Aijun Zhang

Gamma-ray resistance of regulatory CD4+CD25+Foxp3+ T cells in mice.

Abstract CD4+CD25+ regulatory T cells (Treg cells) are an important subset of T cells for keeping proper immune responses and tolerance. However, the effects of γ radiation on CD4+CD25high Foxp3+ Treg cells have not been examined previously. In the present study, we compared the sensitivity of mouse CD4+CD25high Foxp3+ Treg cells and CD4+CD25− T cells to γ radiation in vitro and in vivo. After C57BL/6 mice received a whole-body dose of 5 Gy γ rays, the numbers of lymphocyte subsets in blood, lymph nodes, spleens and thymuses clearly decreased. However, γ radiation significantly enhanced the ratios of CD4+CD25high Treg cells and CD4+CD25high Foxp3+ Treg cells to CD4+ T cells in the blood, lymph nodes, spleens and thymuses of mice. More dead cells were observed in CD4+CD25− T cells than in CD4+CD25high Treg cells or CD4+CD25high Foxp3+ Treg cells when the cells were irradiated in vitro, indicating that CD4+CD25high Foxp3+ Treg cells are more resistant to γ radiation than other T cells. Moreover, a higher expression of Bcl-2 in CD4+CD25high Treg cells was detected compared with that in CD4+CD25− T cells. CD4+CD25+ Treg cells from irradiated mice were functional, though their immunosuppressive ability was somewhat impaired compared to those from nonirradiated mice as determined by an in vitro assay. These results indicate that mouse CD4+CD25+ Treg cells and CD4+CD25− T effector cells have different sensitivities to γ radiation in mice.

The different effects of indirubin on effector and CD4+CD25+ regulatory T cells in mice : potential implication for the treatment of autoimmune diseases

CD4+CD25+ regulatory T (Treg) cells play an essential role in the induction and maintenance of peripheral self-tolerance. Indirubin, a traditional Chinese medicine, was clinically used in the treatment of chronic myelocytic leukemia as well as some autoimmune diseases, including Alzheimer’s disease, diabetes, and so on. The effects of indirubin on CD4+CD25+Treg cells, which play a critical role in controlling autoimmunity, have not been addressed. In the present study, we observed the cell levels, phenotypes, and immunoregulatory function of CD4+CD25+Treg cells in indirubin-treated mice. Treatment with indirubin significantly enhanced the ratios of CD4+CD25+Treg cells or CD4+CD25+Foxp3+Treg cells to CD4+T cells in peripheral blood, lymph nodes, and spleens (P < 0.01 compared with control mice). CD4+CD25+Foxp3+Treg cells to CD4 single positive cells in the thymi of indirubin-treated mice were significantly higher than those in control mice. Furthermore, splenic CD4+CD25+Treg cells in indirubin-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4+CD25+Treg cells in vitro. The present studies indicate that CD4+CD25+Treg cells are more resistant to indirubin than effector T cells in vivo. The selectively enhanced CD4+CD25+Treg cell levels by indirubin made host to be more favorable for immune tolerance induction, which opened one possibility for indirubin to treat autoimmune diseases.

2-Gy whole-body irradiation significantly alters the balance of CD4+ CD25- T effector cells and CD4+ CD25+ Foxp3+ T regulatory cells in mice.

CD4+CD25+ T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4+CD25+Foxp3+ Treg cells has not been determined. The proportion, phenotypes and function of CD4+CD25+ Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4+CD25+ Treg cells and CD4+CD25+Foxp3+ Treg cells to CD4+ T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4+CD25+ Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4+CD25− T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4+CD25+Foxp3+ Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4+CD25+Foxp3+ Treg cells than in CD4+CD25− T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4+CD25+Foxp3+ Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4+CD25+Foxp3+ Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.

The significantly enhanced frequency of functional CD4+CD25+Foxp3+ T regulatory cells in therapeutic dose aspirin-treated mice ☆

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, produced in the thymus or periphery as a functionally mature T cell subpopulation, play pivotal roles in maintenance of self-tolerance and negative regulation of immune responses. Aspirin (ASA) is widely used to reduce pain, the risk of cardiovascular diseases and allo-graft rejection. However, the effect of ASA on CD4(+)CD25(+)Foxp3(+) Treg cells has yet to be determined. The frequency, phenotype and immunosuppressive function of CD4(+)CD25(+)Foxp3(+) Treg cells were detected in BALB/c mice treated with low or high doses of ASA for 4 weeks. ASA significantly decreased the percentage and number of CD4(+) T cells in the periphery, while ASA remarkably increased the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells in CD4(+)T cells. The total cell numbers of thymocytes were significantly decreased in ASA-treated mice, but the number of CD4(+) CD25(+)Fxop3(+) cells and its ratio in CD4(+)CD8(-) thymocytes were markedly enhanced in the thymi of ASA-treated mice. The phenotype of CD4(+)CD25(+) Treg cells, including the expressions of CD44, CD45RB, CD62L, CD69, GITR and CTLA-4, did not show detectable changes in ASA-treated mice. CD4(+)CD25(+) Treg cells in ASA-treated mice exhibited unimpaired immunosuppressive function on CD4(+)CD25(-) T effector cells. ASA significantly enhanced the frequency of functional CD4(+)CD25(+)Foxp3(+) Treg cells in mice in a therapeutic dose range. The different effects of ASA on CD4(+)CD25(+)Foxp3(+) Treg cells and CD4(+)CD25(-) T cells may potentially make hosts susceptible to tolerance induction which would be beneficial for tolerance induction in patients with autoimmune diseases or allo-grafts. This study may have potential impacts in the clinical application of ASA.

Deficiency of mouse CD4+CD25+Foxp3+ regulatory T cells in xenogeneic pig thymus-grafted nude mice suffering from autoimmune diseases.

Xenogeneic thymus transplantation can efficiently induce specific immune tolerance to donor antigens in athymic recipients. However, many nude mice suffer from autoimmune diseases (AID) for over 10 weeks after xenogeneic thymus transplantation. CD4+CD25+Foxp3+ regulatory T (Treg) cells were recently determined to play a pivotal role in keeping immune tolerance in humans and mice. Thus, we investigated this subpopulation of Treg cells in the periphery of pig thymus-grafted nude mice suffering from AID. Our results showed that the expression of Foxp3, CTLA-4 and GITR on mouse CD4+CD25+ T cells and the ratio of CD4+CD25+Foxp3+ Treg cells to CD4+ T cells were significantly decreased in the periphery of pig thymus-grafted nude mice suffering from AID, compared with healthy pig or mouse thymus-grafted nude mice. Furthermore, mouse CD4+CD25+ T cells in pig thymus-grafted nude mice suffering from AID showed more severe deficiency in immunosuppressive function compared with the counterpart in xenogeneic pig or syngeneic thymus-grafted nude mice without AID. Thus, the decreased frequency, altered phenotype and functional deficiency of mouse CD4+CD25+ Treg cells in pig thymus-grafted nude mice may contribute to the development of AID in this model.

Development of mouse CD4+CD25+Foxp3+ regulatory T cells in xenogeneic pig thymic grafts

Xenogeneic thymus transplantation is an effective strategy to induce tolerance to donors mainly by clonal depletion of reactive T cells. Recent studies have shown that functional mouse CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) could efficiently populate in the periphery of athymic mice after grafting with neonatal pig thymus. However, it is still unknown whether xenogeneic thymus grafts could directly support the development of mouse CD4(+)CD25(+)Foxp3(+) Treg cells as an autogeneic counterpart. Our results show that the percentages of mouse CD4(+)CD8(-)CD25(+) thymocytes are similar among auto- and xenogeneic thymic grafts in thymic mouse recipients. Mouse CD4(+)CD8(-)CD25(+) thymocytes maturing in xenogeneic thymic grafts exhibit similar expressions of Foxp3, TCR, CTLA-4 and GITR as those in autogeneic thymic grafts. Moreover, mouse CD4(+)CD8(-)CD25(+) thymocytes maturing in xenogeneic thymic grafts showed a significant immunosuppressive function on the proliferation of CD4(+)CD25(-) T cells stimulated with Con A or allogeneic antigens, although they showed weaker effects than those maturing in autogeneic thymic grafts. Therefore, the present data provides direct evidence for the ability of xenogeneic porcine thymus grafts to support the development of mouse naturally occurring CD4(+)CD25(+)Foxp3(+) Treg cells.