis of ITP.

Dysregulated negative immune regulators in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disease mediated by IgG antiplatelet autoantibodies, resulting in an isolated thrombocytopenia. The mechanism for ongoing antiplatelet antibody production is explained by a ‘pathogenic loop’ model consisting of macrophages in the reticuloendothelial system, platelet‐reactive CD4+ T cells and B cells producing IgG antiplatelet antibodies. In ITP patients, a variety of negative immune regulators including CD4+ T regulatory cells, B regulatory cells and tolerogenic dendritic cells are dysfunctional, resulting in failure to efficiently suppress the pathogenic loop. In addition, Helicobacter pylori infection leads to defective inhibitory FcγRIIB signalling in macrophages and thereby increases susceptibility to ITP. In ITP patients, dysregulation of these negative immune regulators is associated with each other in the impaired immune regulatory network. Thus, strategies that enhance functions of these intrinsic negative immune regulators would be promising future approaches for treating ITP.

CD4+CD25+Foxp3+ Regulatory T Cells in the Pathophysiology of Immune Thrombocytopenia

Regulatory T cells characterized by CD4, CD25, and transcription factor forkhead box P3, called Tregs, are a subpopulation of CD4(+) T cells specialized for immune suppression. Tregs contribute to maintenance of peripheral immune tolerance, and their defects are thought to play a role in the pathogenesis of various autoimmune diseases. Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and reduced platelet production, resulting in decreased platelet count. Recently, a series of studies in adults and children with ITP have found that the frequency of Tregs is reduced in circulation, bone marrow, and spleen, and Treg function is impaired. Treg dysregulation is improved after platelet count is recovered by treatment with dexamethasone, rituximab, or thrombopoietin receptor agonists. In addition, a critical role of Tregs in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in functional Tregs. Thrombocytopenia observed in Treg-deficient mice is mediated through production of IgG anti-platelet autoantibodies, which is analogous to human ITP. Further studies evaluating mechanisms of Treg dysregulation in ITP patients are necessary to elucidate the pathogenesis of ITP and develop novel therapeutic strategies that suppress anti-platelet autoimmune response.

Splenic macrophages maintain the anti-platelet autoimmune response via uptake of opsonized platelets in patients with immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is an autoimmune disease primarily caused by IgG anti-platelet autoantibodies. Activation of autoreactive CD4(+) T cells upon recognition of cryptic GPIIb/IIIa peptides presented by antigen-presenting cells (APCs) is a critical step for triggering and maintaining the pathogenic anti-platelet autoantibody response. We investigated which APCs carry the cryptic peptides of GPIIb/IIIa that activate autoreactive CD4(+) T cells in ITP patients. GPIIb/IIIa-reactive T-cell lines generated from ITP patients were cultured with autologous freshly isolated splenic macrophages, B cells or dendritic cells. To further investigate how the macrophages presented the antigenic GPIIb/IIIa peptides, we prepared macrophages from the peripheral blood monocytes of the same patients during remission. Macrophages induced the proliferation of GPIIb/IIIa-reactive T-cell lines without an exogenous antigen, but B cells and dendritic cells required GPIIb/IIIa peptides to stimulate the T cells. Macrophages derived from peripheral blood during remission required an exogenous antigen to induce the GPIIb/IIIa-reactive T-cell line response, but could elicit a response without added antigen if they were preincubated with platelets from ITP patients with platelet-associated anti-GPIIb/IIIa antibodies or healthy platelets pretreated with ITP platelet eluates. The T-cell response was inhibited by anti-FcgammaRI antibody. Finally, cultured macrophages that captured opsonized platelets promoted anti-GPIIb/IIIa antibody production in mixed cultures of autologous GPIIb/IIIa-reactive T-cell lines and B cells. Splenic macrophages that take up opsonized platelets via FcgammaRI are major APCs for cryptic GPIIb/IIIa peptides, and are central to the maintenance of anti-platelet autoantibody production in ITP patients.

Corticosteroid administration depresses circulating dendritic cells in ITP patients

Idiopathic or immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by low platelet count resulting from antibody-mediated destruction of platelets. The production of these IgG anti-platelet autoantibodies is critically governed by T-lymphocytes which can be activated by antigen-presenting cells (APC) such as dendritic cells. We hypothesized that one of the mechanisms by which corticosteroid administration can suppress the immune system is to depress the number of circulating dendritic cells production in ITP patients. Dendritic cell population was measured in peripheral blood of three ITP patients before and after the administration of prednisone. Both counts of myeloid and lymphoid dendritic cells in the blood of ITP patients were greatly reduced after the administration of prednisone. The decrease in circulating dendritic cells is associated with the increase of platelets in circulation with the treatment of prednisone. These results suggest that corticosteroid therapy may decrease the effects of the autoantibody on platelets in ITP patients by reduce the number of circulating dendritic cells.

Possible presence of enhancing antibodies in idiopathic thrombocytopenic purpura.

It is difficult to detect IgG anti-platelet autoantibodies in idiopathic thrombocytopenic purpura (ITP). Recently, it was reported that reactivity with glycoprotein IIb/IIIa was lost when IgG anti-GPIIb/IIIa antibodies from seven ITP patients were digested with pepsin to yield F(ab')2 fragments. These findings suggested that some IgG antiplatelet autoantibodies in ITP may be of low affinity and thus require the presence of 'enhancing' anti-IgG antibodies (i.e. rheumatoid factors, RFs) for detection. To test this hypothesis, we used a phage display technique to isolate five IgG RFs from an ITP patient (patient 1). Sequence analysis revealed that these RFs consisted of two clones, represented by GG3 and GG48. Both representative RFs bound specifically to IgG Fc fragments with apparent dissociation constants of 8.2 x 10(-8) M and 8.8 x 10(-7) M, respectively. Moreover, IgG RFs were subsequently found in a serum sample from patient 1. Combined, these results suggest that IgG RFs may occur in ITP, and may be required for the detection of some IgG anti-platelet autoantibodies and for the corresponding antibody-mediated platelet destruction in autoimmune ITP.