Antibody-induced transplantation tolerance that is dependent on thymus-derived regulatory T cells

Targeting of the CD45RB isoform by mAb (anti-CD45RB) effectively induces donor-specific tolerance to allografts. The immunological mechanisms underlying the tolerant state remain unclear although some studies have suggested the involvement of regulatory T cells (T-regs). Although their generative pathway remains undefined, tolerance promoting T-regs induced by systemic anti-CD45RB treatment have been assumed to originate in the peripheral immune system. We demonstrate herein that separable effects on the peripheral and central immune compartments mediate graft survival induced by anti-CD45RB administration. In the absence of the thymus, anti-CD45RB therapy is not tolerogenic though it retains peripheral immunosuppressive activity. The thymus is required for anti-CD45RB to produce indefinite graft survival and donor-specific tolerance, and this effect is accomplished through thymic production of donor-specific T-regs. These data reveal for the first time an Ab-based tolerance regimen that relies on the central tolerance pathway.     

Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival

Regulatory T (T(reg)) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Failure of host antitumor immunity may be caused by exaggerated suppression of tumor-associated antigen-reactive lymphocytes mediated by T(reg) cells; however, definitive evidence that T(reg) cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4(+)CD25(+)FOXP3(+) T(reg) cells in 104 individuals affected with ovarian carcinoma, that human tumor T(reg) cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor T(reg) cells are associated with a high death hazard and reduced survival. Human T(reg) cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages. Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of T(reg) cells to the tumor. This specific recruitment of T(reg) cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking T(reg) cell migration or function may help to defeat human cancer.     

Regulatory T cells and immune tolerance

Regulatory T cells (Tregs) play an indispensable role in maintaining immunological unresponsiveness to self-antigens and in suppressing excessive immune responses deleterious to the host. Tregs are produced in the thymus as a functionally mature subpopulation of T cells and can also be induced from naive T cells in the periphery. Recent research reveals the cellular and molecular basis of Treg development and function and implicates dysregulation of Tregs in immunological disease.     

Regulatory conversation between antigen presenting cells and regulatory T cells enhance immune suppression

Regulatory T cells (Treg) were originally described by their suppressive function exerted on effector T cells, but recent evidence also reveals interactions with antigen presenting cells (APCs). In general, all major subpopulations of APCs, i.e., dendritic cells (DC), B cells and monocytes/macrophages (Mvarphi), respond to exposure to Treg by down regulation of their antigen presenting function, upregulation of immunosuppressive molecules and secretion of immunosuppressive cytokines. Thus, Treg gain influence on the innate immune system and are able to augment their immunosuppressive capacities by blocking the effective priming of T effector cells by APCs. Conversely, APCs have an important role in nurturing peripheral Treg populations, since it has been shown that immature DC, as well as alternatively activated Mvarphi, are able to induce Treg de novo. These properties are dependent on the expression of surface molecules (CTLA-4, F4/80) and the production of soluble factors such as IL-10 and Indoleamine 2,3-dioxygenase by the APC subpopulations. On the whole, the mutual interaction of Treg and APCs enables Treg to sustain their immunosuppressive functions which, in healthy individuals, may be crucial for the maintenance of peripheral tolerance.     

A link between PDL1 and T regulatory cells in fetomaternal tolerance

Acceptance of the fetus expressing allogeneic paternal Ags by the mother is a physiologic model of transplantation tolerance. Various mechanisms contribute to fetal evasion from immune attack by maternal leukocytes. We have recently demonstrated that the inhibitory costimulatory molecule PDL1 plays a critical role in fetomaternal tolerance in that PDL1 blockade or deficiency resulted in decreased allogeneic fetal survival rates. CD4(+)CD25(+) T regulatory cells (Tregs) have also been demonstrated to play an important role in fetomaternal tolerance. Since PDL1 is expressed on Tregs, we explored the interactions between PDL1 and Tregs in vivo in a mouse model of fetomaternal tolerance. Depletion of CD25(+) T cells abrogated the effect of anti-PDL1 Ab indicating that the effect of PDL1 is possibly mediated by CD25(+) Tregs. Adoptive transfer of Tregs from wild-type but not PDL1-deficient mice into PDL1-deficient recipients significantly improved fetal survival. The frequency, phenotype and placental trafficking of Tregs from PDL1-deficient mice were similar to those of wild-type controls, but were defective in inhibiting alloreactive Th1 cells in vitro. This is the first report providing evidence for a link between PDL1 and T regulatory cells in mediating fetomaternal tolerance.     

Natural regulatory T cells and de novo-induced regulatory T cells contribute independently to tumor-specific tolerance

Thymus-derived, naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) and Tregs induced in the periphery (iTregs) have both been implicated in regulating immune responses. However, the relationship between these populations in the same host, and their relative contribution to the overall Treg pool, has not been examined. Using a tumor-induced T cell tolerance model, we find that expansion of nTregs and de novo generation of iTregs both contribute to tumor-specific T cell tolerance. In this system in which the number of tumor-specific nTregs can be controlled, the efficiency of nTreg expansion significantly exceeds that of the induction of Tregs from uncommitted progenitors in the tumor-bearing host. However, pre-existing nTregs are neither required for the induction of Tregs nor measurably impact on the extent of their accumulation. Instead, induction of Ag-specific regulatory cells from naive cells is intrinsically influenced by the tumor microenvironment and the presence of tumor Ag.     

Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.     

Regulatory T cells in transplantation tolerance

The identification and characterization of regulatory T (T(Reg)) cells that can control immune responsiveness to alloantigens have opened up exciting opportunities for new therapies in transplantation. After exposure to alloantigens in vivo, alloantigen-specific immunoregulatory activity is enriched in a population of CD4+ T cells that express high levels of CD25. In vivo, common mechanisms seem to underpin the activity of CD4+CD25+ T(Reg) cells in both naive and manipulated hosts. However, the origin, allorecognition properties and molecular basis for the suppressive activity of CD4+CD25+ T(Reg) cells, as well as their relationship to other populations of regulatory cells that exist after transplantation, remain a matter of debate..     

IL-17A-dependent CD4+CD25+ regulatory T cells promote immune privilege of corneal allografts

IL-17A is a proinflammatory cytokine that has received attention for its role in the pathogenesis of several autoimmune diseases. IL-17A has also been implicated in cardiac and renal allograft rejection. Accordingly, we hypothesized that depletion of IL-17A would enhance corneal allograft survival. Instead, our results demonstrate that blocking IL-17A in a mouse model of keratoplasty accelerated the tempo and increased the incidence of allograft rejection from 50 to 90%. We describe a novel mechanism by which CD4(+)CD25(+) regulatory T cells (Tregs) respond to IL-17A and enhance corneal allograft survival. Our findings suggest the following: 1) IL-17A is necessary for ocular immune privilege; 2) IL-17A is not required for the induction of anterior chamber-associated immune deviation; 3) Tregs require IL-17A to mediate a contact-dependent suppression; 4) corneal allograft Tregs suppress the efferent arm of the immune response and are Ag specific; 5) Tregs are not required for corneal allograft survival beyond day 30; and 6) corneal allograft-induced Treg-mediated suppression is transient. Our findings identify IL-17A as a cytokine essential for the maintenance of corneal immune privilege and establish a new paradigm whereby interplay between IL-17A and CD4(+)CD25(+) Tregs is necessary for survival of corneal allografts.     

Stat4 is critical for the balance between Th17 cells and regulatory T cells in colitis

Th17 play a central role in autoimmune inflammatory responses. Th1 are also necessary for autoimmune disease development. The interplay of Th1 signals and how they coordinate with Th17 during inflammatory disease pathogenesis are incompletely understood. In this study, by adding Stat4 deficiency to Stat6/T-bet double knockout, we further dissected the role of Stat4 in Th1 development and colitis induction. We showed that in the absence of the strong Th2 mediator Stat6, neither Stat4 nor T-bet is required for IFN-γ production and Th1 development. However, addition of Stat4 deficiency abolished colitis induced by Stat6/T-bet double-knockout cells, despite Th1 and Th17 responses. The failure of colitis induction by Stat4/Stat6/T-bet triple-knockout cells is largely due to elevated Foxp3(+) regulatory T cell (Treg) development. These results highlight the critical role of Stat4 Th1 signals in autoimmune responses in suppressing Foxp3(+) Treg responses and altering the balance between Th17 and Tregs to favor autoimmune disease.     

IL-2, regulatory T cells, and tolerance

IL-2 is a potent T cell growth factor that for many years was assumed to amplify lymphocyte responses in vivo. Accordingly, IL-2 has been used clinically to enhance T cell immunity in patients with AIDS or cancer, and blocking Abs to the IL-2R are used to inhibit T cell responses against transplanted tissues. It was later shown in mice that, unexpectedly, disruption of the IL-2 pathway results in lymphoid hyperplasia and autoimmunity rather than immune deficiency, indicating that the major physiological function of IL-2 is to limit rather than enhance T cell responses. This apparent paradox has recently been resolved with the discovery that IL-2 is critical for the development and peripheral expansion of CD4(+)CD25(+) regulatory T cells, which promote self-tolerance by suppressing T cell responses in vivo. Our new understanding of IL-2 biology prompts a re-evaluation of how best to clinically manipulate this important immunoregulatory pathway.     

IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression

Both mast cells (MCs) and regulatory T cells (Tregs) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MCs and Tregs have been shown to play a protective role. Transfer of wild-type (wt) Tregs into wt recipients almost completely prevents development of NTS and leads to a profound increase of MCs in the renal draining lymph nodes (LNs). By contrast, transfer of wt Tregs into animals deficient in MCs, which are characterized by an exaggerated susceptibility to NTS, no longer exhibited protective effects. Blocking the pleiotropic cytokine IL-9, known to be involved in MC recruitment and proliferation, by means of a mAb in mice receiving Tregs abrogated protection from NTS. Moreover, transfer of IL-9-deficient Tregs also failed to protect from NTS. In the absence of Treg-derived IL-9, MCs fail to accumulate in the LNs, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Tregs. In summary, to our knowledge, we provide the first direct in vivo evidence that the nephroprotective, anti-inflammatory effects of Tregs critically depend on IL-9-mediated attraction of MCs into kidney-draining LNs.     

Human CD4+ regulatory T cells and activation-induced tolerance

In the past years growing attention has been given to the role of regulatory T (Tr) cells in inducing and monitoring peripheral tolerance. Various subsets of Tr cells have been described based on their surface phenotype and cytokine production. However, presently there are no specific reliable markers for any of the Tr subsets and their classification is based predominantly upon their mode of suppression. In addition, the molecular mechanisms underlying the induction and mode of action of all Tr cell subsets remain to be elucidated. Here we review recent developments regarding human CD4+ Tr cells, their origin, phenotype, antigen specificity and mode of suppression.     

Gammadelta T cells promote anterior chamber-associated immune deviation and immune privilege through their production of IL-10

Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance that is induced by introducing Ags into the anterior chamber (AC) of the eye, and is maintained by Ag-specific regulatory T cells (Tregs). ACAID regulates harmful immune responses that can lead to irreparable injury to innocent bystander cells that are incapable of regeneration. This form of immune privilege in the eye is mediated through Tregs and is a product of complex cellular interactions. These involve F4/80+ ocular APCs, B cells, NKT cells, CD4+CD25+ Tregs, and CD8+ Tregs. gammadelta T cells are crucial for the generation of ACAID and for corneal allograft survival. However, the functions of gammadelta T cells in ACAID are unknown. Several hypotheses were proposed for determining the functions of gammadelta T cells in ACAID. The results indicate that gammadelta T cells do not cause direct suppression of delayed-type hypersensitivity nor do they act as tolerogenic APCs. In contrast, gammadelta T cells were shown to secrete IL-10 and facilitate the generation of ACAID Tregs. Moreover, the contribution of gammadelta T cells ACAID generation could be replaced by adding exogenous recombinant mouse IL-10 to ACAID spleen cell cultures lacking gammadelta T cells.     

Induction of regulatory T cells and dominant tolerance by dendritic cells incapable of full activation

Transplants tolerated through a process known as infectious tolerance evoke continuous recruitment of regulatory T (Treg) cells that are necessary to maintain the unresponsive state. This state is maintained long-term and requires continuous Ag exposure. It is not known, however, whether infectious tolerance operates through sustained recruitment of pre-existing regulatory cells, induction of regulatory cells, or both. Using mice deficient in natural Treg cells, we show here that quiescent donor dendritic cells (DC) laden with histocompatibility Ag can induce Treg cells de novo that mediate transplantation tolerance. In contrast, fully activated DC fail to do so. These findings suggest that DC incapable of delivering full activation signals to naive T cells may favor their polarization toward a regulatory phenotype. Furthermore, they suggest a role for quiescent endogenous DC in the maintenance of the tolerant state.     

Activated CD69+ T cells foster immune privilege by regulating IDO expression in tumor-associated macrophages

Substantial evidence indicates that immune activation at stroma can be rerouted in a tumor-promoting direction. CD69 is an immunoregulatory molecule expressed by early-activated leukocytes at sites of chronic inflammation, and CD69(+) T cells have been found to promote human tumor progression. In this study, we showed that, upon encountering autologous CD69(+) T cells, tumor macrophages (MΦs) acquired the ability to produce much greater amounts of IDO protein in cancer nests. The T cells isolated from the hepatocellular carcinoma tissues expressed significantly more CD69 molecules than did those on paired circulating and nontumor-infiltrating T cells; these tumor-derived CD69(+) T cells could induce considerable IDO in monocytes. Interestingly, the tumor-associated monocytes/MΦs isolated from hepatocellular carcinoma tissues or generated by in vitro culture effectively activated circulating T cells to express CD69. IL-12 derived from tumor MΦs was required for early T cell activation and subsequent IDO expression. Moreover, we found that conditioned medium from IDO(+) MΦs effectively suppressed T cell responses in vitro, an effect that could be reversed by adding extrinsic IDO substrate tryptophan or by pretreating MΦs with an IDO inhibitor 1-methyl-DL-tryptophan. These data revealed a fine-tuned collaborative action between different types of immune cells to counteract T cell responses in tumor microenvironment. Such an active induction of immune tolerance should be considered for the rational design of effective immune-based anticancer therapies.     

Gamma delta T cells are needed for ocular immune privilege and corneal graft survival

It has been recognized for over a century that the anterior chamber of the eye is endowed with a remarkable immune privilege. One contributing component is the Ag-specific down-regulation of systemic delayed-type hypersensitivity (DTH) that is induced when Ags are introduced into the anterior chamber. This phenomenon, termed anterior chamber-associated immune deviation (ACAID), culminates in the generation of regulatory cells that inhibit the induction (afferent suppression) and expression (efferent suppression) of DTH. Since gamma delta T cells play a major role in other forms of immune regulation, we suspected they might contribute to the induction and expression of ACAID. Mice treated with anti-gamma delta Ab failed to develop ACAID following anterior chamber injection of either soluble Ag (OVA) or alloantigens (spleen cells). Additional experiments with knockout mice confirmed that mice lacking functional gamma delta T cells also fail to develop ACAID. Using a local adoptive transfer of DTH assay, we found that gamma delta T cells were required for the generation of regulatory T cells, but did not function as the efferent regulatory cells of ACAID. The importance of gamma delta T cells in corneal allograft survival was confirmed by blocking gamma delta T cells with GL3 Ab before corneal transplantation. While in vivo treatment with normal hamster serum had no effect on corneal graft survival, infusion of anti-gamma delta Ab resulted in a profound increase in corneal allograft rejection. Thus, gamma delta T cells are needed for sustaining at least one aspect of ocular immune privilege and for promoting corneal allograft survival.     

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.