Study On The Number And Function Of CD4 + T Cell Subsets In Peripheral Blood Of Patients With Immune Thrombocytopenic Purpura

Background:Immune thrombocytopenic purpura (ITP) is an organ-specific autoimmune disease. The anti-platelet autoantibodies activate reticular endothelial system (RES) which plays an important role in platelet destruction. Due to disease phases, ITP is categorized into newly-diagnosed ITP, persistent ITP, and chronic ITP. On the basis of immunology developments, it is now argued that cell-mediated immunity also takes an important part in the pathogenesis of ITP. Aberrant T cell function in ITP includes activation of platelet-specific auto-reactive T cells, enhanced cytotoxicity towards platelets, and deletion of Tregs. Our study investigated immune status of helper T cells and its relationship with response to treatment. In order to further reveal the role T cell played in the pathogenesis of ITP and to provide new theoretical evidence for evaluation of treatment in ITP patients, we measured T cell subgrouping (Thl/Th2/Th17/Treg), related cytokines, and their changes within courses of disease. Protein microarray is a multiplex approach to identify protein-protein interactions. Using protein microarray technology, we could measure dozens of T-cell-related cytokines simultaneously, which might give us further insight into pathogenesis of ITP and provide more potential targets for therapeutic use.Objective:By measuring Th cell associated gene expression before and after prednisone therapy, our study was to investigate features of CD4+T cell subgrouping and their relationship with courses of disease. Using protein microarray technology, we measured40related cytokines in patients of different phases of disease in order to reveal cytokines correlating with disease phases.Methods:We enrolled20previously untreated ITP patients (8males,12females) with a median age of41.0yrs (range:20to81) and20normal controls. Using FCM technology, we measured CD4+T cell percentage, CD8+T cell percentage, and CD4/CD8ratio in ITP patients. Using real-time RT-PCR methods, we measured the expression of7Th cell associated genes including T-bet, IFN-y, GATA-3, TGF-β, Foxp3, IL-2, IL-4in PBMC of ITP patients before and after conventional dose of prednisone therapy (lmg·kg-1·d-1) and of normal controls.Furthermore, we applied RayBio(?) Human Inflammation Antibody Array3for measuring of40CD4+T cell related cytokines in30newly-diagnosed patients,30patients undergoing complete remission.Results:All patients completed their prednisone therapy with no severe adverse effects. With a mean platelet count elevation from (17.6±13.8)×109/L to (123.8±52.8)×109/L after treatment,12patients had a complete remission (BPC>100×109/L),7patients partial remission (BPC>50×109/L), and1patient significant clinical improvement but platelet count of45×109/L. T-bet, IFN-y and IL-2were significantly over-expressed in ITP patients (p<0.01), which represented a Thl/Th2inbanlance. After treatment, Foxp3were up-regulated (p<0.05), T-bet, IFN-γ, IL-2and IL-4were markedly down-regulated (p<0.05), while IL-2was still way above normal level (p<0.01).Within Th1-related genes (T-bet、IFN-γ、IL-2), T-bet and IFN-y were down-regulated (p<0.05) after treatment in all subgroups. IL-2was also down-regulated after treatment in all subgroups, but in patients with a pre-treatment platelet count lower than10×109/L or a post-treatment platelet count higher than100×109/L, the difference had no statistical significance. Th2-related genes (GATA-3、 IL-4) had no significant changes in all subgroups. Within Treg-related genes (Foxp3、 TGF-β), Foxp3was significantly up-regulated (p<0.05) after treatment in patients with a pre-treatment platelet count lower than10×109/L. Interestingly, TGF-P showed a different pattern between old and young patients. It was down-regulated (p<0.05) among patients younger than60, while up-regulated in older patients.Compared to normal control, EOTAXIN, EOTAXIN-2, GM-CSF, G-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-11, IL-12p40, IP-10, MCP-1, M-CSF, RANTES, PDGF-B were2-fold over-expressed in newly-diagnosed patients. RANTES, G-CSF, PDGF-B, GM-CSF, M-CSF, IL-4, ICAM-1, IL-8were2-fold over-expressed in patients undergoing complete remission. EOTAXIN, EOTAXIN-2, IFN-γ, IL-1β, IL-2, IL-6, IL-11, IL-12p40, IL-7, IP-10, MCP-1were2-fold over-expressed in newly-diagnosed patients but not in patients undergoing complete remission.Conclusion:We found aberrant Th cell function and unbalanced Th cell subgrouping in ITP patients. Prednisone therapy could effectively tune Th cell subgroups to a suppressive pattern in newly-diagnosed ITP patients, which might involve suppression of Thl and Th2, and induction of Treg. Surveillance of Th cell immune status may become an effective method for evaluation of treatment and prediction of prognosis in ITP patients, and provide evidence for selection of second-line therapies as well. Capable of measuring cytokines in a high throughput mode using very few amounts of samples, protein microarray technology was an ideal method to screen for ITP-related cytokines.