Induction of antigen-specific immune tolerance by TGF-beta-induced CD4+Foxp3+ regulatory T cells

Abstract

Like natural CD4(+)CD25(+) Treg cells, TGF-beta-induced Treg cells also prevent allograft rejection in MHC-mismatched organ transplantation models. In analyzing this effect with greater detail, we determined that injection of TGF-beta-induced, alloactivated CD4(+)CD25(+) cells induces antigen-specific immune tolerance in vivo. Increased CD4(+)CD25(+) cells in recipients contribute to this immune tolerance. In addition, adoptive transfer of TGF-beta-induced CD4(+)CD25(+) cells did not result in significant toxic and side effects in recipients. These results indicate that TGF-beta-induced, alloactivated CD4(+)CD25(+) cells may provide a safe and effective approach to protect MHC-mismatched organ grafts from rejection in a clinical setting.

Keywords: Foxp3; Immunoregulation; TGF-β; regulatory T cells; transplant tolerance.

Figure 1

TGF-β is able to induce alloactivated CD4+ cells to express Foxp3. Naïve CD4+CD25- cells isolated from DBA/2 mice were stimulated with γ–irradiated (2000-rad) non-T cells from C57BL/6 mice in the presence of IL-2 (40 units/ml) with (CD4reg) or without TGF-β (2 ng/ml) (CD4con) for 5 days. Foxp3 and CD25 expression on CD4+ cells were determined by flow cytometry (A), Foxp3 mRNA were determined by RT-PCR and quantitative RT-PCR (B &C). Effect of TGF-β receptor I inhibitor on Foxp3 expression induced by TGF-β (D). TGF-β-induced Foxp3 production in wild type and TGF-β receptor II Knock out mice was determined by flow cytometry (E). Results were representative of three independent experiments (A-C). The values indicate the mean ± SEM of three separate experiments (D-E).

Figure 2

TGF-β-primed, alloactivated CD4+CD25+ cells display potent antigen-specific suppression. CD4con or CD4reg cells were generated as above and CD4+CD25+ subset was positively selected by AutoMACS separator. 20% of these CD4+CD25+ cells were added to syngeneic T cells and stimulated with γ–irradiated (2000-rad) non-T cells from C57BL/6 mice (H-2^b, specific antigen) or C3H (H-2^k, third party antigen) mice for three days. Proliferation was measured as uptake of H³-thymidine in triplicate cultures. Values indicate the mean ± SEM of three separate experiments.

Figure 3

Adoptive transfer of TGF-β-primed, alloactivated CD4+CD25+ cells induces immune tolerance in vivo. 5 million of CD4+CD25+ subset from CD4con or CD4reg cells was intravenously injected to syngeneic DBA/2 mice. After three weeks, T cells isolated from mice received CD4con or CD4reg cells were stimulated with γ–irradiated (2000-rad) non-T cells from C57BL/6 mice (H-2^b, specific antigen) or C3H (H-²k, third party antigen) mice for three days and T cell proliferation was determined as above. Values indicate the mean ± SEM of three mice. The experiment was repeated with similar results.

Figure 4

Increased CD4+CD25+ cells in recipients contribute to immune tolerance in vivo. Experiments were similarly conducted as Fig. 3 and CD4+CD25+Foxp3+ cells on the gate of CFSE- cells were counted in mice received from CD4con or CD4reg cells. T cells in these mice were depleted CD25+ subset and their proliferation against alloantigen was determined as Fig. 2. In some experiments, 10% of CD4+CD25+ subset was added to back and T cell proliferation was determined as above. Values indicate the mean ± SEM of four mice. The experiment was repeated with similar results.

Figure 5

Adoptive transfer of TGF-β-primed, alloactivated CD4+CD25+ cells to syngeneic mice does not lead to evident toxic effects in important organs. 10 million of CD4reg cells were intravenously injected to syngeneic mice and pathological characteristics were evaluated by H&E staining in three weeks after cell injection. Results were representative of two similar experiments.