EMERGING CONCEPTS IN CD8⁺ T REGULATORY CELLS

Summary

CD8⁺ T regs are elicited by unique antigen presenting cells during viral infections, by manipulation of co-stimulatory molecules, or in the development of tumors. CD8⁺ T regs display antigen-specificity, which is most exquisitely manifested by the HLA-E-restricted cytolytic CD8⁺ T regs in MS. There is evidence that some CD8⁺ T regs also express organ specificity. In many cases, IFN-γ is required for either the induction or expression of CD8⁺ T regs. CD8⁺ T regs can produce suppression directly by killing immune cells or indirectly by co-opting other cells to elaborate end-stage suppressive molecules such as TGF-β, IL-10, and indoleamine dioxygenase (IDO).

Introduction

The seminal findings of Sakaguchi and co-workers resurrected the concept of suppressor cells and demonstrated the role of CD4⁺CD25⁺ T cells in self-tolerance[1]. Legitimization of suppressor cells was marked by their new euphemistic moniker, T regulatory cells (T regs). The past decade has witnessed an explosion in the number of studies on both natural and induced CD4⁺CD25⁺ T regs, while research on CD8⁺ T regs has received considerably less attention. In spite of this, significant new insights have come to light and indicate that CD8⁺ T regs play a unique role in restoring immune homeostasis and in maintaining immune privileged sites.

Induction of CD8⁺ T regs via Co-Stimulatory Molecule Interactions

CD4⁺CD25⁺ T regs can arise naturally in the thymus or can be induced by a variety of manipulations. By contrast, CD8⁺ T regs do not normally occur naturally, but are induced under a variety of conditions including manipulation of co-stimulatory molecule interactions[2–4], antigen processing by unique antigen presenting cells (APC)[5,6], during viral infections[7], or in the development of some tumors[8].

The co-stimulatory molecule CD137 (4-1BB) is important for immunity against tumors and viruses, yet it also inhibits experimental autoimmune diseases[3]. Myers and co-workers recently showed that immunization with OVA in combination with polyI:C and anti-4-1BB induced the generation of CD8⁺ T regs that profoundly inhibited CD4⁺ T cells[3]**. The anti-4-1BB-induced suppression was mediated by CD8⁺ T regs and required the presence of IFN-γ, yet IFN-γ alone did not mediate suppression. CD8⁺ T regs did not function unless they produced and responded to IFN-γ. That is, anti-4-1BBinduced CD8⁺ T regs did not develop in IFN-γR ^−/− mice. Binding of IFN-γ to the CD8⁺ T regs stimulated the production of TGF-β, which acted as the end stage suppressive molecule. Moreover, addition of neutralizing anti-TGF-β reversed the suppressive effects of CD8⁺ T regs. Thus, engagement of the co-stimulatory molecule 4-1BB generates CD8⁺ T regs that both produce and respond to IFN-γ, which in turn, induces the elaboration of TGF-β, which suppresses antigen-specific proliferative responses of CD4⁺ T cells.

A recent study demonstrated that blockade of the co-stimulatory molecule, CD40, promoted the indefinite survival of heart allografts in rats that was mediated by CD8⁺ T regs[9]**. Graft survival could be adoptively transferred with spleen cells through four generations of hosts. This “infectious tolerance” was entirely dependent on CD8⁺ T cells and IFN-γ, as treatment with either anti-CD8 antibody or neutralizing anti-IFN-γ antibody resulted in allograft rejection. Allografts contained an abundance of IFN-γ, which is a major stimulus for the induction and expression of indoleamine dioxygenase (IDO). IDO is a potent inhibitor of tryptophan metabolism and it is widely recognized that tryptophan deprivation leads to T cell apoptosis. Blockade of IDO with 1-methyl tryptophan resulted in rapid graft rejection in tolerized rats. Immunohistochemical analysis revealed that IDO was expressed primarily in the vascular endothelial cells of the heart allografts. This demonstrates for the first time, the generation of a novel CD8⁺ T reg population that mediates infectious tolerance through at least 4 generations and promotes allograft survival by co-opting cells within the allograft to produce the potent immunosuppressive molecule IDO. This in turn creates an ad hoc immune privileged site.

Antigens Introduced into an Immune Privileged Site Induce CD8⁺ T regs

One of the more important factors that contributes to ocular immune privilege is the unique immune deviation that is elicited when antigens are introduced into the anterior chamber (AC)[10,11]. Anterior chamber-associated immune deviation (ACAID) culminates in the appearance of CD8⁺ T regs that suppress Th1 immune responses, such as delayed-type hypersensitivity (DTH), and deviates antibody responses from complement-fixing isotypes to non-complement-fixing isotypes. F4/80⁺ ocular APC capture antigens introduced into the AC and then migrate to the spleen where they initiate a series of cellular interactions involving NKT cell emigrants from the thymus[12,13], B cells[5,6,14] CD4⁺ T cells[15,16], CD8⁺ T cells[17], and γδ T cells[18,19]. The process begins when ocular APC release antigenic fragments, which are captured by antigen-specific B cell receptors on splenic B cells. After internalizing and processing the cognate antigen, B cells proliferate thereby expanding the numbers of antigen-specific splenic B cells that are available for capturing and presenting antigen to T cells[5,6,20]. B cells utilize both MHC class I and class II molecules to present peptides to CD8⁺ and CD4⁺ T cells respectively[5]*. Following antigen presentation by B cells, the T cells differentiate into CD4⁺ T regs and CD8⁺ T regs.

Generation of ACAID CD8⁺ T regs also requires the participation of splenic γδ T cells[18,19]. Although γδ T cells can act as APC in other settings[21], they do not participate in antigen presentation in ACAID, and instead they serve as important sources of IL-10 within the splenic milieu[22]. γδ T cell-derived IL-10 is required for the induction of ACAID CD8⁺ T regs, as reconstitution of γδ TCR^−/− mice with γδ T cells from IL-10 ^−/− mice fails to restore ACAID CD8⁺ T regs, while reconstitution with γδ T cells from IL-10 competent mice does.

NKT cells also participate in the generation of ACAID. Within the spleen, CD-1d-reactive NKT cells produce RANTES, which recruits precursor CD8⁺ T regs and additional eye-derived F4/80⁺ APC to the spleen [23]. NKT cells produce IL-10, a crucial cytokine needed for the generation of ACAID CD8⁺T regs[24]. NKT cells also produce urokinase-type plasminogen activator (uPA), which contributes to the generation of CD8⁺T regs by binding to the uPA receptor on on F4/80⁺ APC within the spleen and converting latent TGF-β into its active form.[25]**. Active TGF-β, along with NKT cell-derived IL-10, promotes the generation of CD8⁺ T regs.

ACAID CD8⁺ T regs inhibit DTH by previously sensitized CD4⁺ T cells by a mechanism that requires IFN-γ[26]. Neither ACAID CD8⁺ T regs from IFN-γR^−/− mice nor IFN-γ alone can inhibit DTH. However, IFN-γ acts on CD8⁺ T regs induced in normal mice and “licenses” them to express their suppressive function in situ. The mode of suppression by CD8⁺ T regs remains to be elucidated, but neither perforin-mediated cytolysis nor FasL-induced apoptosis is necessary[26]*.

CD8⁺ T regs in Multiple Sclerosis

Investigations on T regs in multiple sclerosis (MS) have been largely limited to the roles of CD4⁺ T regs, however recent studies have demonstrated the participation of CD8⁺ T regs[27,28]. CD8⁺CD122⁺ Tregs spontaneously arise in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS. In vivo depletion of CD8⁺CD122⁺ cells exacerbates EAE in mice, while adoptive transfer of CD8⁺CD122⁺cells from naïve donors to recipients at the height of clinical disease mitigates EAE [29]. Correale and Villa detected the presence of spontaneously occurring CD8⁺ T regs in the peripheral blood of MS patients[27]*. CD8⁺ T regs isolated from MS patients lysed myelin-reactive CD4⁺ T cells by a cytolytic process that was granule-mediated and restricted by the non-classical MHC Class Ib molecule HLA-E. HLAE-self-peptide complexes can interact with CD94/NKG2 receptors expressed on NK and CD8⁺ T cells to regulate their activities. Interactions between the killer inhibitory complex, CD94/NKG2A, result in the inhibition of CD8⁺ T cell cytotoxicity against target cells, which in this case were myelin-reactive CD4⁺ T cells from MS patients. Consistent with this, Correale and Villa found significantly elevated expression of CD94/NKG2A on CD8⁺ T regs in patients during exacerbations of MS. Moreover, the cytolytic activity of the CD8⁺ T regs from MS patients could be blocked with anti-HLA-E antibody, which further supports the role of HLA-E-restricted CD8⁺ T regs in MS.

Glatiramer acetate (GA) is a synthetic copolymer that was discovered in studies on the treatment of EAE in rodents and shares key amino acid sequences with myelin basic protein. GA has received FDA approval as an immunotherapy for the treatment of MS, yet until recently, GA’s mode of action has been unclear. However, Tennakoon and co-workers reported that injection of GA induced CD8⁺ T regs that killed GA-reactive CD4⁺ T cells from MS patients[28]**. GA-induced CD8⁺ T regs expressed upregulation of perforin and killed GA-loaded CD4⁺ T cells in a GA-specific manner. Moreover, like the study by Correale and Villa[27], this investigation demonstrated that the cytolysis mediated by CD8⁺ T regs could be blocked with anti-HLA-E antibody. Remissions in MS in patients who were treated with GA were correlated with the spontaneous acquisition of GA peptide-induced CD8⁺ T regs that were HLA-E-restricted and acted to eliminate CD4⁺ pathogenic T cells[28,30].

CD8⁺ T regs that Arise Spontaneously in Tumor-Bearing Hosts

A recent report from Jarnicki and co-workers uncovered an unusual compartmentalization of CD8⁺ T reg activity in murine CT26 colon cancer[8]**. Injection of OVA into subcutaneous (SC) tumors resulted in the impaired OVA-specific T cell proliferation and reduced secretion of IFN-γ. CT26 tumor cells injected intravenously produced lung tumors that secreted TFG-β, which induced the production of IL-10 and TGF-β by tumor-infiltrating CD4⁺ and CD8⁺ T cells. In addition, resident APC in the lungs secreted high levels of IL-10, which further drove the in situ generation of IL-10-producing CD8⁺ T regs[31]. Although both lung and SC tumors induced T regs, in vivo depletion of CD8⁺ T cells resulted in a sharp decrease in lung tumors, but increased growth of SC tumors. Depletion of CD8⁺ T regs resulted in enhanced secretion of IFN-γ by CD4⁺ T cells in the lungs and a commensurate reduction in the growth of lung tumors. By contrast, depletion of CD8⁺ T cells in SC tumor-bearing mice led to a steep reduction in tumor-specific CTL and enhanced growth of the SC tumors. This study was the first to report CD8⁺ T regs in the anti-tumor response in the lungs. Generation of CD8⁺ T regs by lung tumors is reminiscent of the CD8⁺ T regs induced in ACAID. The AC of the eye contains high levels of TGF-β which stimulates IL-10 production by resident APC and renders them tolerogenic. In CT26 lung tumors, resident APC already express elevated levels of IL-10 [31], which are driven even higher by TGF-β that is produced by the lung tumors. In both ACAID and pulmonary CT26 tumors, IL-10 secreting APC induce the generation of CD8⁺ T regs. Thus, the local tissue environment can have a profound effect in shaping the immune response through the preferential induction of CD8⁺ T regs.

Neither Fish nor Foul: Tumor-Promoting CD8⁺ T Cells without Immunoregulatory Activity

The association between inflammation and cancer has been recognized for over a century and has led some to suggest that under some circumstances, a weak immune response promotes, rather than inhibits the development of cancer[32–34]. Recent studies demonstrated that αβ T cell deficiency was associated with reduced progression of chemically induced cancers[35,36]. Two explanations come to mind to account for this curious finding: a) eliminating the αβ T cell population removes a population of T regs that restricts innate immune effector cells (e.g., NKT cells and γδ T cells) or b) a subpopulation of αβ T cells express tumor-promoting properties. Roberts and co-workers found that the growth of chemically induced tumors was stunted in CD8^−/− mice compared to CD4^−/− mice and wild-type syngeneic mice[36]**. Tumor growth was similarly reduced in TCRβ^−/− mice. However, TCRβ^−/− mice reconstituted with αβ TCR CD8⁺ T cells exhibited increased tumor growth similar to wild-type mice. Analysis of tumor-infiltrating lymphocytes (TIL) in chemically induced tumors in wild-type mice revealed the presence of CD8⁺ T cells that expressed a surface marker phenotype consistent with effector-memory cells (CD44⁺CD62L⁻). The CD8⁺ TIL produced high amounts of IFN-γ and TNF-α mRNA when stimulated with anti-CD3. The CD8⁺ TIL also produced COX-2, a major enzymatic inducer of inflammatory mediators and a molecule associated with tumor development. Interestingly, it has been reported that TNF-α ^−/− mice[37] and COX-2^−/− mice are resistant to chemically induced carcinomas[38]. Thus, the tumor-promoting activity of CD8⁺ T cells might be mediated via TNF-α- and COX-2-dependent processes. These results are reminiscent of the “immune stimulation” of cancer hypothesis proposed by Prehn over 30 years ago and re-defined last year [33].

Conclusions

IFN-γ plays a critical role in either the induction or the expression of CD8⁺ T reg activity. On first blush this seems counterintuitive considering the pro-inflammatory properties of IFN-γ. IFN-γ does not act as the end stage suppressive molecule, but functions indirectly by co-opting other cells to produce immunosuppressive and anti-inflammatory molecules such as TGF-B, IL-10, and IDO. Local production of these molecules creates ad hoc immune privileged sites. The challenge ahead is to design strategies that mimic CD8⁺ T regs and to directly deliver these suppressor molecules in proper quantities and times to organs under autoimmune attack or organ allografts undergoing rejection.