Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance.

Emerging evidence suggests critical roles for APCs in suppressing immune responses. Here, we show that zymosan, a stimulus for TLR2 and dectin-1, regulates cytokine secretion in DCs and macrophages to induce immunological tolerance. First, zymosan induces DCs to secrete abundant IL-10 but little IL-6 and IL-12(p70). Induction of IL-10 is dependent on TLR2- and dectin-1-mediated activation of ERK MAPK via a mechanism independent of the activation protein 1 (AP-1) transcription factor c-Fos. Such DCs stimulate antigen-specific CD4+ T cells poorly due to IL-10 and the lack of IL-6. Second, zymosan induces F4-80+ macrophages in the splenic red pulp to secrete TGF-beta. Consistent with these effects on APCs, injection of zymosan plus OVA into mice results in OVA-specific T cells that secrete little or no Th1 or Th2 cytokines, but secrete robust levels of IL-10, and are unresponsive to challenge with OVA plus adjuvant. Finally, coinjection of zymosan with OVA plus LPS suppresses the response to OVA via a mechanism dependent on IL-10, TGF-beta, and lack of IL-6. Together, our data demonstrate that zymosan stimulates IL-10+ IL-12(p70)- IL-6low regulatory DCs and TGF-beta+ macrophages to induce immunological tolerance. These data suggest several targets for pharmacological modulation of immune responses in various clinical settings.

Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells.

CD4+CD25+ regulatory T (TR) cells have been described in both humans and mice. In mice, TR are thymically derived, and lack of TR leads to organ-specific autoimmunity. Recently, the forkhead/winged helix transcription factor, FoxP3, has been shown to be important for the function of TR cells in mice. In this study, human TR cells were examined and, in results similar to those of studies done in mice, expression of FoxP3 was found exclusively in CD4+CD25+ T cells and correlated with the suppressive activity of these cells. In contrast to the mouse studies, activation of human CD4+CD25- T cells led to expression of FoxP3. Expression of FoxP3 in activated human CD4+CD25+ cells also correlated with suppression of proliferation by these cells in freshly isolated CD4+CD25- T cells from the same donor. This suppression was cell-contact dependent and cytokine independent. Thus, in humans, during activation of CD4+CD25- T cells in an immune response, two populations of cells may arise, effector CD4+CD25+ and regulatory CD4+CD25+ T cells, with expression of FoxP3 correlated with regulatory activity. These data also raise the possibility that a failure to generate peripheral TR cells properly may contribute to autoimmune disease and suggest a possible therapeutic role for FoxP3 in the treatment of such diseases.

IL-2Rbeta links IL-2R signaling with Foxp3 expression.

Immunological tolerance to self antigens is a tightly regulated process. Recent work has demonstrated that the forkhead family member Foxp3 is a critical element in the differentiation and function of mouse CD4(+)CD25(+) regulatory T cells (Treg). Recent work has suggested an important role for IL-2 in the development and maintenance of Treg. To directly assess the effect of IL-2 signaling on Treg development and function, we analyzed mice that were genetically deficient in components of the IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Ralpha) displayed a slight decrease in Treg within the thymus, while peripheral numbers are unchanged. In contrast, we found that mice deficient in CD122 (IL-2Rbeta) had a profound reduction in both thymic and peripheral Treg, coinciding with more rapid development of a fatal lymphoproliferative disease. Expression of a Foxp3 transgene restored Treg and protected against the onset of autoimmunity. Thus, a signal mediated by IL-2Rbeta is essential for the development and homeostasis of Foxp3(+) Treg in vivo.

Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells.

CD4+CD25+ T regulatory (T(R)) cells are an important regulatory component of the adaptive immune system that limit autoreactive T cell responses in various models of autoimmunity. This knowledge was generated by previous studies from our lab and others using T(R) cell supplementation and depletion. Contrary to dogma, we report here that injection of anti-CD25 mAb results in the functional inactivation, not depletion, of T(R) cells, resulting in exacerbated autoimmune disease. Supporting this, mice receiving anti-CD25 mAb treatment display significantly lower numbers of CD4+CD25+ T cells but no change in the number of CD4+FoxP3+ T(R) cells. In addition, anti-CD25 mAb treatment fails to both reduce the number of Thy1.1+ congenic CD4+CD25+ T(R) cells or alter levels of CD25 mRNA expression in treatment recipients. Taken together, these findings have far-reaching implications for the interpretation of all previous studies forming conclusions about CD4+CD25+ T(R) cell depletion in vivo.

Dynamic regulation of FoxP3 expression controls the balance between CD4+ T cell activation and cell death.

The forkhead-family transcription factor FoxP3 is important for the development and function of CD4+CD25+ regulatory T cells. While the overall phenotypic effects of FoxP3 expression are evident, the mechanism by which FoxP3 regulates T cell activation is not well understood. CD4+ T cells from mice that express a FoxP3 Tg are refractory to TCR-mediated stimulation, failing to proliferate or produce cytokines, but possess suppressive activity towards normal T cells. In this report we show that these T cells express elevated levels of mRNA for pro-apoptotic genes and undergo rapid apoptosis following stimulation. These T cells also display slower cell cycle transit following activation, suggesting that FoxP3 is capable of regulating the ability of T cells to respond to TCR-mediated activation. Lastly, we show that contrary to expected results, under Th1 or Th2 driving conditions, CD4+ T cells from FoxP3 Tg mice differentiate into effector cells. Concomitant with differentiation is a loss of FoxP3 mRNA and protein. These data demonstrate that FoxP3 levels regulate T cell function, and that FoxP3 itself is dynamically regulated during effector T cell differentiation.

Scurfin (FoxP3) controls T-dependent immune responses in vivo through regulation of CD4+ T cell effector function.

Scurfin, the protein product of the FoxP3 gene, is a forkhead-family transcription factor that negatively regulates T cell function. Mice carrying a loss-of-function mutation in FoxP3 (scurfy mice) present with fatal autoimmune-like disease caused by hyperresponsive CD4(+) T cells. Mice that overexpress scurfin (FoxP3 Tg mice) possess fewer mature T cells with reduced functional capabilities compared with normal littermate control mice. We analyzed the ability of CD4(+) T cells and B cells from FoxP3 Tg mice to respond to a T-dependent Ag and found that immunized FoxP3 Tg mice displayed reduced total and Ag-specific serum Ig and disorganized splenic architecture. However, when cultured in vitro, FoxP3 Tg B cells responded normally, suggesting that the poor Ab response was a result of defective T cell help in vivo. When challenged, CD4(+) T cells from FoxP3 Tg mice display reduced up-regulation of CD40 ligand and fewer IFN-gamma-producing cells. Overall, these findings show that overexpression of scurfin reduces T cell responses in vivo such that CD4(+) T cells cannot provide help to B cells during a T cell-dependent Ab response.

Adaptive immunity in mice lacking the beta(2)-adrenergic receptor.

The beta-2-adrenergic receptor (beta(2)AR) is expressed by most lymphocyte populations and binds the sympathetic neurotransmitter norepinephrine (NE). Stimulation of the beta(2)AR is reported to be the primary mechanism by which signals from the sympathetic nervous system influence both cell-mediated and humoral immunity. We report here that body/organ weights, lymphoid organ cell number/phenotype/histology, the contact sensitivity response, and the amount, avidity, and isotype of antibody resulting from a T cell-dependent antibody response in beta(2)AR deficient mice (beta(2)AR-/- mice) were all similar to measures made in beta(2)AR+/+ mice. Other members of the adrenergic receptor family did not appear to compensate for the absence in beta(2)AR expression. In contrast, beta(2)AR-/- B cells cultured in vitro were unable to respond to NE in a manner similar to beta(2)AR+/+ B cells. Thus, mice in which expression of the beta(2)AR gene is defective from early development to adulthood may no longer require that NE stimulate the beta(2)AR to maintain immune homeostasis, and this may be due to a non-adrenergic mechanism that provides compensation in vivo.

The generation of mature, single-positive thymocytes in vivo is dysregulated by CD69 blockade or overexpression.

During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development. Blockade of CD69 led to a reduction in single-positive cells and a concomitant increase in double-positive cells in the thymus. Overexpression of a CD69 transgene in the thymus resulted in a dramatic increase in both CD8SP and CD4SP cells. Coexpression with a TCR transgene demonstrated that both positive and negative selection were enhanced by the increased levels of CD69 on thymocytes. Finally, mice overexpressing CD69 displayed a sharp reduction in the number of T cells in the spleen and lymph node. Taken as a whole, these data suggest the involvement of CD69 in the process of selection and maturation during the trafficking of thymocytes to the medulla.