CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8⁺CD122⁺ cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8⁺CD122⁻ population, were previously shown to include cells with regulatory activity and could be separated into CD49d^low cells and CD49d^high cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122⁺ cells. Regulatory activity was observed in CD8⁺CD122⁺CD49d^low but not in CD8⁺CD122⁺CD49d^high cells, indicating that the regulatory cells in the CD8⁺CD122⁺ population could be narrowed down to CD49d^low cells. CD8⁺CD122⁻ cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122⁺ Tregs. CD122⁺ Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8⁺CD122⁻ cells, indicating that the regulation by CD122⁺ Tregs is Fas/FasL-dependent. CD122⁺ Tregs taken from IL-10–deficient mice could regulate CD8⁺CD122⁻ cells as equally as wild-type CD122⁺ Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122⁺ Tregs in vitro. CD122⁺ Tregs also regulated CD4⁺ cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122⁺ Tregs that kill activated T cells to maintain immune homeostasis.Fas (CD95) and its ligand FasL (CD178) compose a unique system that is strongly related to programmed cell death (1). FasL has been considered one of the effector molecules involved in the killing of target cells by cytotoxic T lymphocytes (CTLs) (2). When Fas binds to FasL, it induces downstream signal transduction pathways that subsequently activate cell death induction pathways (3, 4). Thus, the Fas/FasL system appears to act as an effector for CTL-mediated killing of virus-infected or cancer cells, similar to the perforin–granzyme system (5, 6). However, because Fas-mutant (lpr) and FasL-deficient (gld) mice show lymphoproliferative changes, it has been suggested that the Fas/FasL system is important for suppression/regulation of activated effector T cells (7, 8).Molecular mechanisms after Fas activation have been thoroughly investigated, and the signal transduction pathway has been largely elucidated (9, 10). However, research on the cellular events that use the Fas/FasL system has progressed comparatively slowly. There are only a few relevant reports in this respect, mostly of human CD4⁺Foxp3⁺ Tregs that use the Fas/FasL system for their regulatory activity. [In this article, we use the term “Treg(s)” for any kind of T cells that show immune regulatory activity.] To complicate matters further, some contradictory reports claim that such Fas/FasL-engaging Tregs do not exist (8, 11, 12). No reliable reports suggesting that CD8⁺ T cells use the Fas/FasL system for their regulatory action are available. Therefore, it is not clear precisely which subset of T cells express FasL or where Fas/FasL-dependent CD8⁺ Tregs, if such cells exist, are located and at which point they function. Thus, the ultimate pathophysiological role of the Fas/FasL system is still unknown.Regulation of the immune reaction is of pivotal importance for maintaining health in multicellular organisms. In highly developed animals, Tregs, a subset of T lymphocytes especially known as CD4⁺CD25⁺Foxp3⁺ cells, are the main regulators of the immune response (13–16). However, it is not quite clear whether CD8⁺ regulatory T cells exist; there are only few and contradictory reports on their existence, in contrast to the reports on CD4⁺ Tregs. A population of predominantly CD8⁺ suppressor T cells has been described and debated in the 1980s (17, 18). In the 2000s, we found and reported that the cells of central memory phenotype (CD8⁺CD122⁺) also possess the regulatory function, and some other reports regarding CD8⁺ Tregs with some contradictory findings have been published. To date, more than 10 CD8⁺ Treg populations with different markers have been reported (19).One of the best characterized CD8⁺ Treg populations is the CD8⁺CD122⁺ Treg (122⁺ Treg) population. To confirm the existence of CD8⁺ Tregs, markers that may be possible to distinguish Tregs from other T cells were examined. We previously found that CD49d can separate CD8⁺CD122⁺ cells into at least two subsets (CD49d^low and CD49d^high) (20). To judge which cells have a stronger regulatory activity, we prepared two experimental systems: an in vitro system, based on cell culture, and an in vivo system, based on adoptive transfer of T cells.Interestingly, CD8⁺ Tregs express CD122 (IL-2/IL-15 receptor β chain) in contrast to CD4⁺ Tregs, which express CD25 (IL-2 receptor α chain), indicating the fundamental importance of IL-2 at the development/maturation of both CD4⁺ and CD8⁺ Tregs. Suzuki and coworkers generated CD122-deficient mice using gene targeting (21) and used them to identify 122⁺ Tregs (22). In this study, which is a follow-up study to our previous report, we performed in vitro and in vivo experiments to gain further understanding of these cells. We found that CD49d might be a good marker for classifying CD8⁺ T cells into naïve, resting T cells (CD62L⁺CD122⁻), effector memory T cells (CD62L⁻), and T cells of central memory phenotype (CD122⁺CD49d^low), by using multicolor staining of CD62L, CD122, and CD49d. To our knowledge, this is the first report on the role of the Fas/FasL system in the action of CD8⁺CD122⁺ Tregs (122⁺ Tregs) (22–25). Additionally, we show that CD8⁺ Tregs are included in the CD49d^low population, which corresponds to the central memory T-cell population, and that their mechanism of suppression depends on the cytotoxicity mediated by the Fas and FasL interaction.