Numerical and functional defects in CD8+ CD28- T-suppressor lymphocytes from patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder, and loss of immune tolerance has been implicated in ITP pathogenesis. CD8+ CD28- suppressor (Ts) cells have an immunosuppression function and are involved in several autoimmune disorders. However, the role of Ts cells in ITP is currently not clear. Here, flow cytometry was used to detect the CD8+ CD28- CD127- proportion, which was decreased in active ITP patients compared with that of controls. Function analysis showed that immunosuppression of CD8+ CD28- Ts cells in ITP patients was impaired. Mechanistic studies have shown that CD8+ CD28- Ts cells from controls can downregulate CD80 and upregulate LILRB4 (ITL3) and LILRB2 (ILT4) expression on CD14+ monocytes, whereas these abilities were not found in Ts cells from ITP patients. Furthermore, Inducible T-cell costimulatory (ICOS) expression on the Ts cell surface after activation was decreased whereas programmed death 1 and interleukin 10 expression was not changed in ITP patients compared with those of controls. In summary, the down-regulated quantity and function of Ts cells in active patients indicated that a Ts defect was involved in ITP. Moreover, decreased ICOS expression and the loss of the ability to regulate co-stimulator expression on antigen-presenting cells partly explained the defective Ts-mediated suppression.

Upregulation of CD72 expression on CD19+ CD27+ memory B cells by CD40L in primary immune thrombocytopenia.

CD72 is a co-receptor of B cells and regulates B cell activation. Although aberrant expression of CD72 has been reported in primary immune thrombocytopenia (ITP), it is uncertain whether this aberrant expression is restricted to specific B cell subsets. Furthermore, the mechanisms that regulate CD72 expression are unknown. In this study, we found higher frequency of CD19+ B cells, CD19+ CD27+ memory B cells and lower frequency of CD19+ CD27- naive B cells in active ITP patients compared with controls and patients in remission. CD72 expression on CD19+ CD27+ cells was upregulated in active ITP patients and correlated with platelet count and anti-platelet autoantibodies. In vitro, CD40L could specifically induce CD72 upregulation on CD19+ CD27+ B cells. In combination with CD40L, interleukin (IL) 10 and BAFF (also termed TNFSF13B) further enhanced CD72 expression on CD19+ CD27+ B cells, whereas IL21 reduced CD72 upregulation. CD72mRNA expression after CD40L stimulation was increased in ITP patients and controls. Significant increase of CD40L on CD4+ T cells was correlated with CD72 expression on CD19+ CD27+ B cells in ITP patients. In conclusion, upregulation of CD72 expression on CD27+ memory B cells might take part in the pathogenesis of ITP. Elevated CD40L on CD4+ cells combined with cytokines might contribute to the upregulation of CD72 expression on CD27+ memory B cells.

Increased plasma sCXCL16 levels may have a relationship with Th1/Th2 imbalance in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is a disease of autoimmunity in which there are Th1/Th2 imbalance and disordered cytokine profiles. CXC chemokine ligand 16 (CXCL16) was proved to implicate in some autoimmune diseases. Our research aimed to determine plasma soluble CXCL16 (sCXCL16) levels and its effects in ITP. We used ELISA to measure plasma sCXCL16, IFN-γ and IL-4 and flow cytometry to determine expression of CXCR6 on lymphocyte subsets. We used real-time PCR to detect the CXCL16 and CXCR6 mRNA expression. Additionally, plasma sCXCL16, CXCL16 and CXCR6 mRNA levels of 8 patients were monitored before and after treatment. We found that patients with active ITP had higher circulating sCXCL16 in plasma than healthy controls and patients in remission. Meanwhile, negative relationships between sCXCL16 and platelet count, IL-4 and positive relationships between sCXCL16 and IFN-γ, IFN-γ/IL-4 ratio were observed. Besides, expression of CXCR6 on lymphocyte subsets and mRNA levels of CXCL16 and CXCR6 were all increased in active ITP. Additionally, plasma sCXCL16 and IFN-γ levels and CXCR6 mRNA expression were down-regulated after effective treatment compared with those before treatment. Thus, increased plasma sCXCL16 might be implicated in the pathogenesis of ITP and have a relationship with Th1/Th2 imbalance.

Stromal cell-derived factor-1 rs2297630 polymorphism associated with platelet production and treatment response in Chinese patients with chronic immune thrombocytopenia.

Stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in megakaryopoiesis and platelet production. SDF-1 rs2297630 is a functional polymorphism in linkage disequilibrium with other functional variants in SDF-1. This study aimed to investigate the role of SDF-1 rs2297630 in chronic ITP. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing. Immature platelet fraction (IPF) was performed using Sysmex XE-2100. Anti-platelet autoantibodies were assayed by enzyme-linked immunosorbent assay. The main characteristics at diagnosis and the outcome of chronic ITP in 201 Chinese patients were retrospectively reviewed. There was no significant difference in either genotype or allelic distribution between ITP patients and the controls (p = 0.114; p = 0.787). However, both heterozygote (GA) and homozygote minor allele (AA) patients had significantly increased megakaryocyte quantity compared to homozygote genotype (GG) patients at diagnosis (p = 0.011). The mean IPF values of GA and AA genotype patients were higher than those observed in the GG genotype patients when platelet counts ≤50 × 10(9)/L at diagnosis (p = 0.007). Patients with GA and AA genotype showed a higher response rate to standard treatments than patients with GG genotype (p < 0.001). In particular, GA and AA genotype patients had a significantly increased chance of responding to steroids, intravenous immunoglobulin (IVIG), and thrombopoietin analogs (p = 0.007; p = 0.029; p = 0.034, respectively). No significant difference was found between anti-platelet antibodies and genotypes (p = 0.296). In summary, the SDF-1 rs2297630 was associated with platelet production and treatment response in Chinese patients with chronic ITP.

Decreased TLR4 expression on monocytes may cause regulatory T cells abnormality in patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an autoimmune disease with many immune dysfunctions including T helper type 1 cell (Th1) polarization and regulatory T cells (Tregs) deficiency. This study aimed to determine the effects of TLR4 on Treg differentiation and the cytokine production of peripheral blood mononuclear cells (PBMCs) from patients with ITP. We found that expression of TLR4 on monocytes was significantly decreased in patients with active ITP than that in healthy controls and it had positive correlation with platelet count. However, there was no expression of TLR4 on CD4+ T cells. The result of further experiments in vitro showed that lipopolysaccharide (LPS) stimulation could enhance TLR4 expression on monocytes. Additionally, activation of TLR4 with LPS could promote differentiation of Treg cells and anti-TLR4 attenuated this effect. There was no significant difference about Th17 cells among three subgroups. However, the Th17/Treg cell ratio was decreased after stimulation with LPS and increased with anti-TLR4. Moreover, activation of TLR4 with LPS could significantly promote the secretion of interleukin-10 (IL-10) and transforming growth factor-ß1 (TGFB1), while anti-TLR4 significantly suppressed the secretion of them. Nevertheless, the secretion of IL-17A did not reach the statistical difference among three subgroups. In summary, decreased TLR4 appears to cause Tregs abnormality in ITP by modulating Tregs differentiation and immunoregulatory cytokines.

[Prediction of infections within 6 months of the initial diagnosis in adults with immune thrombocytopenia by absolute lymphocyte count].

OBJECTIVE: To explore incidence, risk factors and prognosis of the first 6 months infectious events in adults with newly diagnosed primary immune thrombocytopenia (ITP), and evaluate the value of initial absolute lymphocyte count (ALC) in predicting infection. METHODS: The initial clinical records and infectious events during 6 months of 217 adult with newly diagnosed ITP were retrospectively analyzed. Statistical methods were used to analyze risk factors of the 6 months infections in adults ITP, the prediction of ALC in risk of infection, and the association of ALC and prognosis. RESULTS: Infection rate of ITP patients accepting therapy within 6 months after the initial diagnosis was 13.8% (30/217), and infection rate of patients ≥ 60 years of age 25% (14/56). Multivariate unconditioned Logistic analysis showed that gender and ALC were independent risk factors for the 6 months infection of ITP patients (P<0.05, 95% CI 1.150-7.298, OR 2.722 and P<0.001, 95% CI 6.802-80.749, OR 23.436). Cutoff value of ALC was 1.225 × 109/L, sensitivity and specificity of its value were 0.866 and 0.700 respectively. Infection rate of ALC>1.225 × 109/L in adult ITP was lower than of ALC ≤ 1.225 × 109/L (5.3% vs 45.7%, χ² = 49.151, P<0.001). Furthermore, persistent recovery and the 1-year mortality rate after diagnosis had no difference among patients of different ALC (28.0% vs 26.0%, χ² = 0.071, P>0.05, and 98.6% vs 97.8%, χ² = 0.095, P>0.05). There were no significant differences in persistent recovery in patients with and without infection (30.0% vs 27.3%, χ² = 0.096, P>0.05). The 1-year mortality rate after diagnosis was significantly lower in those patients who developed an infection (93.3% vs 99.3%, χ² = 4.607, P<0.05). CONCLUSION: Initial ALC was an independent risk factor of 6 months infection in adult ITP. It could be a predictive index of infection within 6 months of the initial diagnosis in ITP patients. Infection as an important factor affected the survival of ITP patients.

Elevated Semaphorin 5A correlated with Th1 polarization in patients with chronic immune thrombocytopenia.

BACKGROUND: Primary immune thrombocytopenia (ITP) is an immune-mediated disorder in which cellular immunity deficiency and disturbed cytokine profiles have been found. Semaphorin 5A (Sema5A) has been showed to be implicated in cellular immune response. We aimed to evaluate the role of Sema5A in patients with chronic ITP. METHODS: Plasma levels of Sema5A, T helper (Th) cytokines (interferon [IFN] -γ,interleukin [IL]-4,IL-17A) were determined by enzyme-linked immunosorbent assay (ELISA) in ITP patients and healthy controls. Using real-time quantitative polymerase chain reaction (RT-PCR), mRNA levels of Sema5A and its receptor plexin-B3, plexin-A1 in peripheral blood mononuclear cells(PBMCs)were studied in all subjects. Specific anti-platelet autoantibodies were measured by the Pak Auto method. The dynamic change of plasma Sema5A and mRNA levels of its receptors was measured in 9 patients after effective therapy. RESULTS: Plasma Sema5A levels were significantly increased in active patients with chronic ITP compared to patients in remission and healthy controls. Elevated levels of Sema5A were found positively correlated with higher levels of plasma IFN-γ, IFN-γ/IL-4 ratio and negatively correlated with lower levels of plasma IL-4, platelet counts in ITP patients. The mRNA plexin-B3 was decreased in active ITP patients and inversely correlated with plasma Sema5A levels. Additionally, plasma levels of Sema5A and IFN-γ were reduced with up-regulation of plexin-B3 expression after effective treatment. CONCLUSIONS: Our data demonstrated elevated plasma Sema5A in chronic ITP patients might be involved in Th1 polarization by down-regulating receptor plexin-B3 expression and correlated with disease activity.

[Application of immature platelet fraction absolute immature platelet fraction and thrombelastograph on assessment of bleeding risk in patients with immune thrombocytopenia].

OBJECTIVE: To explore the clinical value of immature platelet fraction (IPF), absolute immature platelet fraction (A- IPF) and thrombelastograph (TEG) on assessment of bleeding risk of immune thrombocytopenia (ITP). METHODS: two hundred and seventy- one patients with ITP were assessed based on ITP-BAT bleeding grading system. IPF, A-IPF were determined in 271 patients ,TEG in 125 patients. The correlations between bleeding grades and IPF, A-IPF, variables of TEG in subgroups were analyzed by statistical method. The predictive value of IPF, A-IPF, and variables of TEG on bleeding risk of ITP patients was evaluated. RESULTS: There were no significant differences in bleeding degree in all patients with different gender and disease stage (P>0.05). Mild bleeding rate in children was higher than that in adult (P<0.05). PLT inversely correlated with bleeding grade for the entire cohort (P<0.001). In all subjects, PLT< 30 × 109/L and pediatric cohorts with PLT< 30 × 109/L, PLT were negatively correlated with IPF (P<0.05), positive correlated with A-IPF (P<0.001) and the maximum amplitude (MA (P<0.05). Bleeding grades were significantly correlated with IPF, A-IPF, MA in all subjects and patients with PLT< 30 × 109/L (P<0.001). IPF, A-IPF and MA did not correlate with bleeding grades in children with PLT< 30 × 109/L (P>0.05). ROC curve analysis revealed IPF, A-IPF and MA had better predictive value (AUC 0.745, 0.744, 0.813, P<0.001). Multivariate analysis showed that IPF and MA were independence factors for predicting bleeding risk in ITP patients and comprehensive predictive value was higher (AUC 0.846, P<0.001) than single variable. CONCLUSION: IPF, A-IPF and MA could accurately evaluate bleeding risk in ITP patients. It may be considered as reference index of the treatment and observation index of curative effect.

[Clinical significance of ITP-BAT bleeding grading system for patients with immune thrombocytopenia].

OBJECTIVE: To explore the clinical significance, reliability and responsiveness of ITPBAT bleeding grading system for patients with immune thrombocytopenia (ITP). METHODS: One hundred and eighty-three patients with ITP were assessed by using of ITP-BAT bleeding grading system. Test-retest reliability, responsiveness of ITP-BAT bleeding grading system and association between bleeding grades and platelet counts, age, gender, disease stage were analyzed. RESULTS: Bleeding degree of ITP patients and the platelet count were negatively correlated (r=- 0.744, P<0.01) and bleeding degree increased significantly with platelet counts below 20×109/L (χ²=82.40，P<0.01). Mild bleeding rate in children was 68.5%, higher than that in adult(χ²=8.839，P<0.01), and severe bleeding rate in the elderly was 14.3%, higher than that in non-elderly(χ²=7.056，P<0.01). There were no significant differences in bleeding degree in patients with different gender and disease stage (χ²=4.922, P>0.05 and χ²=3.411, P>0.05). Bleeding grades before and after treatment had more significant difference(Z=-6.61, P<0.01). Scoring consistency of two doctors was 66.1% (κ=0.561), and scoring consistency of the same doctor was 94.7% (κ=0.874). CONCLUSION: ITP-BAT bleeding grading system in China has good validity and responsiveness, closely related to clinical indicators. It is sensitive to the variation of the hemorrhage in patients. ITP-BAT could be used as a reference index of the treatment, and also be used as an observation index of curative effect.