Conjunctiva Resident CD4+CD25+ Regulatory T cells Express Toll Like Receptor (TLR)2 and TLR9 and Their Respective Ligands Lipoteichoic Acid and Cytosine-Phosphate-Guanine, Enhance Their Suppressive Capacity

Abstract

Purpose: : We recently reported that functional Foxp3+CD4+CD25+^(Bright) "natural" regulatory T cells (T_reg cells) are abundant in naïve rabbit conjunctiva, and suppress virus-specific CD4+ and CD8+ effector T cells during ocular HSV1 infection. Although TLRs can modulate T_reg function, their role in conjunctiva T_reg function remains to be elucidated.

Methods: : 60 conjunctivas were harvested from naive rabbits. T_reg cells were purified from conjunctivas by immuno-magnetic sorting and TLR profiles examined by FACS. Purified T_reg cells were exposed in vitro to TLR ligands, LTA (TLR2), LPS (TLR4) and CpG (TLR9) and their stimulation was assessed by CFSE. The effect of adding antigen presenting cells (APCs) to TLR-ligand treated T_reg cells was determined. To evaluate the role of T_regs on ocular surface immune responses in vivo, naïve rabbits were depleted of T_regs by sc injections of cyclophosphamide (50 µg; x6) and then immunized by topical ocular applications of 100 ug HSV-gD peptides + 25 ug TLR9 ligand (CpG) every 10 days (x3). Non-depleted, immunized rabbits were used as controls. Ten days later, immune T effector cells (T_eff cells) were harvested and stimulated in vitro with the same HSV-gD peptides and T_eff cell responses measured.

Results: : Conjunctival T_reg cells expressed high levels of TLRs 2 and 9, but not TLRs 3, 4 or 8. T_reg cell proliferation was induced by TLR ligands LTA (TLR2) and CpG (TLR9), but not by LPS (TLR4). Addition of autologous APCs improved TLR-mediated proliferative responses. Cyclophosphamide significantly depleted local (conjunctiva) and systemic (PBMC) T_reg cells, while CD8+ T cell counts were unaffected. Depletion of T_reg cells enhanc

Conclusions: : This is the first report showing that TLRs 2 and 9 are highly expressed on conjunctiva T_reg cells. They were functionally active and acted with APCs to fine tune HSV-specific ocular mucosal T_eff cell responses. Thus, depletion of conjunctival T_reg cells or interference with TLR2 and TLR9 function may be useful to enhance the efficacy of topical ocular immunization.

Keywords: conjunctiva • herpes simplex virus • immunomodulation/immunoregulation