Th Subsets Expression Changed With Antiphospholipid Antibodies

Chapter One The expression of Th cell subtypes in cultured PBMCs with different concentrations of aPLObjective:To evaluate the Thl/Th2/Th17/Treg cells expression in APS, and whether they are related to the aPL titers.Method:Peripheral mononuclear cells (PBMCs) were isolated from health doners, and incubated with different titers of aPL. After incubated48hours, PBMCs were collected, and the Th cell subsets were detected by Flow Cytometry and the transcription factors were detected by Real-time PCR.Results:It was showed that after incubated with aPL, there was a significant decrease in Thl frequencies and T-bet mRNA, a significant increase in Th2frequencies and GATA3mRNA. Th1/Th2ratio was decreased. The Th17frequencies and ROR-yt mRNA was increased, but Treg frequencies and Foxp3mRNA was decreased. And also we found the Th subsets expression were positively correlated with the antibodies titers, especially at higher concentrations.Conclusion:1. aPL may change Thl/Th2/Thl7/Treg expression, result Thl/Th2and Th17/Treg imbalance, and result immune disorders. All these may participate in the aPL mediated tissue damage. 2. The expression of Th cell subsets were correlated with the aPL titers. Chapter Two The expression of costimulatory molecules PD-L1in cultured PBMCs with aPLObjective:To evaluated the costimulatory molecules PD-L1expression with different titers of aPL, and to explore the possible mechanisms about Th cell differentiation disorder with PD-L1.Method:Peripheral mononuclear cells (PBMCs) were isolated from health doners, and incubated with different titers of aPL. After incubated48hours, PBMCs were collected, and the PD-L1was detected by Flow Cytometry and Real-time PCR. And we analysed the relationships between PD-L1expression and Th cells differentiation. We also compared the Th1/Th2/Th17/Treg cells expression with PD-L1-Ig pretreated or not by Flow Cytometry and Real-Time PCR.Results:It was showed that incubated with aPL can induce significant increase in PD-L1proten and mRNA expression (F=9.391, p=0.000; F=15.344,p=0.000), and were positively correlated with the antibodies titers (r=0.713,p=0.000; r=0.756, p=0.000).PD-L1protein expression has negative correlation with Thl frequencies, Treg frequencies and Th1/Th2proportion, but had no correlation with Th2frequencies and Th17frequencies. In the same, PD-Ll mRNA expression has negative correlation with T-bet mRNA, Foxp3mRNA and T-bet/GATA3proportion, but had positive correlation with GATA3mRNA and ROR-yt mRNA. However pretreated with PD-L1-Ig had no function in Th differentiation (p>0.05).Conclusion:1. aPL may result PD-L1upregulated, and were positive correlated with the aPL titers. The costimulatory molecules PD-L1may be play a role in the development of APS.2. The costimulatory molecules PD-L1may participate in Th cell differentiation disorder in APS.3. Block the costimulatory pathway of PD-L1had no function in Th differentiation. Chapter Three The expression of Th cell subtypes in incubated PBMCs with with aPL treated by Polysaccharide nucleic acid fraction of Bacillus Calmette GuerinObjective:To evaluate the positive mechanism of Polysaccharide nucleic acid fraction of Bacillus Calmette Guerin in treating APSMethod:Peripheral mononuclear cells (PBMCs) were isolated from health doners, and incubated with high titers of aPL and Polysaccharide nucleic acid fraction of Bacillus Calmette Guerin. After incubated48hours, PBMCs were collected, and the Th cell subsets were detected by Flow Cytometry and the transcription factors were detected by Real-time PCR.Results:It was showed that after treated with Polysaccharide nucleic acid fraction of Bacillus Calmette Guerin, there was significant increase in Th1frequencies and T-bet mRNA (p=0.000; p=0.000); but no different in Th2frequencies and GATA3mRNA (p>0.05); Th1/Th2ratio was significant increased; Treg frequencies and Foxp3mRNA was significant increased (p=0.016; p=0.024), but no different in Th17frequencies and ROR-yt mRNA (p>0.05).Conclusion:Polysaccharide nucleic acid fraction of Bacillus Calmette Guerin can effectively reversed Th cells differentiation, it could be make sense in treating APS.