The potent immunosuppressive action of rapamycin is commonly ascribed to inhibition of growthfactor-induced T-cell proliferation. However, it is now evident that the serine/threonine protein kinase mammalian target of rapamycin (mTOR) has an important role in the modulation of both innate and adaptive immunity. mTOR regulates diverse functions of professional antigen-presenting cells, such as dendritic cells (DCs), and has important roles in the activation of conventional T cells and the function and proliferation of regulatory T cells. Here, we review our current understanding of the mTOR pathway and the consequences of mTOR inhibition, both in DCs and T cells, including new data on the regulation of FOXP3 expression.Rapamycin was isolated in the early 1970s from a soil sample obtained on Easter Island (Rapa Nui) and identified as a potent anti-fungal metabolite 1 . This macrolide, which is produced by Streptomyces hygroscopicus, was found to inhibit cell proliferation and to have potent immunosuppressive activity. It is used for prevention of kidney transplant rejection 2 . Rapamycin and its derivatives are undergoing clinical testing for prophylaxis of graft rejection 3 and graft-versus-host disease (GVHD) [G] 4 , chemotherapy of some cancers 5 and the prevention of restenosis following angioplasty 6 .Our understanding of the mechanisms that underlie the unique immunosuppressive profile of rapamycin continues to evolve. In line with this, the central and pervasive role of the serine/ threonine kinase 'mammalian target of rapamycin' (mTOR) in innate and adaptive immunity is becoming apparent. Blockade of mTOR by rapamycin impairs dendritic cell (DC) maturation and function, and inhibits T-cell proliferation, a mechanism that underpins its immunosuppressive effect. There is now strong evidence that mTOR is crucial for the regulation of antigen responsiveness in CD4 + T cells. This effect seems to be mediated by an influence of mTOR inhibition on naturally-occurring regulatory T (T Reg ) cells, which have a key role in immunological tolerance. Exciting information has emerged regarding the role of the phosphatidylinositol-3-kinase (PI3K)-AKT-mTOR pathway in regulating DC and T-cell function, particularly in relation to the expression of forkhead box P3 (FOXP3) and the differentiation of T Reg cells. Here, we review the remarkable recent progress in elucidation of the mechanisms by which mTOR inhibition affects intracellular signalling pathways in immune cells, particularly DCs and T cells, and how this influences immunity.

The ability of dendritic cells (DC) to regulate Ag-specific immune responses via their influence on T regulatory cells (Treg) may be key to their potential as therapeutic tools or targets for the promotion/restoration of tolerance. In this report, we describe the ability of maturation-resistant, rapamycin (RAPA)-conditioned DC, which are markedly impaired in Foxp3− T cell allostimulatory capacity, to favor the stimulation of murine alloantigen-specific CD4+CD25+Foxp3+ Treg. This was distinct from control DC, especially following CD40 ligation, which potently expanded non-Treg. RAPA-DC-stimulated Treg were superior alloantigen-specific suppressors of T effector responses compared with those stimulated by control DC. Supporting the ability of RAPA to target effector T and B cells, but permit the proliferation and suppressive function of Treg, an infusion of recipient-derived alloantigen-pulsed RAPA-DC followed by a short postoperative course of low-dose RAPA promoted indefinite (>100 day) heart graft survival. This was associated with graft infiltration by CD4+Foxp3+ Treg and the absence of transplant vasculopathy. The adoptive transfer of CD4+ T cells from animals with long-surviving grafts conferred resistance to rejection. These novel findings demonstrate that, whereas maturation resistance does not impair the capacity of RAPA-DC to modulate Treg, it profoundly impairs their ability to expand T effector cells. A demonstration of this mechanism endorses their potential as tolerance-promoting cellular vaccines.

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2d) mouse bone marrow progenitors in IL-10 and TGF-β, then stimulated the cells with LPS. These “alternatively activated” (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.

Hepatic stellate cells (HSCs) may play an important role in hepatic immune regulation by producing numerous cytokines/chemokines, and expressing Ag-presenting and T cell co-regulatory molecules. Due to disruption of the endothelial barrier during cold-ischemic storage and reperfusion of liver grafts, HSCs can interact directly with cells of the immune system. Endotoxin (LPS), levels of which increase in liver diseases and transplantation, stimulates the synthesis of many mediators by HSCs. We hypothesized that LPS-stimulated HSCs might promote hepatic tolerogenicity by influencing naturally-occurring immunosuppressive CD4+CD25+FoxP3+ regulatory T cells (Tregs). Following their portal venous infusion, allogeneic CD4+ T cells, including Tregs, were found closely associated with HSCs, and this association increased in LPS-treated livers. In vitro, both unstimulated and LPS-stimulated HSCs up-regulated Fas (CD95) expression on conventional CD4+ T cells and induced their apoptosis in a Fas/FasL-dependent manner. By contrast, HSCs induced Treg proliferation, which required cell-cell contact, and was MHC class II-dependent. This effect was augmented when HSCs were pretreated with LPS. LPS increased the expression of MHC class II, CD80 and CD86, and stimulated the production of IL-1α, IL-1β, IL-6, IL-10 and TNFα by HSCs. Interestingly, production of IL-1α, IL-1β, IL-6 and TNFα was strongly inhibited, but that of IL-10 enhanced, in LPS-pretreated HSC/Treg co-cultures. Adoptively transferred allogeneic HSCs migrated to the secondary lymphoid tissues and induced Treg expansion in lymph nodes. These data implicate endotoxins-stimulated HSCs as important immune regulators in liver transplantation by inducing selective expansion of tolerance-promoting Tregs, and reducing inflammation and allo-immunity.

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8α− (myeloid) and CD11c+CD8α+ (“lymphoid-related”) DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-γ) and Th2 (IL-4) responses. When higher LPS concentrations (≥100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.