Characterization of Ocular –MSH–Induced Regulatory T Cells

Abstract

Purpose: : Healthy aqueous humor promotes the induction of antigen–specific primed CD25⁺ CD4⁺ regulatory T (Treg) cells. This mechanism of immunoregulation is mediated by the neuropeptide α–melanocyte stimulating hormone (α–MSH) through the melanocortin 5 receptor (MC5r) on primed T cells. There are several types of Treg cells reported in the literature; therefore, we characterized the α–MSH–induced Treg cells to see whether they are similar to these reported Treg cells.

Methods: : The draining lymph nodes were collected from wild type or MC5r^(–/–) C57BL/6 mice immunized 7 day prior to desiccated M tuberculosis. The T cells were isolated by either a CD3 or a CD4 enrichment column. The T cells were stimulated through their T cell receptor (TcR) with anti–CD3ε antibody while being treated with 30 pg/ml of α–MSH under serum–free conditions. The expression of Treg cell markers CD25, CD44, CD62L, GITR, and CTLA4 were assayed 4 days later by two color flow cytometry gating on CD25⁺ cells and determining the percentage of the CD25⁺ cells expressing the other markers. The expression of FoxP3 protein was assayed by immunoblotting lysates of sorted α–MSH–induced Treg cells. Message for FoxP3 was assessed in CD4⁺ T cells by real–time PCR 4 hours after α–MSH treatment and TcR–stimulation.

Results: : There was 8–times more CD25⁺ CD4⁺ Treg cells in cultures of primed T cells TcR–stimulated and treated with α–MSH than in cultures of TcR–stimulated primed T cells not treated with α–MSH, and there was no effect of α–MSH on the CD4⁺ population of stimulated primed MC5r^(–/–) T cells. The α–MSH–induced CD25⁺ CD4⁺ Treg cells were CD44⁺, CD62L⁺, CTLA4⁺, but GITR^–. The α–MSH–induced Treg cells expressed FoxP3 protein, and had a 5–fold expression of FoxP3 mRNA 4 hours after α–MSH treatment to the 2.5–fold increase in T cells only TcR–stimulated.

Conclusions: : The α–MSH–induced Treg cells share some of the characteristics of other Treg cells described in the literature, but differ significantly to be considered unique. In addition, the enhancement of FoxP3 mRNA expression by α–MSH in the CD4⁺ T cells implies that the immune privileged ocular microenvironment has a mechanism to promote the expression of regulatory genes in activated T cells.

Keywords: immunomodulation/immunoregulation • immune tolerance/privilege • neuropeptides