Notch signalling regulates cytokine production by CD8+ and CD4+ T cells.

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.

Retinoic acid generates regulatory T cells in experimental transplantation.

Regulatory T cells play a key role to inhibit effector lymphocytes, avoid, autoimmunity, and restrain allogeneic immunity. Retinoic acid is an important cofactor that stimulates the generation and expansion of regulatory T cells. Naive T cells, coincubated with allogeneic antigen-presenting cells and retinoic acid, in conjunction with transforming growth factor (TGF) ß and interleukin (IL) 2, generated allogeneic regulatory T cells de novo. These cells were able to inhibit skin rejection in adoptive transfer experiments. The generation of regulatory T cells ex vivo with retinoic acid, TGF-ß, and IL-2 represents a new step toward specific regulation of allogeneic immune responses.

Differential regulation of Notch ligands in dendritic cells upon interaction with T helper cells.

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta-like and Jagged, in antigen-presenting cells. However, whether stimuli other than pathogen-derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta-like 4 and Jagged 2 expression in bone marrow-derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta-like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL-4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2-promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL-4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL-4. These data suggest that Notch ligands are differentially regulated in DCs: Delta-like 4 is regulated by T helper cells and by pathogen-derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen-derived Th2-promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs.

Dendritic cells and B cells cooperate in the generation of CD4(+)CD25(+)FOXP3(+) allogeneic T cells.

BACKGROUND: CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an essential role in immune tolerance, suppressing responses against self-antigens. Additionally, Treg play an important role in maintaining immunosuppression to alloantigens as well as to other antigens. It is well known that in the gut, a subset of dendritic cells produces retinoic acid (RA), which together with transforming growth factor (TGF-beta) is able to differentiate naïve T cells into Treg. The aim of this study was to establish the role of antigen-presenting cells (APC) in the differentiation of allogeneic Tregs under the effect of RA and TGF-beta. METHODS: Splenic CD4(+)CD25(-) naïve T cells from C57BL/6 mice were co-cultured with splenic CD11c-enriched APC from Balb/c mice in the presence of TGF-beta, RA, and interleukin (IL-2). After 6 days of culture, cells were analyzed for the expression of Foxp3 by flow cytometry. Additionally, we investigated the role of B cells and dendritic cells (DCs) and their stimulatory capacity in the generation of Tregs. RESULTS: Our results showed that co-culture of naive T cells with the appropriate level of stimulation by APC in the presence of TGF-beta, RA, and IL-2 provided a new powerful approach to generate allogeneic Treg cells. We demonstrated that although B cells and DCs can generate Tregs by themselves, a mixure of both APC improved their capacity to efficiently generate Tregs. Also, we observed that although the addition of IL-2 to the cultures was not crucial to generate Tregs, it was required to optimize their expansion and cell survival.

Transforming growth factor-beta and all-trans retinoic acid generate ex vivo transgenic regulatory T cells with intestinal homing receptors.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) mediate immunologic self-tolerance and suppress immune responses. In the gut, a subset of dendritic cells is specialized to induce Treg in a transforming growth factor-beta (TGF-beta)- and retinoic acid (RA)-dependent manner. The aim of this study was to establish if RA synergizing with TGF-beta induced antigen specific CD4(+) CD25(high) Foxp3(+) Treg portraying gut homing receptors. Splenic CD4(+)CD25(-) Foxp3(-) naïve T cells from DO11.10 mice were cocultured with splenic CD11c(+) dendritic cells from Balb/c mice in the presence of TGF-beta, RA, and low levels of an antigenic peptide. After 5 days of culture, cells were analyzed for the expression of Foxp3 and the gut homing receptors CCR9 and alpha4beta7. The number of Foxp3(+) T cells generated with TGF-beta and RA was at least 3 times higher than in the cultures with TGF-beta alone and 15 times higher than in controls without exogenous cytokines. Also, supplementation of the cultures with RA induced the expression of the intestinal homing receptors CCR9 and alpha4beta7. Our results showed that coculture of naïve T cells with antigen-presenting cells in the presence of TGF-beta and RA represents a powerful approach to generate Treg with specific homing receptors.

Interleukin-4 selectively inhibits interleukin-2 secretion by lipopolysaccharide-activated dendritic cells.

Dendritic cells (DCs) generated in vitro from bone marrow precursors using granulocyte-macrophage colony-stimulating factor (GM-CSF) secrete interleukin-2 (IL-2) upon activation, an event probably associated to the initiation of adaptive immune responses. Additionally, they produce IL-12, a cytokine related to T-cell polarization. To analyse the effect of IL-4 on DC differentiation and function, we assessed the capacity of murine bone marrow dendritic cells (BMDCs) differentiated with GM-CSF in the presence or absence of IL-4 to produce IL-2 and IL-12 upon lipopolysaccharide (LPS) activation. We found that although IL-4 enhanced DC IL-12p70 production, it strongly impaired IL-2 secretion by BMDCs. This inhibition, which depends on the presence of IL-4 during LPS activation, is DC specific, as IL-4 did not affect IL-2 secretion by T cells. Interestingly, inhibition of DC IL-2 production did not prevent DC priming of T lymphocytes. These results illustrate a new putative role for IL-4 on the regulation of the immune response and should help clarify the controversial reports on the effect of IL-4 on DCs.

Cyclosporine preconditions dendritic cells during differentiation and reduces IL-2 and IL-12 production following activation: a potential tolerogenic effect.

The mode of action of cyclosporine (CsA) has been ascribed to its capacity to inhibit IL-2 and IFNgamma production by T cells, two cytokines implicated in allograft rejection. Recently, it has been reported that upon activation, dendritic cells (DCs) exhibit transient production of IL-2, a property that appears to be related to their capacity to initiate immune responses. On the other hand, DCs can generate signals determining Th1/Th2 polarizing effects, an effect that can drastically influence the outcome of organ transplant. The purpose of the present study was to investigate the effect of CsA on cytokine production by immature and mature DCs. DC precursors from mouse bone marrow were induced to differentiate by incubation with GM-CSF for 5 days followed by activation with LPS for 4 hours. CsA was added at different times during this process. Our results show that when CsA is added during the differentiation period following activation with LPS, IL-2 and IL-12 secretion are significantly reduced without affecting the evolution of the DC. Conversely, CsA had no effect when added during the LPS activation period. These results show that CsA affects DCs before they receive the final activation stimulus, preconditioning them to antigen stimulation. This preconditioning of DCs by calcineurin-inhibiting drugs conceptually integrates the mode of action of CsA with the tolerogenic and T-cell polarization function ascribed to DCs. These results may be especially meaningful for the future design of immunosuppressive protocols.