Abstract
Purpose: :
We recently reported that functional Foxp3+CD4+CD25+(Bright) "natural" regulatory T cells (nTreg cells) are abundant in naïve rabbit conjunctiva and suppress virus-specific CD4+ and CD8+ conjunctival effector T cells (Teff) during ocular HSV1 infection (Nesburn A. B. et al., J. Virol. 81:7647-61, 2007) but little is known about the regulatory mechanisms of these nTreg cells in ocular HSV-1 infection. Although there are reports that Toll-like receptors (TLRs) modulate Treg function, the identification and biological role of TLRs, particularly on conjunctiva resident nTreg cells, are unexplored.
Methods: :
60 conjunctivas from naive rabbits were used to purify nTreg cells by immuno-magnetic sorting. The existence of TLRs on purified nTreg cells were identified by FACS and compared with autologous conjunctiva resident antigen presenting cells (APCs). The in vitro proliferation of stimulated nTreg cells were measured by CFSE labeling of nTreg cells and tested in the presence and absence of TLR2 ligand LTA and TLR9 ligand CpG2007. The in vitro suppressive property of nTreg cells were measured by co-culturing nTreg with CFSE labeled Teff cells at a ratio of 1:2 and tested in the presence and absence of TLR2 ligand LTA and TLR9 ligand CpG2007. The IFN-γ, IL-10 and TGF-β expression profile of LTA treated nTreg cells were determined by real time PCR.
Results: :
We found: (1) conjunctiva resident nTreg cells express high levels of TLR2 and TLR9 and relatively low levels of TLR3, TLR4 and TLR8. (2) The expression level of TLR2 and TLR9 on conjunctiva nTreg cells are comparable with the autologous CD11b+ antigen presenting cells (APCs), which are well known for TLR expression. (3) TLR2 ligand, Lipoteichoic Acid (LTA), induced proliferation of nTreg cells in vitro and the addition of autologous APCs further improved the LTA-mediated proliferation. (4) In contrast, TLR9 ligand, CpG2007, inhibited the proliferation of nTreg cells in vitro, and the addition of autologous APCs did not improve the proliferation of nTreg cells. (5) LTA partially released the suppressive activity of nTreg cells on conjunctival Teff cells. (6) Finally, TLR2 ligand LTA induced the expression of IFN-γ and IL-10 mRNA, but not TGF-β mRNA in proliferated nTreg cells.
Conclusions: :
This is the first report describing that conjunctiva resident nTreg cells express high levels of TLR2 and TLR9 and the ability of the TLRs to interact with their specific ligand and modulate the ocular mucosal Teff cell responses in vitro.
Keywords: herpes simplex virus • immunomodulation/immunoregulation • receptors