Shortly after the role of T cells in protective immunity was recognized, Gershon and coworkers1 proposed that a subpopulation of T cells might be capable of suppressing the immune response. Accordingly, the term “suppressor T cell” was coined to describe T cells that were responsible for processes that were previously categorized as tolerance, antigen competition, or feedback regulation. Initially, suppressor cells were defined largely by their in vitro functional properties. However, the suppressor cell concept quickly fell out of favor due to the absence of distinct surface markers or transcription factors unique to this cell population along with the inability of suppressor cell investigators to identify genes in the putative I-J region in the immune response region of the murine major histocompatibility complex (MHC) implicated in suppressor cell development.2 As a result, suppressor cell research was largely abandoned for over a decade until it was resurrected by Sakaguchi et al3 who provided compelling evidence for the presence of CD4 + CD25+ suppressor cells. However, this time around, suppressor cells wore the euphemistic moniker T regulatory cells (Treg). Since Sakaguchi's report, hundreds of publications have described and analyzed CD4+ Treg and the overwhelming majority of these publications have focused on conventional CD4 + CD25 + Foxp3+ Treg. Because numerous review articles have addressed the biology and immunologic properties of CD4 + CD25 + Foxp3+ Treg, this review will focus on non-CD4 + CD25 + Foxp3+ Treg in the context of organ transplantation.
CD8+ Treg Phenotypes
The phenotype of Gershon's original suppressor cell was defined by its expression of the CD8 (Lyt-2) surface marker.4 However, in the 40 years since their original description, CD8+ Treg have been categorized into several distinct phenotypes including: (a) Qa-1/HLA-restricted Treg5-7; (b) CD8 + CD122+ Treg8; (c) CD8 + CD28− Treg9; (d) CD8 + Foxp3+ Treg10; (e) CD8 + CD103+ Treg11; (f) CD8 + LAG-3 + Foxp3 + cytotoxic T lymphocyte-associated protein (CTLA-4)+ Treg12; (g) CD8 + IL-10 + CCR7 + CD45RO+ Treg13; (h) CD8 + CD45RClow Treg14; (i) CD8 + CD122 + PD-1+ Treg,15 and (j) CD8 + CD11chigh Treg16 (Table 1). CD8+ Treg can arise within the thymus as naturally occurring CD8+ Treg or they can be induced in peripheral tissues by a variety of maneuvers including donor-specific transfusion,21 injection of antigen into the anterior chamber (AC) of the eye,22 anti-idiotype immunization with allospecific T cells or MHC-derived peptides,23,24 or by in vivo immunization combined with blockade of CD40 costimulatory molecules.25 Expression of the IL-2 receptor β subunit, CD122, is crucial for CD8+ Treg development and function.7 Although CD122+ is also expressed on classical CD8+ memory T cells, expression of PD-1 on CD8 + CD122+ T cells is limited to Treg and distinguishes Treg from memory T cells.26 In addition to the surface markers mentioned above, CD8+ Treg can also express CD44 and the natural killer (NK) cell inhibitory marker, Ly49.
CD8+ Treg Mechanisms of Suppression
CD8+ Treg suppression can be mediated by either contact-dependent27 or contact-independent mechanisms28 (Table 2). Contact-dependent suppression by CD8+ Treg involves the direct killing of CD4+ T effector cells by perforin-mediated cytolysis29,30 or Fas ligand (FasL)-induced apoptosis.16 In addition to suppressing effector T cells, CD8 + Foxp3+ Treg are capable of inducing the de novo generation of CD4 + Foxp3+ Treg by a process that is contact-dependent and requires the production of soluble TGF-β,31 and is reminiscent of a phenomenon that has been previously described as “infectious tolerance.” When CD8+ Treg are in contact with CD4+, T cell suppression is supported by IFN-γ, indoleamine 2,3 dioxygenase, and fibroleukin-2.28 However, indoleamine 2,3 dioxygenase can also mediate suppression when CD8+ Treg are not in direct contact with CD4+ effector T cells by inhibiting T cell proliferation.28 CD8+ Treg can also suppress immune effector responses by elaborating a variety of soluble factors, such as TGF-β and IL-10, which inhibit T cell activation and proliferation.32,33
Recent investigations on CD8+ Treg have demonstrated the importance of the nonclassic MHC Ib molecules Qa-1 (mouse) and HLA-E (human) in the induction of CD8+ Treg.7 Engagement of Qa-1-Qdm peptides expressed on CD4+ Th1 cells with its receptor, NKG2A/CD94 on CD8+ T cells leads to the generation of CD8+ Treg.7 Qa-1–restricted CD8+ Treg are believe to arise in the thymus and play a central role in the maintenance of self-tolerance.7 They can also be induced in the periphery by T-cell vaccination34-36 or by introducing alloantigens into the AC of the eye.37,38
CD8+ Treg and Transplantation
The majority of studies on Treg have focused on CD4 + CD25+ Treg and it has been assumed by many that CD8+ Treg were minor players in the maintenance of self-tolerance and in the promotion of allograft survival. However, recent evidence suggests that CD8 + CD122+ naturally occurring Treg play a crucial role in immune homeostasis and contribute to allograft survival15,26,39-41 (Table 3). In fact, there is compelling evidence that CD8+ Treg are more potent than conventional CD4 + CD25+ Treg in suppressing pancreatic islet allograft rejection.39 In 1 study, CD8 + CD122+ Treg underwent swifter expansion, produced more IL-10, and suppressed T cell proliferation in vitro more effectively than their CD4 + CD25+ Treg counterparts.39 Importantly, adoptively transferred CD8 + CD122+ Treg were able to prolong the survival of pancreatic islet allografts, whereas similar adoptive transfers of CD4 + CD25+ Treg were unable to prolong allograft survival.39 CD8 + CD28− Treg can promote the generation of tolerogenic dendritic cells (DCs) and are expanded in heart allograft recipients.44 A mouse study of allogenic stem cell transplantation revealed that in addition to the conventional role of CD4+ Foxp3+ Treg in this model, CD8 + Foxp3+ Treg were able to prevent graft-versus-host disease mortality.57
CD8+ Treg may also be one of the key elements sustaining immune privilege in the eye. Nominal antigens or alloantigens introduced into the AC of the eye elicit the generation of antigen-specific CD8+ Treg through a process called AC-associated immune deviation.61 Interestingly, 2 populations of Treg are induced after AC injection of alloantigens, CD4+ Treg and CD8+ Treg.22 The CD8+ Treg that are generated in the spleen after AC injection of antigen suppress both ocular and systemic immune responses by elaborating TGF-β, IL-10, and IFN-γ.61,62 Additionally, CD8+ Treg are also induced in situ by cells lining the AC of the eye. Corneal endothelial cells express membrane-bound TGFβ2, which directly inhibits the activation of effector CD8+ T cells in situ and also induces the conversion of effector CD8+ T cells to Treg63 that elaborate TGFβ1. The pigmented epithelial cells of the iris also induce the generation of CD8+ Treg by B7-1/B7-2 interaction with CTLA-4 on effector T cells.64,65 It bears noting that injection of donor-specific alloantigenic cells into the AC of the eye produces AC-associated immune deviation and dramatically enhances corneal allograft survival.66,67
While most studies have focused on CD4 + CD25 + Foxp3+ Treg in promoting allograft survival, there is a sizeable body of evidence that CD8+ Treg can also enhance the survival of skin,25,42 heart,21,25,28,44,45 pancreatic islet,25,39 and kidney53 allografts and heart25 and corneal24 xenografts, and hematopoietic allografts57 (Table 3).
T Cell Receptorαβ + CD4−CD8− Double Negative Treg
Double Negative Treg Phenotypes
In 1989, Strober and colleagues68 described and cloned a population of spleen cells that did not express either CD4 or CD8, but could suppress T cell proliferation in vitro. The CD4−, CD8− T cell population was termed double negative (DN) T cells. The DN T cells express the αβ T cell receptor (TCR), yet do not express CD4, CD8, or NK cell markers and represent 1% to 5% of the αβ TCR positive T cells in mice and humans.17,69 Although no specific marker has been identified for DN Treg, a lack of Foxp3 expression and patterns of surface markers have been reported70 (Table 1). The origin of DN T cells is still unclear. Some studies suggest that DN αβ TCR+ T cells can be derived from CD8+ T cells,71 whereas others have shown that CD4+ T cells can convert to DN Treg both in vitro and in vivo.18 Cloned DN Treg from TCR transgenic mice are identified as CD25 + CD30 + CD28low,17 whereas DN Treg arising from CD4+ precursors are CD28 + CD25 + CD44 + CD69 + .18 Isolated human DN Treg are CD27 + CD28lowCD25 − .19 Subsequent studies reported that DN Treg displayed antigen-specific suppressive activity both in vitro and in vivo.17 The DN Treg not only suppress CD8+ T cell responses but also inhibit CD4+ T cells,43 B cells,55 NK cells,58 and DCs.72
DN Treg Mechanisms of Suppression and Impact on Transplantation
The DN Treg have a unique capacity to acquire the entire MHC-alloantigen complex from antigen presenting cells' (APCs) cell membrane via a cell-contact-dependent interaction with the TCR on the DN Treg via a process termed “trogocytosis” and to express the captured MHC-alloantigen complex on their cell surface.73 The effector CD8+ T cell binds to the captured MHC-alloantigen complex that is now presented on the DN Treg, which culminates in the transmission of a death signal to the CD8+ T cells.73-75 Accordingly, suppression by DN Treg is antigen-specific (Figure 1).
There is evidence that the death signal delivered by the DN Treg to the CD8+ T cells in mice is through Fas/FasL,75 whereas other studies suggest a role for perforin-mediated cytolysis18 (Table 2). The suppression of CD8+ T cells by DN T cells via recognition of alloantigens and the killing of CD8+ effector T cells is reminiscent of the “veto cell” concept that was proposed 35 years ago.76 Adoptive transfer of DN Treg after a cardiac allograft in mice prolonged graft survival as well as augmented the Foxp3+ Treg population.46 There is also evidence that murine DN Treg regulate immune responses at the level of antigen-presenting DCs. Gao and co-workers72 demonstrated that murine DN Treg expressed high levels of CTLA-4 and downregulated costimulatory molecules CD80 and CD86 on antigen-presenting mature DCs. Moreover, DN Treg killed syngeneic antigen-loaded DCs or allogeneic DCs through a Fas-FasL pathway.
The DN Treg can also suppress B-cell and NK-cell responses. Ma and coworkers55 found that adoptively transferred DN Treg prolonged rat heart xenograft survival in mice and induced B cell apoptosis via a perforin-dependent process. As might be expected, antidonor IgM and IgG antibody titers were significantly diminished in recipients of adoptively transferred DN Treg.55 In a model of murine allogenic bone marrow transfer, DN Treg supported graft survival through suppressing NK cells by perforin and Fas-FasL–dependent pathways.58
In contrast to murine DN Treg, human DN Treg do not kill effector T cells.77 Although cell-cell contact is required for human DN Treg to function, the suppressive activity of human DN Treg is not Fas/FasL or perforin-mediated. Suppression by DN Treg is reversible, and the function of previously suppressed effector T cells can be restored once DN Treg are removed. Soluble factors, such as IL-10 and TGF-β, are not involved in suppression mediated by DN Treg.77 In a study of 40 human patients who received a hematopoietic stem cell allograft, the percent of DN Treg in the peripheral blood was inversely correlated with risk of graft rejection.59
The DN Treg have been shown to enhance the survival of skin,17,18,43 pancreatic islet,18,49 and heart allografts46-48 and heart xenografts55,56 and hematopoietic stem cell allografts58,59 (Table 3).
Type 1 Regulatory T Cells
Type 1 Treg Phenotypes
Two categories of CD4+ Treg have been described: conventional CD4 + CD25 + Foxp3+ Treg78 and type 1 Treg (Tr1 cells).79 The Tr1 cells are found in both humans and mice and are characterized by their copious secretion of IL-10 and their lack of Foxp3 expression. Human and mouse Tr1 clones coexpress CD49b and lymphocyte activation gene 3 (LAG-3), which distinguishes Tr1 cells from other CD4+ T cells, including Th1, Th2, Th17, and Foxp3+ Treg20 (Table 1). The Tr1 cells are induced in vivo after chronic antigenic stimulation in the presence of IL-1080 or in vitro by activating naive T cells through their TCR in the presence or IL-10 alone or IL-10 in combination with immunosuppressive drugs, such as dexamethasone or rapamycin (RPM).50,79,81
A growing body of evidence indicates that IL-27 produced by tolerogenic DCs is a crucial differentiation factor for the development of IL-10–producing Tr1 cells in mice82-84 and humans.85 However, Jin and coworkers86 have recently shown that IL-6 can induce the differentiation of murine IL-10–producing Tr1-like Treg from naive CD4+ T cells in the absence of IL-27, suggesting that IL-6 produced in inflammatory conditions might serve as a feedback mechanism for generating Tr1 cells that dampen inflammation and restore homeostasis.
Tr1 Mechanisms of Suppression and Impact on Transplantation
The Tr1 cells mediate immune suppression by secreting IL-10 and by killing antigen-presenting cells via a perforin/granzyme-dependent process that requires MHC class I recognition87 (Table 2). The Tr1 also suppress Th17 cells in murine model of colitis by an IL-10–dependent process.88
The first indication that Tr1 cells might contribute to allograft tolerance came from severe combined immunodeficiency patients who developed long-term tolerance to stem cell allografts and expressed cells with Tr1-like properties.89 Subsequent studies showed that Tr1 cells were associated with the induction of mixed chimerism in patients receiving hematopoietic stem cell transplants.60 Investigations in murine allograft models have demonstrated a role for Tr1 cells in tolerance in pancreatic islet transplantation.50,51 In humans, Tr1 cells can also contribute to transplantation tolerance as Tr1 cells have been detected in patients who spontaneously developed tolerance to kidney or liver allografts54 and also occur in mice treated with IL-10 in combination with RPM a means of establishing tolerance for pancreatic islet transplants.50 In human subjects, Tr1 cells have been associated with tolerance in kidney,54 pancreatic islet,52 and liver54 allografts (Table 3).
Effect of Immunosuppressive Agents on Treg Generation and Function
Immunosuppressive drugs, such as cyclosporine A (CsA) and RPM, have made organ transplantation a feasible and effective therapeutic option for treating end-stage organ failure. By contrast, the clinical application of Treg in promoting the long-term survival of organ transplants is gaining traction but much remains to be improved before it becomes a reality. It is important to address the impact of immunosuppressive drugs in the context of various organ transplantations because they may have varied effects depending on dose, combination treatments, or the type of organ affected. A key issue is whether conventional antirejection agents can be combined with Treg for promoting graft acceptance or if immunosuppressive drugs have untoward effects on Treg function. Cyclosporine A and RPM have been used successfully to prevent transplant rejection. Although both CsA and RPM target IL-2, they have remarkably different effects on the generation and function of CD4 + CD25 + Foxp3+ Treg. Cyclosporine A inhibits IL-2 transcription and synthesis, whereas RPM acts downstream by blocking T-cell responses to IL-2. Using a murine heart allograft model, Coenen and co-workers found that CsA treatment resulted in a sharp reduction in peripheral CD4 + CD25 + Foxp3+ Treg, whereas RPM treatment did not reduce the generation of these Treg.90 Moreover, RPM, but not CsA, induces de novo generation of CD4 + CD25 + Foxp3+ Treg which potentiate murine skin allograft survival in an alloantigen-specific manner.91 Similar findings were seen in another skin allograft model where RPM treatment supported Treg and CsA antagonized Treg expansion.92
Immunosuppression by preventing the egress of T cells from lymphoid organs can be achieved by FTY720 (Fingolimod), a sphingosine-1-phosphate receptor agonist. In a murine corneal allograft model, graft survival was prolonged by topical treatment with CsA or FTY720, with an increase in CD4+ Treg found in the FTY720-treated mice.93 A model using adoptive transfer for allograft rejection found that either RPM or FTY720 treatment significantly enhanced conversion of CD4 + CD25 + Foxp3+ Treg.94
Histone deacetylase inhibitors (HDACs) may be a novel route for promoting Treg presence by preventing the conversion of Treg into effector T cells through enhancing the access to Foxp3 within the chromatin. An HDAC, suberoylanilide hydroxamic acid, synergized with low doses of tacrolimus to prolong cardiac allograft survival in mice by promoting expression of Treg molecules, Foxp3 and CTLA-4, as well as increasing apoptosis of T effector cells.95 Furthermore, if the cardiac graft was introduced into Foxp3-deficient recipient mice, suberoylanilide hydroxamic acid treatment was still able to marginally increase allograft survival, suggesting that non-Foxp3 regulatory cells are also impacted by HDACs. Another HDAC, trichostatin A, in combination with donor-cell transfusion increased CD4 + CD25 + Foxp3+ Treg numbers and promoted mouse pancreatic islet graft survival.96
There are still many clinical drugs that could potentially impact Treg numbers and functions. A high-throughput screen, flow cytometry based assay using Foxp3-GFP reporter mice evaluated the in vitro effects of 640 FDA-approved drugs and found that after 3 days in culture 75 drugs significantly increased Treg numbers.97 This study measured Foxp3+ T cell numbers, thus it warrants future investigations to determine if the same drugs, or a different combination of drugs, have similar impacts on non-Foxp3+ Treg.
Few studies have been conducted with regard to the impact of immunosuppressive agents on the generation or function of non-CD4 + CD25 + Foxp3+ Treg. In vitro studies using human peripheral blood mononuclear cells found that RPM caused an increase in the numbers of CD103 + CD8+ alloreactive T cells with immunosuppressive properties, whereas CsA had no significant effect on the percentage of these cells and prednisolone diminished the numbers of these cells.98 CD8 + CD28- Treg function was improved in rheumatoid arthritis patients that received TNF-α inhibitor therapy, whereas patients treated with methotrexate had no effects on the defective CD8 + CD28− Treg activity normally found in these patients.99 There is a dearth of published reports on the effects of immunosuppressive agents on Tr1 Treg and DN Treg activity in the context of allograft and xenograft survival. Thus, there are significant gaps in our knowledge about the effects of immunosuppressive agents and the non-CD4 + CD25 + Foxp3+ Treg.
Since Sakaguchi's discovery of CD4 + CD25+ Treg, over 25,000 publications have dealt with the general topic of Treg. The overwhelming majority of these publications have focused on the role of CD4 + CD25+ Treg. However, in recent years, there has been a growing awareness of the importance of CD8+ Treg, DN Treg, and Tr1 cells in controlling autoimmune diseases and in enhancing allograft survival. The presence of multiple populations of Treg is a reflection of the remarkable redundancy and plasticity of the immune system. The importance of CD4 + CD25 + Treg for immune homeostasis is well recognized. Deficiencies in Foxp3 expression invariably lead to lymphoproliferative and multiorgan autoimmune diseases in both humans and mice. It has been suggested that CD4 + CD25+ Treg are generated in response to the initial priming stage of the immune response and act to limit immune-mediated inflammation that inflicts damage to juxtaposed tissues in various organs. By contrast, CD8+ Treg, and perhaps DN Treg, are generated from previously activated T cells. In both cases, Treg act to suppress immune-mediated inflammation and restore immune homeostasis. In certain conditions, organ allografts and xenografts benefit from the development of these nonconventional Treg. Harnessing CD4 + CD25+, CD4− CD8+, DN Treg, and Tr1 cells as a comprehensive means of enhancing the survival of allografts and xenografts in patients at high risk of rejecting their grafts is an appealing goal that is still in its early stages of development.
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