Thrombospondin-1-Dependent Regulation of Foxp3-Positive Regulatory T Cells by TGFβ-Treated Antigen Presenting Cells

Abstract

Purpose: : Immune deviation induced by eye-derived TGFβ-exposed antigen presenting cells (APCs) is mediated by regulatory T cells. These regulatory T cells (Treg) include CD4⁺ cells capable of secreting increased amounts of TGFβ and decreased levels of IFNγ and are known to suppress the proliferation of proinflammatory T cells as well as delayed type hypersensitivity response (DTH). Increased expression of thrombospondin-1 (TSP1) by TGFβ-exposed APCs is essential for their ability to induce immune deviation and contributes significantly to the generation of Tregs. We now examine Foxp3 expression by these Tregs and determine role of TSP1 in their generation.

Methods: : Thioglycollate-elicited peritoneal exudate cells (PECs) from C57BL/6 (Wild-type) or TSP1null mice were used as APCs. These cells, pulsed with ovalbumin, were cultured overnight in the presence or absence of TGFβ (5 ng/ml) and after washing with fresh medium were co-cultured with T cells from OT-II mice. Expression of Foxp3 and intracellular IL-17 was evaluated in CD4⁺CD25⁺ T cells by flow cytometry. Culture supernatants collected at 48 hr were analyzed for the presence of IL-17 by ELISA. Expression of TSP1 by peritoneal macrophages after TGFβ treatment was examined by flow cytometry.

Results: : Intracellular expression of TSP1 by TGFβ-treated macrophages was significantly increased as compared to that detected in untreated macrophages. The number of CD4⁺CD25⁺Foxp3⁺ T cells increased among T cells co-cultured with TGFβ-treated wild-type APCs as compared to untreated APCs. Also, T cells activated with TGFβ-treated wild-type APCs secreted decreased amount of IL-17 as compared to those activated by untreated APCs. In the absence of TSP1, TGFβ-treated APCs failed to increase the number of CD4⁺CD25⁺Foxp3⁺ T cells as compared to those detected in T cells cultured with untreated TSP1deficient APCs. Also, an increased number of IL-17 expressing cells were detectable in T cells activated by TGFβ-treated TSP1 deficient APCs than those activated by untreated APCs. These results are consistent with the failure of TSP1 deficient APCs to suppress DTH response upon their exposure to TGFβ.

Conclusions: : Regulatory T cells involved in immune deviation induced by TGFβ-treated APCs include immunosuppressive CD4⁺CD25⁺Foxp3⁺ Tregs and APC-derived TSP1 was found to regulate their presence while preventing pathogenic Th17 effectors. Our results indicate a significant role of TSP1 in the generation of Tregs capable of suppressing an inflammatory response.

Keywords: immunomodulation/immunoregulation • antigen presentation/processing • ACAID