| Objective
To investigate whether CD4+CD25+ regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID).
Materials and methods
Firstly, we investigated the dynamic frequency changes of the CD4+CD25+ T cells in the spleen and draining lymph nodes (LNs) after intracameral injection of OVA. The percentage of CD4+CD25+ T cells in the CD4+ T cells from the spleen and draining LNs was identified by flow cytometry (FCM) after cells were labeled with Fluorescence-conjuncted anti-CD3, anti-CD4 and anti-CD25 antibodies.
Secondly, we analyzed the expression of Foxp3 (the specific molecular marker for Treg cells) of purified CD4+CD25+ T cells and CD4+CD25– T cells from the spleen of mice with ACAID, positive and naïve control on day 7 after s.c. immunization using quantitative real-time PCR.
Thirdly, we evaluated the suppressive effect of MACS-purified CD4+CD25+ T cells from naïve mice on the anti-CD3-induced proliferation of CD4+CD25－ T cells by in vitro proliferation assay, The cell proliferation was determined by [3H] thymidine incorporation and MTT test. We further characterized the suppressive effect of purified CD4+CD25+ T cells from mice with ACAID on the proliferation of CD4+CD25－ T cells.
Fourthly, a blocking experiment was performed to further address the role of CD4+CD25+ T cells in the induction and maintenance of ACAID. Normal BALB/C mice were treated with anti-CD25 antibody to deplete CD25+ T cells immediately followed by injection of OVA into the anterior chamber, the obtained PBL, spleens and draining LNs were used for confirmation of the accuracy in depleting CD25 molecules, evaluation of ACAID and analysis of the frequency of CD4+CD25+ T cells after OVA inoculation.
Finally, to evaluate the expression of TGF- , IL-10 by purified CD4+CD25+ T cells and CD4+CD25– T cells, studies on this issue may greatly shed light on the regulatory mechanisms involved in ACAID. Freshly isolated CD4+CD25– and CD4+CD25+ cells from the spleen of each group were stimulated with plate-bound anti-CD3 and soluble anti-CD28. Culture supernatants were assayed for TGF-β and IL-10 expression by ELISA.
Statistics Data were analyzed using the SPSS11.5. Analysis of Variance (ANOVA)－dunnett-t test .Values are presented as means ± SEM. p values <0.05 were considered to show a significant difference.
Results
The induction and maintenance of ACAID in BALB/c mice were associated with increased frequency of Treg cells, enhanced suppression effect of CD4+CD25+ T cells on CD4+CD25－T cells. Moreover, expression of the mRNA for Foxp3 was higher in mice with ACAID as compared with negative and positive control. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber could significantly inhibit the induction of ACAID, indicating that CD4+CD25+ T cells potently suppressed Th1 response development and did play an important role in the development of ACAID. Furthermore, purified CD4+CD25+ T cells secreted IL-10, but not TGF-β1, indicating that except Th2-type cells, Treg cells may be another crucial source of this cytokine and IL-10 may be involved in the regulatory mechanism by which Treg cells regulate ACAID.
Conclusion
Altogether, the data reported here is the first demonstration that CD4+CD25+ T cells are involved in the development of ACAID, and play an important role in ACAID, which may have important implications with regard to the possible therapeutic manipulation of Treg cells in vivo.
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