By
From the Department of Immunology, University of Washington, Seattle, Washington 98195
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Abstract |
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Fas ligand (FasL/CD95L) is best known for its role in delivering apoptotic signals through its
receptor, Fas (APO-1/CD95). In this study, we present evidence that FasL has a second role as
a signaling receptor. Alloantigen-specific proliferation by multiple FasL murine CTL lines is
depressed compared to that of FasL+ CTL lines. FasL
CTLs kill efficiently on a per recovered
cell basis and can achieve wild-type levels of proliferation upon stimulation by optimal doses of
anti-CD3, suggesting the lack of a costimulatory signal during antigen stimulation. To test this
hypothesis directly, soluble FasIgG, a fusion protein of murine Fas and human IgG1, was added
to FasL+ CTLs to demonstrate that blocking cell surface Fas-FasL interactions mimics the depression observed for FasL
CTLs. In addition, plate-bound FasIgG in conjunction with suboptimal anti-CD3 stimulation augments proliferative signals in FasL+ but not FasL
CTLs. In
contrast to these results with CD8+ T cells, alloantigen-stimulated FasL
CD4+ T cells proliferate vigorously compared to FasL+ cells. These data demonstrate that reverse signaling
through FasL is required for CTLs to achieve maximal proliferation and may provide clues to
differences in the homeostatic regulation of activated CD4+ and CD8+ T cells during an immune response.
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Introduction |
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Fas ligand (L) belongs to the TNF superfamily, a group of type II transmembrane or secreted molecules whose interactions with their receptors, members of the TNF receptor superfamily, regulate important functions in immune cell proliferation, activation, differentiation, and survival (1). FasL exists mainly in membrane-bound form, is generally expressed on cells of the T cell lineage, and is induced during T cell activation (1). FasL and members of this ligand superfamily primarily interact with their receptors by direct cell-cell contact (3). This observation, coupled with the cross-species sequence conservation of the cytoplasmic domains of these ligands, has led to the suggestion that signaling occurs in both directions for this family of ligand-receptor pairs (3). In support of this argument, family members including CD27L, 4-1BBL, OX40L, CD30L, and CD40L have recently been shown to transduce internally costimulatory signals to activated peripheral lymphocytes (2, 4, 5). As of now, no data exist concerning the reverse signaling capacity of ligands with known negative regulatory functions, such as FasL. To explore this added twist to the picture of reverse signaling, we used CTL lines generated from B6 wildtype and B6.lpr mice, which both express FasL, and from B6.gld mice, which lack FasL function, to demonstrate that FasL shares this capacity for reverse signaling in CD8+ but not in CD4+ T cells. Our data reveal that FasL, a molecule long associated with the induction of apoptosis in Fas+ target cells (6) can also positively regulate the proliferative capacity of CD8+ T cells that express it. These data also offer some insight into how CD4+ and CD8+ T cells may be differentially regulated during an immune response.
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Materials and Methods |
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Mice and Reagents.
C57BL/6 (B6), B6.MRL-Faslpr (B6.lpr),
B6Smn.C3H-Faslgld (B6.gld), B6.C-H2bm1/By (B6.bm1), B6.C-
H2bm12/KhEg (B6.bm12), C3H/HeJ (C3H), C3H/HeJ-Faslgld
(C3H.gld), and C57BL/6-scid/SzJ (B6.SCID) mice were all purchased from The Jackson Laboratory (Bar Harbor, ME) and used
at 6-9 wk of age. Reagents include rat antibodies specific for murine CD4 (RL172.4R6; reference 7) and for murine CD8
(3.168.8; reference 8), a hamster antibody specific for murine
CD3- (145-2C11; PharMingen, San Diego, CA), and a mouse
antibody specific for murine H-2Kk (16-3-1N; reference 9). NIH
(Harvard Medical School, Boston, MA) 3T3 transfectants expressing the FasIgG fusion protein, which consists of the extracellular domain of murine Fas joined to the hinge and constant regions of human IgG1 (10), were derived by Dr. Ben Stanger and
provided by Dr. A. Marshak-Rothstein (Boston University, Boston, MA). Experiments included either the supernatant of this cell
line or sera from B6.SCID mice injected 5 wk previously with
the transfectants.
Generation and Maintenance of T cell lines.
Alloreactive H-2k-
specific CTLs were originally generated from age-matched B6 wild-type, B6.lpr, and B6.gld mice by incubating donor spleen and LN
cells with an equal number of 3,000 rad C3H splenocytes in
RPMI 1640 supplemented with 10% FCS (RP10). Lines were
maintained by restimulation every 8-10 d for 1-12 rounds. RP10
was supplemented after the third stimulation with 50 mM -methyl mannoside and 5% supernatant from rat cells stimulated for 2 d with 3 µg/ml Con A. CD8+ T cells for generating anti-Kbm1
CTLs were purified from spleen and LN cells from age-matched B6 wild-type and B6.gld mice by nonadherence to nylon wool,
treatment with anti-CD4 plus complement, and adherence to
plate-bound anti-CD8 antibodies. Purity as monitored by flow
cytometry was >99%. All CTL lines were routinely monitored
by flow cytometry and CTL assay. CD4+ T cells for generating
primary anti-H-2bm12 responders were similarly nylon wool-purified,
anti-CD8 killed, anti-CD4 panned spleen and LN cells, with >95%
purity by flow cytometry.
Cytotoxicity and Proliferation Assays.
Serial dilutions of effector
cells and 104 tumor targets labeled with 51Cr sodium chromate
were plated in round-bottomed 96-well plates in a final volume
of 200 µl. After a 4 h incubation at 37°C, 100 µl of supernatant
was collected and specific lysis was determined as follows: percentage of specific lysis = 100 × [(cpm released by CTL spontaneous release)/(cpm released by detergent
spontaneous release)]. Spontaneous release in the absence of CTL was <20% in
all experiments. For antigen-specific proliferation, 2 × 104 CTL
from long-term lines or 2 × 105 primary T cells were cultured
with 5 × 105 irradiated allogeneic stimulator cells in a total volume of 200 µl of RP10. For long-term lines, media were supplemented with 5% rat Con A supernatant or 50 U/ml recombinant
human IL-2. For optimal anti-CD3 stimulation, responders were
added to wells coated at 4°C overnight with 15 µg/ml goat anti-
hamster IgG (GahIg) in PBS and then for 4 h with 5 µg/ml anti-CD3 antibodies. For proliferation with FasL costimulation, responders were added to wells coated overnight at 4°C with both
15 µg/ml GahIg and 15 µg/ml goat anti-human IgG and then
for 4 h with a mixture of 1.0 µg/ml anti-CD3 (suboptimal dose)
with either 4.6 µg/ml FasIgG or isotype-matched human IgG1
(huIgG1). Proliferation levels were measured by thymidine incorporation 18 h after pulsing with 1 µCi [3H]TdR/well on day 3. Background counts (responders with only first stage antibodies)
were subtracted from the values. Antigen-specific proliferation was
also assayed for viable cells by trypan blue exclusion over the
course of the culture period. Where appropriate, APCs were
removed before counting viable cells by anti-H-2k plus complement. For the FasIgG blocking assay, FasIgG was added to cultures at 2.8-10 µg/ml. HuIgG1 and/or B6.SCID preimmune sera were used as negative controls.
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Results and Discussion |
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Proliferation assays using
CTL lines derived from wild-type B6, B6.lpr, and B6.gld
mice revealed that FasL+ CTLs proliferate better in response to allogeneic stimulation than do FasL CTLs (Fig.
1, a and c). APC titration from 5 × 104 to 1 × 106 cells
cocultured per well with CTLs did not alter the relative depression in proliferation of B6.gld CTL lines (data not
shown). In addition, kinetic analysis of viable cell counts
revealed that the lack of FasL function also influences the
ability of CTLs to increase in number over the course of an
MLC (Fig. 1 b), and that this expansion coincides with
FasL expression by the responding T cells. As determined
by flow cytometry, FasL expression on the CTL lines is upregulated by 3 h after antigenic stimulation, peaks on day 3, and begins to decrease by day 4 (data not shown). This pattern of depressed proliferative capacity and number of recovered cells was reproducible for three independent CTL
lines and was not reversed by the addition of exogenous
IL-2. The depressed proliferation and recovered cell counts
were also seen upon coculture of B6.gld CTLs with APCs
from C3H.gld mice, demonstrating that this phenomenon
is not caused by Fas-mediated killing of gld CTLs by FasL
expressed on the APCs (Fig. 1 d). Autocrine Fas-mediated suicide (10) can also be ruled out as a mechanism for
depressed proliferation because gld CTLs do not express
functional cell surface FasL. Analysis by flow cytometry
(data not shown) also ensured that there is not an accumulation of Thy1+CD8
CD4
cells analogous to those found
in vivo in gld and lpr mice (1).
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To test the potential involvement of CD4+ T cells in initiating or maintaining the
depressed proliferative capacity of FasL compared to wild-type CTLs, purified populations of CD8+ T cells from B6
and B6.gld mice were tested for their response against the
MHC class I molecule Kbm1. The same pattern of depression was observed for gld relative to wild-type CTLs in
proliferation and expansion of cell numbers over the course
of an MLC (Fig. 1 c). These data indicate that the submaximal proliferation by FasL
CTL lines is not initiated or
maintained by the lack of CD4+ T cell help and further
emphasize that the observed depression in proliferation is
not merely an idiosyncracy of a single B6.gld-derived CTL
line. In addition, this decreased proliferative capacity is not
restricted to long-term lines, as primary cultures of anti-Kbm1 lines also showed the same pattern of proliferation
(data not shown).
In contrast to their CD8+ counterparts, CD4+ T cells lacking functional FasL proliferate vigorously upon alloantigenic stimulation. Purified CD4+ cells from B6.gld mice cultured with class II different B6.bm12 stimulators proliferated better (Fig. 2 a) and generated more cells over the course of the 5 d of MLC (Fig. 2 b) than did CD4+ cells from wild-type B6 mice. This observation demonstrates the differential requirement for functional FasL expression in the promotion of maximal proliferation by CD8+ but not CD4+ T cells.
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Despite their submaximal proliferation in response to antigenic stimulation, gld CTLs do not differ from wild-type CTLs in their ability to proliferate to an optimal dose of anti-CD3 antibody (Fig. 3 a) or to lyse target cells (Fig. 3 b). Therefore, the depression seen with antigenic stimulation of gld CTLs cannot simply be explained as a characteristic of a weak line or a general defect in TCR signaling. Instead, it implies that gld CTLs are missing a costimulatory signal required to achieve maximal proliferation during an antigenic response.
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To pinpoint FasL as the source of the proliferative signal, soluble FasIgG fusion protein was added in an
effort to block cell surface FasL-Fas interactions. A dose-dependent decrease in the proliferation of B6 wild-type CTLs
was observed upon the addition to the culture of increasing
amounts of soluble FasIgG but not isotype-matched huIgG1
(Fig. 4 a). Kinetic analyses also revealed that recovered cell
numbers of both FasL+ CTLs (B6 wild-type and B6.lpr) were
reduced to those of FasL CTLs over the course of the
MLC upon the addition of soluble FasIgG but not huIgG1
(Fig. 4 b). The fact that Fas
CTL lines are susceptible to
blocking by soluble FasIgG demonstrates that Fas-mediated
killing is not involved, which implicates FasL in positive
signaling. When plate-bound FasIgG fusion protein was used
in conjunction with suboptimal amounts of anti-CD3, a costimulatory signal was delivered for proliferation by FasL+
but not by FasL
CTLs (Fig. 4 c). These data demonstrate
that the source of this positive signal is the FasL expressed
on the wild-type and lpr CTL lines. Although it is important to note that the molecules mediating these signals have
yet to be identified, the striking sequence conservation
across species of the cytoplasmic domain of FasL suggests
the presence of important functional domains. A proline-rich region of the cytoplasmic tail, in which 25 out of the
78 residues are prolines, including a stretch of 7 sequential proline residues (13, 14), is reminiscent of an SH3 binding site (15).
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These results provide insights into the observed differences in the regulation of activated CD8+ and CD4+ T
cells. Apoptosis of CD4+ T cells is primarily mediated
through Fas (16), possibly both by fratricide and autocrine
suicide (10). On the other hand, CD8+ T cells are less
sensitive to Fas-mediated death (16, 17) and primarily undergo a slower death induced by the interaction of TNF
with its receptor (16). However, neither mechanism operates exclusively in one T cell subpopulation or the other;
TNFR-mediated apoptosis is measurable in CD4+ T cells
in the absence of Fas expression (18) and CD8+ T cells are
not completely resistant to Fas-mediated death (16). FasL
CD4+ T cells proliferate more extensively to superantigenic (19, 20) and class II-specific (Fig. 2) stimulation than
do wild-type cells, while FasL
CD8+ T cells proliferate
less vigorously than do wild-type cells (Fig. 1). The difference in proliferation observed for these T cell subpopulations may be the sum of two separate events, positive reverse signaling through FasL in CD8+ T cells and the
sensitivity of CD4+ T cells to Fas-mediated death. In addition, FasL and Fas are both found on the surface of CTLs,
leading to a scenario in which both positive and negative
signals may be received by the same cell. The perceived
"reduced sensitivity" of CD8+ T cells to Fas-mediated
death may then be explained by the maintenance of a balance in the CD8+ T cell population in which cell proliferation induced by the FasL signal offsets the number of cells
undergoing Fas-mediated death. Survival of each cell may
depend upon which signal occurs first, or the FasL signal
may be able to override the Fas negative signal, for example
through intracellular competition for signaling molecules.
The role of TCR engagement in conjunction with the
FasL signal remains unclear. Other molecules known for
their positive signaling capabilities have recently been implicated in the death of cells in the absence of a concomitant antigen receptor signal. For example, signaling through
CD40 without concurrent engagement of the B cell receptor leads to Fas-mediated cell death (21, 22), and may serve
an immunoregulatory role by removing nonspecific B cells. It will be interesting to determine if FasL can still signal
without engagement of the CD3/TCR complex and to
analyze the consequences of such uncoupled signaling.
Should FasL fail to deliver a positive signal without the coengagement of the TCR, the death signal through Fas may
serve to remove activated T cells after antigen has been eliminated. In light of the discovery that CD40 signals can
direct germinal center B cells to become memory B cells
(23), one could speculate on the role of FasL in the clonal
expansion of antigen-specific CTLs and the generation of
memory T cells. Adoptive transfer of OT-1 (reference 24;
class I-restricted OVA-specific TCR transgenic B6) spleen
and LN cells into B6 wild-type, B6.lpr, and B6.gld mice
followed by intraperitoneal injection of OVA peptide daily for 3 d revealed that antigen-specific donor cells injected
into B6.lpr mice were defective in their expansion compared to that demonstrated in the other two recipients
(Martin, S., and M. Bevan, unpublished data). These data
are consistent with the inability of the Fas B6.lpr host to
deliver a positive signal through FasL for clonal expansion.
It will be interesting to determine whether this difference in expansion is related to the development of memory cells
and if the FasL signal during an immune response influences the entry of CTLs into the memory pathway.
Several models that allow arrangement of the positive/ negative cell surface receptors into three categories have recently emerged: (a) surface receptors capable of both positive (survival or proliferation) and negative (death) signals such as Fas (25, 26) and TNFR1 (27); (b) positively signaling receptors that augment the death receptor signal (28, 29); and (c) induced receptors paired with constitutive receptors with the opposite signaling capacity, such as CD28 and CTLA-4 (30). Our data place Fas-FasL signaling into this third category. FasL, induced upon activation of CD8+ T cells, sends a negative signal outward through its constitutively expressed receptor while also receiving a positive signal.
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Footnotes |
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Address correspondence to Dr. Pamela J. Fink, University of Washington, Department of Immunology, Box 357650, Rm H574A, Seattle, WA 98195. Phone: 206-685-3608; Fax: 206-616-7237; E-mail: pfink{at}u.washington.edu
Received for publication 4 August 1997 and in revised form 29 October 1997.
We thank Drs. M. Bevan and S. Martin for sharing unpublished observations, our colleagues for helpful comments on the manuscript, and Dr. A. Marshak-Rothstein for FasIgG transfectants.
This work was supported by the following grants from the National Institutes of Health: research grant AG-13078 and Basic Immunology training grant CA-09537.
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