CD8+CD122+CD49dlow regulatory T cells maintainT-cell homeostasis by killing activated T cells via
Fas/FasL-mediated cytotoxicity
Kazuyuki Akanea,b, Seiji Kojimab, Tak W. Makc,1, Hiroshi Shikud,e, and Haruhiko Suzukia,1aDepartment of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan; bDepartment of Pediatrics, Nagoya
University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan; cCampbell Family Institute for Breast Cancer Research, Princess Margaret Cancer
Centre/University Health Network, Toronto, Ontario, Canada M5G 2M9; dDepartment of Cancer Vaccine, Mie University Graduate School of Medicine, Tsu,
Mie 514-8507, Japan; and eDepartment of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan

Contributed by Tak W. Mak, January 15, 2016 (sent for review October 5, 2015; reviewed by Toshiaki Ohteki and Heiichiro Udono)

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 CD49dlow cells and CD49dhigh cells. We established in vitro and in vivo experimental systems to evaluate theregulatory activity of CD122+ cells. Regulatory activity was observed in CD8+CD122+CD49dlow but not in CD8+CD122+CD49dhighcells, indicating that the regulatory cells in the CD8+CD122+ population could be narrowed down to CD49dlow 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.

||

Tregs Fas/FasL cytotoxicity T cells
central memory phenotype| immune homeostasis |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 theFas/FasL system is still unknown.
Regulation of the immune reaction is of pivotal importancefor 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 reportson 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 foundand 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 havebeen published. To date, more than 10 CD8+ Treg populations withdifferent 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 thatCD49d can separate CD8+CD122+ cells into at least two subsets(CD49dlow and CD49dhigh) (20). To judge which cells have astronger regulatory activity, we prepared two experimental systems:
SignificanceF

as (CD95) and its ligand FasL (CD178) compose a uniquesystem 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 systemappears 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) miceshow lymphoproliferative changes, it has been suggested that theFas/FasL system is important for suppression/regulation of activatedeffector 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 ofhuman CD4+Foxp3+ Tregs that use the Fas/FasL system for theirregulatory 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 reportssuggesting that CD8+ T cells use the Fas/FasL system for their2460–2465 | PNAS | March 1, 2016 | vol. 113 | no. 9

Our study demonstrated that CD8+CD122+CD49dlow regulatory
T cells induced apoptosis in target T cells depending on death
receptor Fas (CD95)–FasL (CD178) interaction. This in vitro
phenomenon reflected the regulatory activity of these cells
in vivo, which was also reproducible in the in vivo experimental
system of adoptive T-cell transfer. By using natural mutant miceof Fas or FasL and the gene-targeted mice of IL-10, we confirmed that the regulatory activity of CD8+CD122 +CD49dlow
cells was mediated by Fas–FasL but not by IL-10. In addition, the necessity of MHC class I molecules on the target T cells
was also confirmed by analysis using CD8+ T cells that lack β2microglobulin and consequently class I MHC.
Author contributions: T.W.M. and H. Suzuki designed research; K.A. and H. Suzuki performed research; K.A., S.K., H. Shiku, and H. Suzuki analyzed data; and H. Suzuki wrote
the paper.
Reviewers: T.O., Medical Research Institute, Tokyo Medical and Dental University; andH.U., Okayama University.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1

To whom correspondence may be addressed. Email: tmak@uhnresearch.ca or harusuzu@
med.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1525098113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1525098113

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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 agood 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+CD49dlow),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 CD49dlow population, which corresponds to the central
memory T-cell population, and that their mechanism of suppression depends on the cytotoxicity mediated by the Fas andFasL interaction.
Results
CD8+CD122+ Cells Are Divided into Subpopulations Expressing Different
Sets of Cell-Surface Markers. Although it has been shown that the

CD8+CD122+ population contains Tregs, CD8+CD122+ cells are
generally considered memory T cells. Thus, we needed to identify
a better marker to focus on regulatory cells in the CD8+CD122+
population. We chose CD62L and CD49d; CD62L allows separation of effector memory T cells. CD122 is also frequently used aswell as CD44 and CD62L for the analysis of memory and regulatory
cells (26). We examined the expression of these cell-surface markers
in murine splenic T cells (Fig. 1 A and B). At least four different
subsets of CD8+ cells could be distinguished by this analysis.
Their profiles were CD62L+CD122−, CD62L+CD122+CD49dlow,
CD62L+CD122+CD49dhigh, and CD62L−. The first subset is usually referred to as CD122− cells and consists mostly of naïve, resting
CD8+ cells (Fig. 1B). The last three subsets could be classified intomemory or activated cells and may also contain regulatory cells.

CD8+CD122+CD49d low Cells Show Regulatory Activity in Vitro andin Vivo but CD8+CD122+CD49dhigh Cells Do Not. We found that

CD8+CD122+ cells can be divided into two populations by theirexpression level of CD49d. Then, we performed an in vitro experiment in which candidate Tregs were cocultured with theirtarget cells. As the mechanism of regulation by CD8+ Tregs isthought to involve elimination of the target cells (27), candidate
Tregs should reduce the number of target (regulated) cells during
coculture. Representative results after 72 h of coculture are presented in Fig. 1 C and D. When CD49dlow cells (CD45.1−) weremixed with CD8+CD122− cells (CD45.1+) at a 1:1 ratio at the
beginning of coculture, the percentage of CD8+CD122−CD45.1−
cells markedly decreased during coculture in a time-dependent
manner (Fig. 1C). When CD122− cells were cocultured with
CD49dhigh cells, the decrease in the percentage of CD122− cells
was much less marked than when they were cocultured with
CD49dlow cells (Fig. 1 C and D). These results of relative change
between two different cells cocultured were the reflection of theabsolute number of live cells, which is presented in Fig. S1A.
These results indicate that CD8+CD122− cells reduce their
number by some effect of CD8+CD122+ cells, and such an effectis much stronger in CD49dlow cells than in CD49dhigh cells.
However, this interpretation should be adapted only if the proliferation speed of CD49dlow cells and CD49dhigh cells is identical. Therefore, we examined the growth of separately cultured
CD49dlow and CD49dhigh cells. As a result, both cells grew atvirtually the same rate (Fig. 1E, not significantly different, as
Akane et al.

F

Fig. 1. (A) Spleen cells were analyzed for the expression of CD8, CD122,
CD62L, and CD49d. (B) CD62L+ cells (inside of the rectangle in A) were analyzed for the expression of CD122 and CD49d. (C and D) Analysis of regulatory
activity in coculture. (C, Left) CD8+CD122−CD45.1+ cells and (CD8+CD122+)
CD49dlowCD45.1− cells. (C, Right) CD8+CD122−CD45.1+ cells and (CD8+CD122+)
CD49dhighCD45.1− cells. Representative results obtained after 72 h of coculture
are shown. (D) The percentages of CD8+CD122− on total live cells obtained
from three independent experiments are shown. **Significantly different at
the same time point (P < 0.01, Student’s t test). (E) Cell growth analysis of
CD49dlow and CD49dhigh T cells. For all time points, the values obtained forCD49dlow (red line) and CD49dhigh T cells (blue line) did not significantly differ(P > 0.05, Student’s t test). (F) Analysis of adoptive T-cell transfer to RAG-2−/−
mice. Transfer of a mixture of CD49dlow and CD122− T cells (purple line) effectively prolonged survival of the mice; survival of these mice was significantly
higher than that of mice receiving CD122− cells alone (red line; log-rank test,
P = 0.0003; Wilcoxon test, P = 0.0014). In contrast, transfer of a mixture ofCD49dhigh and CD122− T cells did not significantly affect survival (green line)
compared with that of mice receiving CD122− cells alone (log-rank test,
P = 0.5271; Wilcoxon test, P = 0.8964).

determined by Student’s t test, P > 0.1 for any culture duration),
supporting that CD49dlow cells, but not CD49high cells, possessan activity to reduce the number of cocultured CD8+CD122−
cells, which is possibly achieved by the elimination of target cells(i.e., cytotoxic activity).
For the in vivo experiment, we essentially followed the method
established by Rifa’i et al. (22), with minor modifications. We
performed T-cell adoptive transfer into RAG-2−/− mice and followed their survival. The CD49dhigh cells did not support survival
of RAG-2–deficient mice that had received CD8+CD122− cells. In
contrast, the CD49dlow cells clearly reversed the survival of mice tothe level of those that had not received T cells (Fig. 1F, blue line).
Together with the results of the in vitro experiment, these results
PNAS | March 1, 2016 | vol. 113 | no. 9 | 2461

confirmed that CD8+ Tregs are encompassed in the CD49dlow
subset. Thus, in the following text, CD49dlow cells are describedas “CD8+ Tregs” to reduce the text volume.
CD8+CD122+CD49dlow Tregs Cannot Eliminate or Suppress Fas-Mutated
CD8+CD122− Cells from lpr Mice. We tested the regulatory activity of

CD8+ Tregs under Fas-related conditions. CD8+ Tregs, taken fromwild-type mice, were cocultured with CD8+CD122− cells, taken from
Fas-mutant lpr mice, and the percentage of target CD8+CD122−
cells among total live cells was determined at various time points.
The ratio of CD8+CD122− cells from lpr mice to wild-type CD8+
Tregs decreased as coculture continued (Fig. 2 A and B). However,
the rate and extent of decrease were significantly higher when CD8+
CD122− cells and CD8+ Tregs, both taken from wild-type mice, were
cocultured (Fig. 2 A and B). These changes in the ratio of cellnumber were also observed in the data of absolute cell numbers (Fig.
S1B). The proliferation rate of CD8+CD122− cells from lpr mice wasAB

not different from that of CD8+CD122− cells from wild-type mice
(Fig. 2C). In addition, we examined the proliferation of cocultured
cells by the dilution of prelabeled CFSE and further confirmed thatany types of cells used in this study show a similar pattern of CFSE
peaks (Fig. 2D). This result confirmed that the difference observed
between CD8+CD122− cells from lpr mice and those from wild-typemice in the coculture experiment was not due to a difference inproliferation rate but due to resistance to apoptosis of the lpr micederived CD8+CD122− cells. In the in vivo experiment, RAG-2–deficient mice that had received CD8+CD122− cells taken from lpr
mice died, similar to those injected with CD8+CD122− cells takenfrom wild-type mice. Mice that had received a mixture of
CD8+CD122− cells from lpr mice and CD8+ Tregs obtained
from wild-type mice died, similar to those that had received
CD8+CD122− cells from lpr mice alone (Fig. 2E). Together, thesefindings indicated that Fas-mutant CD8+CD122− cells are resistant
to regulation by CD8+ Tregs, which have a marked effect on wildtype CD8+CD122− cells and prolong the survival of CD8+CD122−
cell-recipient mice.
FasL-Mutant CD8+ Tregs Cannot Eliminate or Suppress Conventional+
CD8+CD122− T Cells. First, the expression of FasL in CD8 Tregs

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Fig. 2. (A and B) Analysis of regulatory activity in coculture of CD49dlow cells(CD45.1) and CD8+CD122− cells (CD45.2) derived from lpr mice and wild-type
mice. (A) Representative results after 72 h of coculture obtained from three
independent experiments are shown. (Left) CD122− cells (lpr) cocultured with
CD49dlow cells. (Right) CD122− cells (WT) cocultured with CD49d− cells. (B) The
percentages of CD8+CD122− cells among total live cells are shown. *Significantly different (P < 0.05, Student’s t test). (C) Absolute numbers of CD122−
T cells derived from WT mice (red line) and lpr mice (blue line) in simple culture
were determined. At all time points, the values obtained for lpr mice-derived
cells and WT mice-derived cells were not significantly different (P > 0.05,
Student’s t test). (D) CD8+CD122− T cells (WT or lpr) labeled with CFSE were
cocultured for 48 h and analyzed. The dilution patterns of CFSE are shown as
representative results of three independent experiments. (E) Analysis ofadoptive T-cell transfer of CD122− T cells from WT or lpr mice mixed with
CD49dlow T cells of WT mice into RAG-2–deficient mice. Addition of WT micederived CD49dlow T cells to lpr mice-derived CD122− cells (blue line) did notaffect survival; survival of these mice was not significantly different from thatof mice receiving lpr mice-derived CD122− cells alone (red line; log-rank test,
P = 0.0878; Wilcoxon test, P = 0.2537).

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was confirmed by real-time PCR (Fig. 3A). Furthermore, the existence of the FasL product on the cell surface was confirmed byflow cytometry (Fig. 3B). This result indicated that FasL is acandidate mediator of killing of Fas-expressing target cells.
Next, we examined the activity of FasL-mutant CD8+ Tregs. CD8+
Tregs taken from gld mice were cocultured with CD8+CD122−
cells taken from wild-type mice, and the percentage of target cells(CD8+CD122−) among CD8+ Tregs was determined at various
time points. The percentage of wild-type CD8+CD122− cells
among CD8+ Tregs from gld mice decreased over time; however,
the rate and extent of decrease were significantly lower thanwhen both types of cells were taken from wild-type mice (Fig. 3 C
and D). The difference was also observed in the absolute cellnumber (Fig. S1C). The proliferation rate of CD8+ Tregs takenfrom gld mice was not different from that of wild-type CD8+ Tregs(Fig. 3E). This result confirmed that an insufficient decrease ofcells cocultured with CD8+ Tregs taken from gld mice, comparedwith those cocultured with wild-type CD8+ Tregs, was not due
to a relative decrease in the proliferation rate of CD8+ Tregs
taken from gld mice but rather due to insufficient elimination of
CD8+CD122− cells by FasL-mutated CD8+ Tregs.
In the in vivo experiment, RAG-2–deficient mice that had received a mixture of wild-type CD8+CD122− cells and CD8+ Tregs
from gld mice died at the same rate as those that had received
wild-type CD8+CD122− cells alone (Fig. 3F). Thus, CD8+ Tregs
taken from FasL-mutant gld mice demonstrated no ability tocontrol CD8+CD122− cells and could not prolong the survival ofmice that received these cells.
CD8+ Tregs Induce Apoptosis in Target-Activated CD8+CD122− T Cells.

To confirm the cytotoxic mechanisms underlying the regulatory
action of CD8+ Tregs, we examined cell death during coculture
by staining the cells with annexin V and 7-amino actinomycin D(7-AAD) (Fig. 4 A and B). Although we could not detectannexin V+7-AAD− cells, we observed annexin V+7-AAD+ cells.
CD8+CD122− cells cocultured with CD8+ Tregs showed a higher
percentage of 7-AAD+ cells than those cocultured with CD49highcells, indicating that CD8+ Tregs induced apoptosis in CD8+CD122−
T cells. CD8+CD122− cells taken from lpr mice did not show anincrease in 7-AAD–stained cells after coculture with CD8+
Tregs, indicating that Fas-mutant CD8+CD122− cells are resistant to apoptosis induced by CD8+ Tregs (Fig. 4B).
CD8+CD122+CD49dlow T Cells Induce Apoptosis via Caspase 8 Pathway.

Judging from the experimental results both in vitro and in vivo, it
is strongly suspicious that the regulatory mechanism performed by
Akane et al.

Acells were significantly increased in lpr and gld mice but not in IL10–deficient mice, compared with wild-type mice (Fig. 4 E and F).
This finding indicates the existence of a negative feedback mechanism that controls the activity of CD8+ Tregs.

B

CD8 + Tregs Cannot Eliminate or Suppress MHC Class I-Deficient
CD8+CD122− Cells. Next, we asked whether recognition of target

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A
Fig. 3. (A) Analysis of RNA expression level of FasL in each population of
CD8+ T cells by real-time PCR. For each sample, FasL mRNA levels were normalized to β-actin and are expressed as fold change relative to the control sample(1.00). †Not significantly different (P > 0.05, Student’s t test); **significantly
different (P < 0.01, Student’s t test). (B) Analysis of FasL expression of flow
cytometory. (C and D) Analysis of coculture of CD8+CD122−CD45.1+ T cells andCD49dlow from gld or WT mice. (C ) Representative results obtained fromthree independent experiments. (D) *Significantly different (P < 0.05,
Student’s t test). (E) Cell growth analysis of CD49dlow T cells from gld miceand WT mice. At all time points, the results obtained from two different
mice were not significantly different (P > 0.05, Student’s t test). (F) Analysisof adoptive T-cell transfer of CD122− T cells (WT) mixed with CD49dlow T cells(WT or gld) into RAG-2–deficient mice. Addition of gld mice-derived
CD49dlow T cells to CD122− cells (blue line) had no significant effect onsurvival compared with transfer of CD122− cells alone (red line; log-rank test,
P = 0.7608; Wilcoxon test, P = 0.6859).

the CD49dlow cells is elimination of cells by using the Fas/FasL
system. To prove the involvement of the Fas/FasL system in our Tcell coculture assay, we introduced the assay system of PhiPhiLux
and CaspaLux. It is well known that caspases are the key moleculeof apoptosis, and caspase 8 is especially directly linked to the Fasderived signal. When caspase 8 is active, Caspalux-8, a substrate forcaspase 8, is digested and becomes a fluorescent substance that canbe detected by flow cytometry. As a result, more caspase 8-active
cells were observed in the cells cocultured with CD49dlow cells(Fig. 4 C and D), indicating that Fas and its downstream caspase,caspase 8, was activated during the process of apoptosis induction.
CD8+ Tregs Are Increased in Fas/FasL-Mutant Mice but Not in IL-10–
Deficient Mice. We demonstrated that CD8+ Tregs, but not

CD49dhigh cells, display regulatory activity that can eliminate
cocultured CD8+CD122− cells in vitro and prolong survival of
CD8+CD122− T-cell–recipient RAG-deficient mice in vivo. The
cause of death in CD8+CD122− T-cell–recipient RAG-deficient
mice was not evident but might involve failure of multiple organs,including bone marrow, lung, liver, etc. Additionally, we foundthat lack of Fas/FasL function affects the regulatory activity of
CD8+ Tregs both in vitro and in vivo. Therefore, we expected
some changes in CD8+regs of Fas/FasL-mutant mice. The absolute number of CD8+ Tregs and their percentage on total CD8+
Akane et al.

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Fig. 4. (A) Result of 7-AAD analysis after 48 h of coculture. Percentages of
7-AAD–positive cells among total cultured cells are shown in the histogram.
(B) Percentages of 7-AAD–positive cells in coculture. *Significantly different(P < 0.01, Student’s t test); †not significantly different (P > 0.05, Student’s
t test). (C and D) Analysis of caspase 8 activity in CD122− (CD45.1+) cells
using CaspaLux8 after coculture with CD49dlow or CD49dhigh (CD45.1−) cells.
(C) Representative results obtained from three independent experiments.
(D) Percentages of cells positive for CaspaLux8 in CD122− CD45.1+ cells
are shown. The data are averages of three independent experiments.
(E) Spleen cells taken from WT, IL-10–deficient, lpr, and gld mice wereanalyzed by flow cytometry. Representative results obtained from three
independent experiments for each mouse strain are shown. (F) Percentagesof CD49dlow cells in total CD8+ cells in each group are shown. Statisticallysignificant differences between the WT and each mutant strain were determined. **Significantly different (P < 0.01, Student’s t test); †not significantly different (P > 0.05, Student’s t test).

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C

cells by CD8+ Tregs depends on T-cell receptor (TCR)–MHC class I
interaction. To address this question, we used β2-microglobulin–
deficient instead of MHC class I-deficient mice; β2-microglobulin–
deficient mice have very few CD8+ cells because of a lack ofpositive selection of CD8+ T cells. This problem can be addressedby generating wild-type mice with bone marrow reconstituted
with MHC class I-deficient bone marrow. When CD8+CD122−
cells, taken from the bone marrow-reconstituted mice, were
cocultured with CD8+ Tregs (Fig. 5 A and B). The rate and extent
of reduction were lower than when they were cocultured with wildtype CD8+CD122− cells (Fig. 5 A and B). The proliferation of
CD8+CD122− cells from mice in which the bone marrow had
been reconstituted with that of β2-microglobulin–deficient mice
was not significantly different from that of CD8+CD122− cells

that CD8+ Tregs recognize their target cells by interaction between a MHC class I molecule (plus an antigen) andthe TCR.

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CD8+ Tregs Can Eliminate CD4+ Cells in a Fas-Dependent Manner.

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Fig. 5. (A and B) Analysis of coculture of CD49d low cells (CD45.1) with
CD8+CD122− cells (CD45.2) derived from mice in which the bone marrow had
been replaced with that from β2M (β2-microglobulin)-deficient mice. (A, Left)
CD49dlow (CD45.1+) cocultured with CD122−(β2MKO) for 72 h. (A, Right)
CD49dlow (CD45.1+) cocultured with CD122− (WT) for 72 h. Representative
results after 72 h of coculture obtained from three independent experiments
are shown. (B) The percentages of CD8+CD122− cells among total live cells areshown in three independent experiments. *Significantly different (P < 0.05,
Student’s t test). (C) Absolute numbers of CD8+CD122− T cells derived from WT
mice or from mice (β2MKO) in simple culture. At all time points, no significant
differences were found (P > 0.05, Student’s t test). (D and E) Analysis of coculture of CD49dlow T cells (CD45.1+) with CD4+CD25− T cells derived from lpr
or WT mice. (D, Left) CD49dlow(CD45.1+) cocultured with CD4+ (lpr) for 72 h.
(D, Right) CD49dlow(CD45.1+) cocultured with CD4+ (WT) for 72 h. Representative results after 72 h of coculture obtained from three independent
experiments are shown. (E) Percentages of CD4+ cells among total live cells
are shown. Data were obtained from three independent experiments. *P <0.05, Student’s t test; **P < 0.01, Student’s t test. (F) CD4+CD25− T cells derived from WT and lpr mice were harvested, and the absolute numbers of live
cells were determined. Average values of three independent experiments areshown. At all time points, the values were not significantly different (P > 0.05,
Student’s t test).

taken from wild-type mice (Fig. 5C). This result confirmed that therecovery of the decrease in CD8+CD122− cells from the bonemarrow-reconstituted mice, compared with that in cells from wildtype mice, was not due to a difference in the proliferation rate
between the CD8+CD122− cells taken from the two different
types of mice. Taken together, these results strongly indicate
2464 | www.pnas.org/cgi/doi/10.1073/pnas.1525098113

One remaining question was whether, besides CD8+ cells, CD8+
Tregs can also suppress and/or eliminate CD4+ cells. CD4+ cells
taken from wild-type and lpr mice were cocultured with CD8+
Tregs, and the numbers of surviving cells were determined using
flow cytometry. CD8+ Tregs were indeed able to reduce thenumber of CD4+ cells. This activity was evident for wild-type aswell as lpr mouse-derived CD4+ cells; however, the activity of lprderived cells was significantly lower than that of wild-type CD4+
cells, indicating that it is Fas-dependent (Fig. 5 D and E). The
proliferation rate of CD4+ cells from lpr mice and that of wild-type
cells was not significantly different (Fig. 5F), indicating that it isFas-dependent. The proliferation rates of CD4+ cells from lpr miceand that of wild-type cells were not significantly different (Fig. 5F),
indicating that the difference in the reduction speed in coculture
was not due to a difference in proliferation rate per se but was dueto the elimination of activated CD4+ cells by CD8+ Tregs.
Discussion
The precise cellular events involved in the process of immune
regulation related to Fas/FasL systems are largely unknown. Because Fas/FasL-mutated animals are prone to develop autoimmune
diseases rather than immunodeficiency and FasL is known to be
expressed in CD8+ cytotoxic T cells, it has been predicted thatthese cells are involved in immune regulation. Additionally, activated CD8+ T cells have been reported to kill clonally establishedT-cell lines; however, there is no firm evidence that the Fas/FasL
system on particular CD8+ T cells in fact regulates the immune
response (28). This is the first report describing that CD8+ Treg
cells directly control CD8+CD122− T cells both in vivo and in vitro
by using the Fas/FasL system.
CD8+CD122+ Tregs were first reported by Rifa’i et al. (22).
However, studies of CD8+CD122+ Tregs have been complicated by the concept of memory T cells marked as CD8+CD122+.
Some investigators have tried to separate Tregs from memory
T cells by staining the CD8+CD122+ cells with a third marker,
such as PD-1 (29), whereas such trials have not succeeded well inpersuading other investigators. However, our experimental results
obtained in the former studies (20–30) and the present studies
have definitely shown the regulatory activity in this T-cell subset.
Our current interpretation is that the regulatory and the memory
functions are compatible. These cells with regulatory activity may
belong to CD8+ memory T cells, possibly recognize self-antigens
by their TCR, and induce apoptosis of Fas-expressing activatedT cells. Based on our new understanding, it should be possible thatcells with memory phenotype (CD8+CD122+) possess the regulatory activity at the same time.
Rifa’i et al. and Endharti et al. (31, 32) reported the regulatory
activity of CD8+CD122+ T cells in both in vivo and in vitro experiments. We first examined which subset of the CD8+CD122+
cells possessed regulatory activity. Okuno et al. (20) found that
CD8+CD122+ cells in murine peripheral immune organs can bedivided into at least two populations according to their expression level of CD49d. The current study indicated that the final
suppression mechanism used by the CD8+CD122+ Tregs appears
to be killing of the activated T cells, which depends on the Fas/
FasL pathway, because Fas-deficient T cells derived from lpr/lpr
mice were resistant to killing (Fig. 2 A and B) and FasL-deficient
Tregs derived from gld/gld mice could not perform this regulatory activity (Fig. 3 C and D).
The molecular mechanism underlying immune suppression by
CD8+ Tregs and the chemical pathway involved in the regulatory
effect of these cells are still largely unknown. We previously
reported that CD8+CD122+ Tregs produce IL-10 and that the
Akane et al.

cytokine could be the effector molecule of these CD8+ Tregs.
Although both the mechanism of recognition of target cells andthe effector molecule of regulation by CD8+ Tregs remain to beelucidated, this study yielded some important clues: Activationof Fas via interaction with FasL is largely associated with thekilling of T cells that are no longer needed and rather harmful
for survival. Another important molecular interaction is that of
MHC class I and TCR. β2-microglobulin–deficient cells areneither regulated in the previous study (33) nor in the present
study, suggesting the importance of specific recognition between“the regulatory cells” and “the regulated cells.” Another important molecule involved in the action of CD8+ Tregs might
be Qa-1, a nonclassical MHC class I molecule (MHC-Ib), as
H. Cantor and his colleague found and reported a CD8+ T-cell
subset that showed a regulatory activity under a Qa-1–restricted
manner (30). In their recent publications, however, the Qa-1–
restricted CD8+ Tregs they proposed were apparently shown to
be CD122+CD44+ (34, 35). The Qa-1–restricted CD8+ Treg isoverlapped with or involved in the CD8+CD122+ Treg reportedby H. Suzuki and his colleagues (22). Two CD8+ Tregs that wereformerly thought to be distinct cell populations turned out to be
the same one, further confirming the existence of CD8+ regulatory T cells, regardless of whether they are the naturally arisingtype or the induced type. Although the relation between CD4+
Tregs and CD8+ Tregs has not been clarified in detail, both Tregs
are indispensable to maintain T-cell homeostasis, and their cooperative work has been suggested in the prevention and cure of an
autoimmune disease (31).

Adoptive T-Cell Transfer to RAG-2−/− Mice. Sorted CD8+CD122− T cells (4 × 105
per recipient mouse) simply or mixed with CD8+CD122+ T cells (49dlow or
CD49high) (1 × 105 per recipient mouse) were i.v. transferred into 8–10-wkold RAG-2–deficient mice that had received a sublethal dose of irradiation(5.5 Gym). The survival of the T-cell–recipient mice was followed up after
adoptive T-cell transfer, and the results were analyzed using Kaplan–Meiersurvival curves.

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Akane et al.

This study forms a basis for continued research of immune
suppression by CD8+CD122+ Tregs. Elucidation of the effector
mechanisms of CD8+ and CD4+ Tregs may pave the way to
improve immune regulation by Tregs in clinical practice.
Materials and Methods
Mice. Animal care was performed according to the guidelines of Nagoya
University (Nagoya, Japan). Experimental protocols were approved by the
Ethics Committee of the Nagoya University Graduate School of Medicine(Nos. 27015, 27017, and 27365).

T-Cell Culture. Activated T cells as described above were either cocultured
or cultured alone in RPMI-1640 media containing 10% (vol/vol) FBS and
20 ng/mL of interleukin-2 (PeproTech Inc.) in a round-bottom 96-well platefor 72 h (0.5 × 105 cells per well).
For other information, see SI Materials and Methods.

PNAS | March 1, 2016 | vol. 113 | no. 9 | 2465

IMMUNOLOGY AND
INFLAMMATION

Activation of CD8+ T Cells. CD8+ T cells in any desired population were collected from murine spleen and lymph node cells by using a FACS SORP Aria II
cell sorter (BD Biosciences) and cultured in an anti-mCD3 antibody-coated96-well plate for 24 h (1.0 × 105 cells per well).

Supporting Information
Akane et al. 10.1073/pnas.1525098113
SI Materials and Methods
Mice. Six- to 8-wk-old C57BL/6 mice and gld and lpr mice ofa C57BL/6 genetic background were purchased from SLC
Japan. Breeding pairs of the C57BL/6CD45.1/CD45.1 congenic
strain and RAG-2–deficient mice on a C57BL/6 genetic background (B6.129S6-Rag2tm1FwaN12) were purchased from Taconic. β2-microglobulin–deficient mice and IL-10–deficient miceon a C57BL/6 genetic background (B6.129P2-Il10tm1Cgn/J)
were originally obtained from the Jackson Laboratory. All mice
were maintained in our animal facility.
Antibodies. FITC (fluorescein isothiocyanate)-conjugated anti-

mouse CD122, PE (R-phycoerythrin)- or APC (allophycocyanin)conjugated anti-mouse CD49d, PE-conjugated anti-mouse FasL,
APC-conjugated anti-mouse CD62L, PE/Cy7-conjugated antimouse CD8a, and FITC- or APC-conjugated anti-mouse CD45.1
antibodies were purchased from BioLegend.
Cytotoxicity Assay. We used APC-conjugated anti-mouse CD45.1
antibody (BioLegend) and annexin V–FITC (BioLegend) with
7-AAD (eBioscience).
+

Activation of CD4+ Naïve T Cells. CD4

T cells in any desired
population were collected from murine spleen and lymph nodecells by using an FACS Aria II cell sorter (BD Biosciences) andcultured and activated for 24 h in a 96-well plate (1.0 × 105 cells
per well) using Dynabeads mouse T activator CD3/CD28 foractivation of mouse T cells (In Vitrogen).
Real-Time PCR Primers. The following real-time PCR primers wereused: CD163L1 A5, 5′-gtctggaaactgtcggagat-3′; CD163L1 A3,
5′-agtcttgtgacccctaaggt-3′; FasL A5, 5′-cgtgagttcaccaaccaaagc-3′;
FasL A3, 5′-cccagtttcgttgatcacaag-3′; β-actin A5, 5′-agtgtgacgttgacatccgt-3′; and β-actin A3, 5′-gcagctcagtaacagtccgc-3′.
PhiPhiLux and CaspaLax. PhiPhiLux-G2D2 kit and CaspaLux8-L1D2
kit systems that can detect the activity of caspase 3 and cspase 8,
respectively, were purchased from OncoImmunin, Inc.

SI Results
CD8+CD122+CD49dlow Cells Taken from IL-10–Deficient Mice Have a
Normal Capacity to Suppress or Eliminate Other Conventional
CD8+CD122− T Cells. Next, we wanted to clarify whether the cyto-

toxic mechanism of the CD8+CD122+CD49dlow cells is related to
IL-10. Because IL-10 has been shown to be important for the
regulatory activity of CD8+CD122+ Tregs (32), we expected that

Akane et al. www.pnas.org/cgi/content/short/1525098113

CD8+CD122+CD49dlow cells taken from IL-10–deficient micewould have a reduced ability to regulate other CD8+ T cells.
However, we found that CD8+CD122+CD49dlow cells from IL10–deficient mice had a normal ability to reduce the number of
cocultured wild-type CD8+CD122− cells (Fig. S2 A and B). Furthermore, CD8+CD122+CD49dlow cells from IL-10–deficient mice
showed apparent activity in supporting the survival of RAG-2–
deficient mice when they were transferred (R-phycoerythrin)
into mice together with CD8+CD122− cells (Fig. S2C).
CD8+CD122+CD49dlow T Cells Induce Apoptosis via a GranzymeIndependent Pathway. Our findings indicated the involve-

ment of the Fas/FasL system in the regulatory mechanism of
CD8 + CD122 +CD49d low Tregs. Another well-known mechanism of cytotoxicity of CD8+ T cells is perforin/granzymeinduced apoptosis. To test whether this mechanism is involved in
the cytotoxic activity of CD8+CD122+CD49dlow Tregs, we added agranzyme inhibitor to the culture medium during coculture of
CD8+CD122+CD49dlow Tregs and their target CD8+CD122− cells.
The granzyme inhibitor did not affect the elimination of target cells by Tregs, suggesting that the mechanism of regulation by CD8 +CD122+CD49dlow Tregs is independent of theperforin/granzyme system (Fig. S2 D and E).
CD163L1, Granzyme B, and FasL Show Characteristic Expression
Profiles in Subpopulations of CD8+ Cells. CD49d clearly separated

CD8+CD122+ cells into two subpopulations and as such is a goodcandidate second marker for CD8+ Tregs (20). To identify abetter marker or a “master gene” for CD8+ Tregs, we performed
microarray analysis. By comparing the expression profile of
CD8+CD122+CD49dlow cells with that of CD8+CD122+CD49highcells and that of CD8+CD122− cells, we identified three different
expression patterns with higher expression in at least one subpopulation of CD8+CD122+ cells than in CD8+CD122− cells:(i) genes with elevated expression in both CD49dlow andCD49dhigh, (ii) genes with elevated expression in CD49dlow but
not in CD49dhigh, and (iii) genes with elevated expression in
CD49dhigh but not in CD49dlow cells. We selected one gene fromeach group [(i) FasL, (ii) CD163L1, (iii) granzyme B], indicatedin the scatter plot (Fig. S3A) and interpretation of the results(Fig. S3B). The expression pattern of the unique gene CD163L1
that was highly expressed in CD8+CD122+CD49dlow cells (36),weakly expressed in CD8+CD122+CD49dhigh cells, and not expressed in other cells including CD4+ cells (Fig. S3C) was confirmed by real-time PCR results (Fig. S3D).

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Fig. S1. (A) Experiment was performed as described for Fig. 1D, and the data were exchanged to absolute numbers of cells by calculating the total numbersof cells obtained by counting cells in a counting chamber times the percentages of the cell of interest obtained by the flow-cytometric analysis. 122−(49dhigh),
CD8+CD122− cells cocultured with CD8+CD122+CD49dhigh cells; 122–(49dlow), CD8+CD122− cells cocultured with CD8+CD122+CD49dlow cells. *Significantly
different (P = 0.0096, Student’s t test). (B) Experiment was performed as described in Fig. 2B, and the data were exchanged to absolute numbers of cells as in A. 49dlow(lpr122–), CD8+CD122+CD49dlow cells cocultured with C57BL/6lpr/pr mice-derived CD8+CD122− cells; 49dlow(WT122–), CD8+CD122+CD49dlow cells cocultured withwild-type C57BL/6 mice-derived CD8+CD122− cells. †Significantly different (P = 0.0175, Student’s t test). (C) Experiment was performed as described in Fig. 3D, andthe data were exchanged to absolute numbers of cells as in A. 122–(gld49dlow), CD8+CD122− cells cocultured with C57BL/6gld/gld mice-derived CD8+CD122+
CD49dlow cells; 122–(WT49dlow), CD8+CD122− cells cocultured with wild-type C57BL/6 mice-derived CD8+CD122+CD49dlow cells. §Significantly different (P = 0.0165,
Student’s t test). (D) Experiment was performed as described in Fig. 5B, and the data were exchanged to absolute numbers of cells as in A. 122–(IL-10KO49dlow),
CD8+CD122− cells cocultured with C57BL/6IL-10KO mice-derived CD8+CD122+CD49dlow cells; 122–(WT49dlow), CD8+CD122− cells cocultured with wild-type C57BL/6
mice-derived CD8+CD122+CD49dlow cells. {Not significantly different (P = 0.5071, Student’s t test). (E) Coculture assay was performed as described in Fig. 1, andafter 48 h of coculture, cells were subjected to the assay for PhiPhiLux. Cells were treated with the PhiPhiLux system according to the protocol provided by the
manufacturer and subsequently analyzed by FACSCalibur flow cytometer. Cells stained with anti–CD45.1-PE (originally CD122−) are shown as histogram analysis.
Percentages of cells positive for PhiPhiLux are shown. Data are representative of three independent experiments. (F) Analysis of PhiPhiLux, indicating activity of
caspase 3, is shown as a bar graph. Statistically significant difference was observed between the cells cocultured with different cells indicated in the panel. The data
are averages of three independent experiments.

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Fig. S2. (A) After 24 h of culture with the stimulation by anti-CD3 Ab and 72 h of coculture of WT CD8+CD122− T cells (CD45.1) and CD8+CD122+CD49dlow T cells(CD45.2), derived from IL-10–deficient mice (Left) or wild-type mice (Right), the cells were stained and analyzed. Representative results obtained after 72 h ofcoculture are shown. Percentages of cells in the upper or lower rectangle among total remaining live cells are shown. (B) CD8+CD122− T cells (CD45.1) andCD8+CD122+CD49dlow T cells (CD45.2) from WT mice and IL-10–deficient mice were cocultured and analyzed. The percentages of CD8+CD122− cells among total
live cells are shown as the averages and SDs obtained from three independent experiments. At all time points, the values were not significantly
different (P > 0.05, Student’s t test) between data obtained from IL-10–deficient mice-derived cells and those obtained from WT mice-derived cells.
(C ) CD8 +CD122− T cells (4 × 105 per recipient mouse) obtained from wild-type mice were mixed with CD8+CD122 +CD49d low T cells (1 × 105 per recipientmouse) taken from WT mice and IL-10–deficient mice and were then i.v. transferred into RAG-2–deficient mice. The survival of the T-cell–recipient
mice was followed up to 140 d after adoptive T-cell transfer, and the results are presented as Kaplan–Meier curves. Addition of IL-10 − /− –derived
CD8 + CD122 + CD49d low T cells to CD122 − cells (red line) led to significantly higher survival of mice compared with transfer of CD122 − cells alone(blue line; log-rank test, P = 0.0166; Wilcoxon test, P = 0.0287), but survival of these mice was not significantly different from that obtained by addition of WT
CD8+CD122+CD49dlow cells (green line; log-rank test, P = 0.4891; Wilcoxon test, P = 0.3927). (D and E) Analysis of granzyme B activity in coculture using
granzyme B Inhibitor I (Calbiochem, Merck Millipore). Representative results of simultaneous experiments with (Right) and without granzyme inhibitor (Left;control) are shown (D). Red line, granzyme inhibitor (+); blue line, granzyme inhibitor (–). At all time points examined, the values in the culture containing the
granzyme inhibitor did not differ significantly from that without the granzyme inhibitor at the same time point (P > 0.05, Student’s t test) (E). The data areaverages of three independent experiments.

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Fig. S3. Search for genes specifically expressed in a subpopulation of CD8+ T cells. (A) Scatter plots comparing gene expression in CD8+CD122+CD49dlow cells(vertical axis) versus CD8+CD122+CD49high cells (horizontal axis in Left panel) and in CD8+CD122− cells (horizontal axis in Right panel). Arrows indicate thepositions of CD163L1, Fas, and granzyme B. (B) Relative expression intensity of FasL, CD163L1, and granzyme B in CD8+CD122+CD49dhigh and CD8+CD122+CD49dlow
cells compared with that in CD8+CD122− cells is summarized. Upward arrows illustrate higher expression in the indicated cells than in CD122− cells, andhorizontal arrows illustrate no difference in expression between the indicated cells and CD122− cells. (C) RT-PCR analysis of CD163L1 in various lymphocytes
and CD8+ T-cell subsets. cDNA was prepared by reverse transcription of RNA extracted from the indicated T-cell subsets and PCR-amplified with CD163L1specific primers. The PCR products were electrophoresed in agarose gels and stained with ethidium bromide. PCR products were normalized by the intensitiesof β-actin. (D) Quantification of CD163L1 expression in various CD8+ T-cell subsets by real-time PCR analysis. RNA extracted from the indicated T-cell subsets wasused to quantify the mRNA levels with SYBR Premix Ex Taq II (TAKARA). The amount of PCR products is shown on a relative scale setting, with that of CD62L−
set as 1.00. Differences in expression between CD122− and other cells (CD62L−, CD49dhigh, and CD49dlow) were statistically analyzed. †Not significantly different.
**Significantly different (P < 0.01, Student’s t test).

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