Elizabeth Adams

Induction of foxP3+ Regulatory T Cells in the Periphery of T Cell Receptor Transgenic Mice Tolerized to Transplants

Transplantation tolerance can be induced in mice by grafting under the cover of nondepleting CD4 plus CD8 or CD154 mAbs. This tolerance is donor Ag specific and depends on a population of CD4+ regulatory T cells that, as yet, remain poorly defined in terms of their specificity, origin, and phenotype. Blocking of the Ag-specific response in vitro with an anti-CD4 mAb allowed T cells from monospecific female TCR-transgenic mice against the male Ag Dby, presented by H-2Ek, to express high levels of foxP3 mRNA. foxP3 induction was dependent on TGF-β. The nondepleting anti-CD4 mAb was also able to induce tolerance in vivo in such monospecific TCR-transgenic mice, and this too was dependent on TGF-β. As in conventional mice, acquired tolerance was dominant, such that naive monospecific T cells were not able to override tolerance. Splenic T cells from tolerant mice proliferated normally in response to Ag, and secreted IFN-γ and some IL-4, similar to control mice undergoing primary or secondary graft rejection. High levels of foxP3 mRNA, and glucocorticoid-induced TNFR superfamily member 18 (GITR)+ CD25+ T cells were found within the tolerated skin grafts of long-term tolerant recipients. These data suggest that regulatory T cells maintaining transplantation tolerance after CD4 Ab blockade can be induced de novo through a TGF-β-dependent mechanism, and come to accumulate in tolerated grafts.

Both CD4+CD25+ and CD4+CD25− Regulatory Cells Mediate Dominant Transplantation Tolerance

CD4+CD25+ T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4+CD25+ T cells do have a suppressive role in transplantation tolerance, so do CD4+CD25− T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4+CD25+ T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in “tolerized” mice. Serial analysis of gene expression confirmed that cells sorted into CD4+CD25+ and CD4+CD25− populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.

Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells

Recently, agonist antibodies to glucocorticoid-induced tumor necrosis factor receptor (GITR) (tumor necrosis factor receptor superfamily 18) have been shown to neutralize the suppressive activity of CD4+CD25+ regulatory T cells. It was anticipated that this would be the role of the physiological ligand. We have identified and expressed the gene for mouse GITR ligand and have confirmed that its interaction with GITR reverses suppression by CD4+CD25+ T cells. It also, however, provides a costimulatory signal for the antigen-driven proliferation of naïve T cells and polarized T helper 1 and T helper 2 clones. RT-PCR and mAb staining revealed mouse GITR ligand expression in dendritic cells, macrophages, and B cells. Expression was controlled by the transcription factor NF-1 and potentially by alternative splicing of mRNA destabilization sequences.

Infectious tolerance via the consumption of essential amino acids and mTOR signaling

Infectious tolerance describes the process of CD4+ regulatory T cells (Tregs) converting naïve T cells to become additional Tregs. We show that antigen-specific Tregs induce, within skin grafts and dendritic cells, the expression of enzymes that consume at least 5 different essential amino acids (EAAs). T cells fail to proliferate in response to antigen when any 1, or more, of these EAAs are limiting, which is associated with a reduced mammalian target of rapamycin (mTOR) signaling. Inhibition of the mTOR pathway by limiting EAAs, or by specific inhibitors, induces the Treg-specific transcription factor forkhead box P3, which depends on both T cell receptor activation and synergy with TGF-β.

Cutting Edge: Anti-CD154 Therapeutic Antibodies Induce Infectious Transplantation Tolerance

Nondepleting anti-CD154 (CD40 ligand) mAbs have proven effective in inducing transplantation tolerance in rodents and primates. In the induction phase, anti-CD154 Ab therapy is known to enhance apoptosis of Ag reactive T cells. However, this may not be the sole explanation for tolerance, as we show in this study that tolerance is maintained through a dominant regulatory mechanism which, like tolerance induced with CD4 Abs, manifests as infectious tolerance. Therefore, tolerance induced with anti-CD154 Abs involves not only the deletion of potentially aggressive T cells, but also a contagious spread of tolerance to new cohorts of graft-reactive T cells as they arise.

Mechanisms of peripheral tolerance and suppression induced by monoclonal antibodies to CD4 and CD8.

Over the last five years it has become increasingly clear that the peripheral immune system can maintain tolerance to both self and non-self antigens through a variety of mechanisms. Although clonal deletion may play an important part in limiting rapidly expanding responses, there are many examples where antigen reactive T cells remain. It has been proposed that tolerance is maintained in this situation either by the induction of anergy or by ongoing suppression. The phenomenon known as immune deviation, where non-inflammatory Th2 responses could suppress Th1 and positively reinforce themselves provided an attractive explanation for infectious tolerance, where tolerant T cells could guide further naive T cells also to tolerance. However, experiments to test this hypothesis in the models of CD4 and CD8 antibody-induced tolerance have given conflicting data, with no clear evidence of Th2 responses in tolerant mice. In this paper we review recent data that IL-4 plays a role in suppression, but that the source of IL-4 may not be the tolerant/suppressor T cell. We also discuss how infectious tolerance can operate on third party antigens if they are linked on the same antigen presenting cell and how CD4+ T cells can suppress CD8+ T-cell responses. Finally, we suggest a model of infectious anergy that is compatible with the available data.

Regulatory T Cells Overexpress a Subset of Th2 Gene Transcripts

There is now compelling evidence for subpopulations of CD4+ T cells whose role is to prevent immune pathology in both autoimmunity and transplantation. We have cloned CD4+ T cells against a male transplantation Ag that, unlike Th1 or Th2 clones, suppresses the rejection of male skin grafts and are therefore considered examples of regulatory T cells. We have identified, using serial analysis of gene expression, transcripts that are overexpressed in regulatory T cells compared with Th1 and Th2 clones. Some of these transcripts are increased in tolerated rather than rejecting skin grafts and in addition are expressed by the natural regulatory CD4+CD25+ subpopulation of naive mice. These genes include prepro-enkephalin, GM2 ganglioside activator protein, glucocorticoid-induced TNFR superfamily member 18, and integrin αEβ7. They seem to represent a subset of transcripts shared with Th2 cells, suggesting that transplantation tolerance and normal immunoregulation may represent a unique form of Th2-like differentiation.

Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance.

A new genetic mouse model demonstrates the necessity of Foxp3+ T reg cells for infectious tolerance.

Infectious tolerance and the long- term acceptance of transplanted tissue

Summary:  Short courses of antibody treatment aimed at blocking the coreceptors CD4 and CD8 and/or costimulatory molecules such as CD40L are able to bring about long‐term acceptance and tolerance of allogeneic transplants. This tolerant state is operational, in that potential effector cells remain but are tightly regulated through the induction of antigen‐specific CD4+ regulatory T cells (Tregs). CD4+CD25+FoxP3+ Tregs appear to play a prominent role, although other categories of Tregs have been documented. Transforming growth factor β (TGFβ) has been found to play a major role in the induction of the tolerant state with therapeutic antibodies as well as promoting the induction of FoxP3+ T cells from naïve populations. The observation that Tregs can be found in tolerated grafts has led to the idea that they may interact with the grafted tissue to establish a state of acquired privilege symmetrical with a similar privileged microenvironment around antigen‐presenting cells in lymphoid tissues. Dampening of aggressive immune responses by Tregs allows antigen to persist and be presented in an innocuous way to promote tolerance in new cohorts of T cells throughout the life of the tolerated graft. Regulation may operate at many stages of an immune response, even as a censor at the terminal differentiation stages of effector function.

Regulatory T cells and dendritic cells in transplantation tolerance: molecular markers and mechanisms.

Summary:  Transplantation tolerance can be induced in adult rodents using monoclonal antibodies against coreceptor or costimulation molecules on the surface of T cells. There are currently two well‐characterized populations of T cells, demonstrating regulatory capacity: the ‘natural’ CD4+CD25+ T cells and the interleukin (IL)‐10‐producing Tr1 cells. Although both types of regulatory T cells can induce transplantation tolerance under appropriate conditions, it is not clear whether either one plays any role in drug‐induced dominant tolerance, primarily due to a lack of clear‐cut molecular or functional markers. Similarly, although dendritic cells (DCs) can be pharmacologically manipulated to promote tolerance, the phenotype of such populations remains poorly defined. We have used serial analysis of gene expression (SAGE) with 29 different T‐cell and antigen‐presenting cell libraries to identify gene‐expression signatures associated with immune regulation. We found that independently derived, regulatory Tr1‐like clones were highly concordant in their patterns of gene expression but were quite distinct from CD4+CD25+ regulatory T cells from the spleen. DCs that were treated with the tolerance‐enhancing agents IL‐10 or vitamin D3 expressed a gene signature reflecting a functional specification in common with the most immature DCs derived from embryonic stem cells.