Significance Of CD4⁺CD25⁺ CD127^low Regulatory T Cells And Notch1 Pathway In The Pathogenesis Of Aplastic Anemia

Objectives1.To examine the amount of CD4⁺CD25⁺CD127^lowregulatory T cells in peripheral blood of aplastic anemia patients in pre-treatment and post-treatment period.2.To detect the level of Notch1 mRNA and Foxp3 mRNA in peripheral blood of AA patients with RT-PCR.3.To analyze the correlations of Treg cells,Foxp3 mRNA and Notch1 mRNA.4.To investigate the significance and pathogenesis of Treg cells in patients with AA.Methods1.Peripheral blood were select from 29 aplastic anemia patients at active phase,14 AA patients at recovery phase and 11 AA patients at unrecovery phase,then the number of CD4⁺CD25⁺CD127^lowT cells and CD4⁺CD25⁺ T cells were examined with FACS and compared with normal controls.2.Detect the level of Foxp3 mRNA and Notch1 mRNA with semi-quantitative RT PCR:The mononuclear cells were separated from peripheral blood by ficoll.Total RNA of T cells was extracted.Inverse transcription and amplification of Foxp3 mRNA and Notch1 mRNA were done using one-step method.The production was detected using electrophoresis method and then was analyzed using imaging system and analysis software. The relative level of Foxp3 mRNA or Notch1 mRNA were accessed by the ratio of Foxp3 or Notch1 absorption factor andβ-actin absorption factor.3.Data were statistically treated by SPSS 13.0 and the correlations of Treg cells,Foxp3 mRNA and Notch1 mRNA were analyzed.Results1.The percentages of actived CD4⁺CD25⁺T cells in peripheral blood of AA patients at active phase and unrecovery phase(4.277%±0.680%,4.165%±0.598%,respectively)were significantly higher than those of normal controls(2.436%±0.834%)(P＜0.05);But the proportions of actived CD4⁺CD25⁺T cells in AA patients at recovery phase decreased to (2.576%±0.698%)(P＜0.05),no significal difference from those of control group.The number of CD4⁺CD25⁺CD127^lowT cells in AA patients at active phase and unrecovery phase(2.368%±1.166%,2.495%±1.097%,respectively)were significantly lower compared with that in normal controls(7.056%±2.676%)(P＜0.01)and AA patients at recovery phase (5.314%±1.024%)(P＜0.01);there is no significant difference between the latter two groups.2.The expression of FOXP3 mRNA in AA patients at active phase(0.260±0.011)is obviously lower compared with that in control group(1.307±0.011)(P＜0.01).After treatment,the expression of FOXP3 mRNA increased to(1.287±0.012)(P＜0.01),but there is no difference with that of normal controls.3.RT-PCR analysis showed that the expression of Notch1 mRNA in AA patients at active phase(0.018±0.005)is lower compared with that in control group (0.308±0.028)(P＜0.01).After treatment,the expression of Notchl mRNA increased to (0.281±0.013)(P＜0.01),but there is no difference with that of normal controls.4.In AA patients the percentages of peripheral CD4⁺CD25⁺CD127^lowT cells and the expression of Foxp3 mRNA were positively related to that of Notch1 mRNA(P＜0.01).ConclusionsThe decreased number and suppressive activity of CD4⁺CD25⁺CD127^lowregulatory T cells in the peripheral blood of patients with AA cause over-activation of autoreactive T cells and suppression of haematopoiesis.One of the pathogenesis maybe involve in the reduced expression of Notch1 in the target cells.The treatment with CsA contributes to increase the percentage of Treg cells obviously and suppress the activation of effective T cells,in order to help the recovery of haematopoiesis of bone marrow.