Singal Regulatory Mechanism Of Eye-derived Foxp3~+Treg Cells And The Relationship With Eye Immune Privilege

Purpose:To study whether CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells) will be induced within eye environment and the effect of c-Rel on eye-derived Treg cells.MethodsThe study of eye environment induce CD4+T cells to Treg cells in vitro and in vivo1) In vitro experiment:Using the spleen of WT B6 mice, prepare naive CD4+T cells, in different culture medium with anti-CD3 aqueous humor (3.0×)、0ng/ml、0.5 ng/ml、1ng/ml、4ng/ml、20ng/ml、100ng/ml TGFβ-1, naive CD4+T cells were were cultured. CD4-PE CD25-Cy5.5 Foxp3-FITC staining were performed. Flow cytometry was performed to test the percentage of Treg cells Mean fluorescence intensity (MFI) was observed considerable differences in the levels.2) In vivo experiment:OVA-TCR-Tg B6 mice. Inject 2ul of the 20 mg/ml ovalubmin (OVA) into the anterior chamber of the right eye, The eye (including the cornea iris, ciliary body, retina, choroids, aqueous humor etc.) was minced with scissors and shaken in medium supplemented with collagenase type D. CD4-PE CD25-Cy5.5 Foxp3-FITC staining were performed. Flow cytometry was performed to test the Treg cells of treated eye and draining LN, blood and spleen.The function of c-Rel in eye-derived Treg cells1) In vitro experiment:Aqueous humor of WT B6 cultured c-Rel-/- /B6 mice naive CD4+T cells and WT B6 mice naive CD4+ T cells. Flow cytometry was performed to test the percentage of Treg cells in different group.2) In vivo experiment:c-Rel -/-/OVA-TCR-Tg B6 mice. Inject 2ul of the 20 mg/ml ovalubmin (OVA) into the anterior chamber of the right eye, The eye (including the cornea iris, ciliary body, retina, choroids, aqueous humor etc.) was minced with scissors and shaken in medium supplemented with collagenase type P. Flow cytometry was performed to test the Treg cells and Th17 cells.real-time PCR test Foxp3 mRNA、IL-17 mRNA in knockout and WT group.The realtationship of c-Rel singal pathway with immune rejection after PKPA total of 60 BALA/c mice received penetrating keratoplasty (PKP) were randomly divided into two groups—PKP group(A)、rotationalautokeratoplasty(RAP)(B). The donor buttons were from C57BL/6 mice. After surgery, the rejection time of allograft was observed by slit-lamp. The eye’s Foxp3mRNA、Th17mRNA、c-relmRNA of rejector group and acceptor group were detected by Real-time RT-PCR.Result1. In aqueous humor (AqH) group the percentage of Treg cells is 8.4%±1.2%. The function of induction of Tregs in 3×AqH is comparable to 0.5 ng/ml TGFβ-1 group.The Foxp3 MFI in AqH is arrive to70±2, more than 20ng/ml TGFβ-1 group (MFI=66±3). Very few CD4+T cell and CD4+Foxp3+ cells present within WT normal eye, and the percentage of Treg cells in normal eye is 5.4%±0.2%. After OVA treatment 2d,4d,6d, the percentage of Treg cells is 60.2%±3.5%,32.2%±2.8%、28.4%±2.2%. The percentage of Treg cells in draining LN is:5.8%±0.8%、5.1%±0.6%,3.8±0.3%.After OVA treatment the percentage of eye’s Treg cells increased significantly (p<0.05). The eye’s Treg cells were higher than LN’s Treg cells (p<0.05)2. After 48h culture with 6×AqH, the percentage of Treg cells in WT group was 2.4%±0.3%, the percentage of Treg cells in c-Rel-/- group was 1.0%±0.2%, the difference is significantly (p<0.05). In vivo experiment showed the Treg cells, CD4+IL-17+ cells decreased in c-Rel-/- group(p<0.01). Real-time PCR demonstrated the Foxp3 mRNA and IL-17 mRNA decreased significantly in c-Rel-/- group (p<0.01)。3. Real time PCR analysis of IL-17mRNA Foxp3 mRNA and Foxp3 mRNA expression in acceptor is higher than in rejector. The ratio of IL-17mRNA/Foxp3mRNA in acceptor (0.25) is lower than in rejector (0.57). The Foxp3 mRNA expression changed as the c-RelmRNA expression. ConclusionIn eye immune response, there are a lot of CD4+ CD25+ Foxp3+ regulatory T cells in the eye. The Treg cells come from not only draining LN but also induced in the eye. Tregs can be induced in the eye, and with higher function of immunosuppression. c-Rel play an important role in eye-derived Treg cells and regulate the eye immune privilege.