Stephen P. Cobbold

Identification of Regulatory T Cells in Tolerated Allografts

Induction of transplantation tolerance with certain therapeutic nondepleting monoclonal antibodies can lead to a robust state of peripheral “dominant” tolerance. Regulatory CD4+ T cells, which mediate this form of “dominant” tolerance, can be isolated from spleens of tolerant animals. To determine whether there were any extra-lymphoid sites that might harbor regulatory T cells we sought their presence in tolerated skin allografts and in normal skin. When tolerated skin grafts are retransplanted onto T cell–depleted hosts, graft-infiltrating T cells exit the graft and recolonize the new host. These colonizing T cells can be shown to contain members with regulatory function, as they can prevent nontolerant lymphocytes from rejecting fresh skin allografts, without hindrance of rejection of third party skin. Our results suggest that T cell suppression of graft rejection is an active process that operates beyond secondary lymphoid tissue, and involves the persistent presence of regulatory T cells at the site of the tolerated transplant.

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.

Different Roles for L3T4 + and Lyt 2 + T Cell Subsets in the Control of an Acute Herpes Simplex Virus Infection of the Skin and Nervous System

Rat monoclonal antibodies were used to deplete selectively Lyt 2 (cytotoxic) and L3T4 (helper) T cell populations in vivo. These antibodies produced greater than 95% depletion of the respective T cell subset as determined by fluorescent antibody and cytofluorographic analyses. Antibody-treated mice were infected in the ear pinna with herpes simplex virus (HSV) and the induction of virus-specific T cell and antibody responses were monitored during the acute infection. Lyt 2-deficient mice produced delayed hypersensitivity and HSV-specific antibodies comparable to those in untreated animals. However, major histocompatibility complex class I-restricted T cell killing was abolished. In contrast, L3T4-deficient animals failed to produce either primary delayed hypersensitivity response or specific antibodies to the virus, but cytotoxic T cell responses were induced and even augmented in comparison with infected, normal animals. This observation clearly demonstrates that Lyt 2 cytotoxic T cells can be induced in a helper T cell-deficient environment. The ability of T cell subset-deficient mice to clear infectious virus was investigated in the skin of the ear and the part of the nervous system innervating the site of infection. L3T4-deficient animals showed a markedly delayed clearance of virus from the ear and also had a more florid infection of the nervous system. However, Lyt 2-deficient mice cleared the infection in the ear normally, but a severe infection of the nervous system was still observed. The implication of these observations to the pathogenesis of this virus is discussed.

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.

Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression

Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other (“linked”) epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10+ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.

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.