Deficiency in NOD antigen-presenting cell function may be responsible for suboptimal CD4+CD25+ T-cell-mediated regulation and type 1 diabetes development in NOD mice.

Various defects in antigen-presenting cells (APCs) and T-cells, including regulatory cells, have been associated with type 1 diabetes development in NOD mice. CD4(+)CD25(+) regulatory cells play a crucial role in controlling various autoimmune diseases, and a deficiency in their number or function could be involved in disease development. The current study shows that NOD mice had fewer CD4(+)CD25(+) regulatory cells, which expressed normal levels of glucocorticoid-induced tumor necrosis factor receptor and cytotoxic T-lymphocyte-associated antigen-4. We have also found that NOD CD4(+)CD25(+) cells regulate poorly in vitro after stimulation with anti-CD3 and NOD APCs in comparison with B6 CD4(+)CD25(+) cells stimulated with B6 APCs. Surprisingly, stimulation of NOD CD4(+)CD25(+) cells with B6 APCs restored regulation, whereas with the reciprocal combination, NOD APCs failed to activate B6 CD4(+)CD25(+) cells properly. Interestingly, APCs from disease-free (>30 weeks of age), but not diabetic, NOD mice were able to activate CD4(+)CD25(+) regulatory function in vitro and apparently in vivo because only spleens of disease-free NOD mice contained potent CD4(+)CD25(+) regulatory cells that prevented disease development when transferred into young NOD recipients. These data suggest that the failure of NOD APCs to activate CD4(+)CD25(+) regulatory cells may play an important role in controlling type 1 diabetes development in NOD mice.

Conversion of CD4+ CD25- cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus.

The CD4+ CD25+ regulatory T cells play a critical role in controlling autoimmunity, but little is known about their development and maintenance. In this study, we investigated whether CD4+ CD25- cells can convert to CD4+ CD25+ regulatory T cells in vivo under natural conditions. CD4+ CD25- cells from CD45.1+ mice were sorted and transferred into congenic CD45.2+ mice. Converted CD4+ CD25+ cells could be detected in lymphoid organs as early as 1 wk after transfer and by 6 wk after transfer, 5-12% of transferred CD4+ cells expressed CD25. Converted CD4+ CD25+ cells themselves failed to proliferate after stimulation, but could suppress proliferation of responder cells in vitro, and also expressed high levels of Foxp3 mRNA. In addition, CD4+ CD25- cells transferred into thymectomized congenic mice converted to CD4+ CD25+ cells that also suppressed responder cell proliferation in vitro, and expressed high levels of Foxp3 mRNA. Finally, CD4+ CD25- cells transferred into B7-/- mice failed to convert into CD4+ CD25+ cells that exhibit the regulatory phenotype. These data indicate that CD4+ CD25- cells convert into CD4+ CD25+ regulatory T cells spontaneously in vivo and suggest that this conversion process could contribute significantly to the maintenance of the peripheral CD4+ CD25+ regulatory T cell population.

Mechanisms of tolerance induced by transforming growth factor-beta-treated antigen-presenting cells: CD8 regulatory T cells inhibit the effector phase of the immune response in primed mice through a mechanism involving Fas ligand.

Transforming growth factor (TGF)-beta-treated antigen-presenting cells [(APC) adherent peritoneal exudate cells] induce a profound tolerance in primed mice that is thought to be mediated by regulatory T cells induced in the spleen. In the current study, we investigated the mechanism(s) involved in tolerance induced in primed mice by TGF-beta-treated APC. Interestingly, TGF-beta-treated APC from class II knockout mice were unable to mediate tolerance in primed mice and failed to induce not only CD4, but also CD8 regulatory T cells. However, the results of several experiments indicated that it was the CD8 regulatory T cells that were required for tolerance induced in primed mice. Using neutralizing antibody, we found that TGF-beta-treated APC-induced CD8 regulatory T cells did not suppress effector T cell function in vivo through the production of IL-4, TGF-beta or IL-10. On the other hand, our data showed that the Fas-Fas ligand (FasL) pathway was involved in this form of tolerance since TGF-beta-treated APC could not mediate tolerance in primed FasL-deficient mice and CD8 T cells from FasL-deficient mice were unable to suppress effector T cell responses. Moreover, the targets of FasL-mediated suppression were found to be the effector T cells as suggested by the data showing that Fas-deficient effector T cells were not susceptible to suppression mediated by CD8 regulatory T cells induced by TGF-beta-treated APC. In conclusion, our data indicate that TGF-beta-treated APC effect tolerance in primed mice via a Fas-FasL-mediated mechanism that requires CD8 cells.

Mechanisms of tolerance induced by TGF beta-treated APC: CD4 regulatory T cells prevent the induction of the immune response possibly through a mechanism involving TGF beta.

Transforming growth factor beta (TGF beta)-treated antigen-presenting cells (APC) pulsed with antigen induce tolerance in mice, i.e. inhibition of IFN-gamma production and delayed type hypersensitivity response. Although evidence suggests that regulatory T cells are involved, their mechanism of action is currently unknown and is the subject of the present study. Both CD4 and CD8 splenic T cells from mice injected i.v. with adherent thioglycolate-elicited peritoneal exudate cells cultured with TGF beta(2) and antigen (TGF beta-treated APC) transferred tolerance to naive recipients. Interestingly, TGF beta-treated APC from class II knockout mice were unable to induce tolerance in wild-type mice, whereas wild-type TGF beta-treated APC could induce tolerance in CD8 knockout mice. TGF beta was detected in cultures of lymphoid cells from mice injected with TGF beta-treated APC, and treatment with anti-TGF beta antibody in vivo impaired tolerance induction. TGF beta appeared to be involved in both the development of CD4 regulatory T cells and the effector function of the CD4 regulatory T cells. In summary, the important findings in this study are that CD4, and not CD8, regulatory T cells are required for tolerance induced by TGF beta-treated APC in naive mice, and tolerance appears to be mediated by a mechanism involving TGF beta.

Deletion, but not anergy, is involved in TGF-beta-treated antigen-presenting cell-induced tolerance.

Intravenous injection of transforming growth factor (TGF-)-beta-treated antigen-presenting cells (APC) pulsed with antigen induces antigen-specific tolerance in both naive and previously primed mice. Although TGF-beta-treated APC-induced tolerance is associated with induction of regulatory T cells and impaired delayed-type hypersensitivity (DTH) responses, the specific mechanisms that mediate this tolerance are not currently known. The goal of the present report was to study the mechanisms involved in TGF-beta-treated APC-induced tolerance by determining the fate of the antigen-specific effector T cells that are regulated. Using a well-characterized system that allows tracking of small numbers of TCR transgenic T cells, we have found that antigen-specific T cell expansion, either in vivo or in vitro, is inhibited in mice that have been injected with TGF-beta-treated APC. The failure of antigen-specific effector T cells to expand did not appear to be due to the induction of anergy, since carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells divided normally in response to antigen and adjuvant in vivo, and addition of exogenous IL-2 was unable to restore T cell expansion in in vitro assays. Interestingly, the percentage of CFSE-labeled cells was decreased after >7-8 divisions following culture in vitro, which correlated with a significant increase in cell death. Cell death was prevented and the ability to expand in vitro was restored by treatment with anti-Fas ligand (FasL) antibody. In conclusion, tolerance induced by TGF-beta-treated APC appears to be associated with deletion of antigen-specific T cells involving the Fas-FasL pathway.