Full text of DOI:10.1111/j.1348-0421.2003.tb03397.x

Microbiol. Immunol., 47(4), 285–293, 2003
Cell-Mediated Fas-Based Lysis of Dendritic Cells
Which Are Apparently Resistant to
Anti-Fas Antibody
Aya Yokota, Atsuhiko Oikawa, Chieko Matsuda, Nobukata Shinohara*, and Koji Eshima
Department of Immunology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228–8555, Japan
Received October 16, 2002; in revised form, January 10, 2003. Accepted January 15, 2003
Abstract: In this report, the controversy concerning the sensitivity of dendritic cells (DCs) to Fas-dependent
induction of apoptosis was examined using murine DCs. Although DCs could not be lysed when exposed
to an anti-Fas antibody, Jo2, the observed resistance turned out to reflect their lack of the expression of FcγR
necessary for crosslinking the antibody, rather than their intrinsic resistance. Thus, at least a fraction of
DCs was sensitive to Jo2 in the presence of FcγR-expressing by-standers. Consistently, a significant frac-
tion of DCs was sensitive to Fas-dependent lysis mediated by T cells including the antigen-specific killing
ꢀ
by CD4 T cells. Both immature (class II MHClow) and mature (class II MHChigh) DCs were sensitive to the
Fas-based induction of apoptosis.
ꢀ
Key words: Dendritic cell, Fas, CD4 T cell
ꢀ
Activated CD4 T cells, particularly Th1 type cells,
frequently express cytolytic activity (3, 19, 22, 25, 28, 29,
36, 37). These cells express Fas-ligand (FasL) upon
activation (33) and their lytic activity, unlike that of
reported that the expression of FLIP is enhanced in
mature DCs, providing the explanation for their resistance
(17). Wang et al. showed that apoptosis of DCs could be
induced through TRAIL instead of Fas (40). On the
contrary, reports by Matsue et al. and Kawamura et al.
indicated the sensitivity of DCs to the Fas-based induc-
tion of cell death (12, 20). In this report, we have direct-
ly examined the putative sensitivity of conventionally
obtained DCs to Fas-mediated induction of apoptosis.
ꢀ
CD8 CTL, is almost exclusively attributable to the Fas-
based pathway because of no or very low expression
of perforin (6, 34, 35). Such a lytic activity of CD4 T
cells suggests their possible involvement in the negative
regulation of immune responses, since professional anti-
gen-presenting cells are their likely specific targets (16,
ꢀ
ꢀ
35). A possible role of cytolytic CD4 T cells in specific
Materials and Methods
suppression of antibody responses and surveillance of
autoantibody production has been discussed elsewhere
(31). Among the cells with antigen-presenting func-
tion, dendritic cells (DCs) are highly specialized cells for
antigen presentation and stimulation of T cells, particu-
larly virgin T cells (2, 4). Differentiated DCs express Fas
on their surface, but their sensitivity to the Fas-based
induction of apoptosis has been controversial. A number
of studies have reported that DCs are totally resistant to
the Fas-based induction of apoptosis. Ashany et al.
reported that DCs are totally insensitive to Fas-mediated
lysis (1). Rescigno et al. claimed that the treatment of
DCs with soluble FasL facilitated further maturation
rather than induction of apoptosis (27). Leverkus et al.
Animals. BALB/c (BALB/c Cr Slc), C3H (C3H/HeJ
Yok Slc) and C3H-lpr (C3H/HeJ JMS Slc-lpr) mice
were purchased from Japan SLC (Shizuoka, Japan).
ꢀ
Cells. BK1, a BALB/c-derived KLH-specific CD4 T
cell clone with lytic activity (34), 4CK, a KLH-specific
ꢀ
CD4 bulk T cell line established from a C3H/HeJ
ꢀ
mouse, and P0K, an H-2k-specific allogeneic CD8 per-
forin (ꢁ/ꢁ) T cell line (14), were established by us and
were maintained in vitro with regular antigenic stimula-
tions. All three T cell lines had been shown to be capa-
ble of exclusively Fas-dependent antigen-specific cell
Abbreviations: CML, cell-mediated lysis; DCs, dendritic cells;
ELISA, enzyme-linked immunosorbent assay; FasL, Fas-ligand;
FcγR, Fcγ-receptor; FCS, foetal calf serum; FITC, fluorescein
isothiocyanate; GM-CSF, granulocyte macrophage colony stim-
ulating factor; IFN-γ, interferon-gamma; MHC, major histo-
compatibility complex; PE, phycoerythrin.
*Address correspondence to Dr. Nobukata Shinohara, Depart-
ment of Immunology, Kitasato University School of Medicine,
1–15–1 Kitasato, Sagamihara, Kanagawa, 228–8555, Japan. Fax:
ꢀ81–42–778–9266. E-mail: nobu@med.kitasato-u.ac.jp
285

286
A. YOKOTA ET AL
ꢀ
lysis. A CD8 allogeneic cytolytic T cell clone, OE4,
specific for class I MHC antigens of H-2d (32) was also
maintained in vitro. A20.2J was a BALB/c-derived
class II positive B lymphoma (5). A transfectant T lym-
phoma line expressing mouse Fas, mFas/L5178Y
(referred to as Fas/LY in this paper) (6), was kindly
provided by Dr. S. Yonehara, Kyoto University.
cultured overnight. The floating cells recovered from the
second culture were treated with anti-Thy1.2, anti-B220
and complement. Eighty-five to ninety-five percent of the
resulting cell preparations were DCs as judged by a
staining with anti-CD11c monoclonal antibody.
Cell-mediated lysis (CML) assays. CML assays were
performed as described previously (30). Briefly, 5ꢂ
51
Cell culture. Cells were cultured in Dulbecco’s mod-
ified essential medium (DMEM: GIBCO, Grand Island,
N.Y., U.S.A.) supplemented with 200 µM non-essential
amino acids, 1 mM sodium pyruvate, 2 mM extra gluta-
103/well of Cr-labeled target cells were mixed with
graded numbers of effector cells in a total volume of 200
µl/well. After centrifugation at 1,000 rpm, the assay
plates were incubated at 37 C for 6 hr in 10% CO2.
Radioactivities released in the supernatant were deter-
mined and percent specific lysis was calculated as (exper-
imental releaseꢁspontaneous release) / (maximal release
ꢁspontaneous release)ꢂ100.
ꢁ2
mine, 5ꢂ10 mM 2-mercaptoethanol (2-ME), penicillin
(100 units/ml), streptomycin (100 units/ml), 20 mM
HEPES buffer (pH 7.2), and 10% prescreened heat-
inactivated fetal calf serum (FCS). For the maintenance
of T cell lines, the medium was further supplemented
with 5% of 48 hr supernatant of Con A-stimulated LEW
rat spleen cell culture which was further passed through
a Sephadex G-10 column to remove Con A.
Antibodies. Monoclonal antibodies to FcγR (2.4G2),
I-Ak (10-2-16) and B-220 were obtained as culture super-
natant and titers were predetermined.
Anti-Fas-mediated cytolysis. Target cells labeled with
51Cr (5ꢂ103 cells/well) in 200 µl of 10% FCS culture
medium with indicated concentrations of Jo2 with or
without 2.4G2 were incubated for 8 hr at 37 C. Specif-
51
ic Cr release was calculated according to the above
mentioned formula.
Flow cytometry. Flow cytometric analyses were per-
formed as described previously (15). 2–10ꢂ105 cells
were stained in and washed with ice-cold Hanks’ bal-
anced salt solution containing 0.5% bovine serum albu-
min and 0.02% sodium azide. Stained cells after wash-
ing were examined by flow cytometric analyses on
FACSCalibur (Becton Dickinson, Mountain View, Calif.,
U.S.A.). PE (phycoerythrin)-labeled anti-CD11c (21),
FITC (fluorescein isothiocyanate)-labeled anti-Fas anti-
body Jo2 (23), PE-anti-B7-2 (CD86) (7), PE-anti-hamster
IgG, and FITC-annexin V (39) were purchased (Pharmin-
gen, San Diego, Calif., U.S.A.). FITC-labeled anti-I-Ak,
10-2-16 (24), purified anti-FcγR (Fcγ receptor) antibody
2.4G2 (38) and FITC-labeled anti-rat Ig antibody were
prepared in this laboratory.
Preparation of splenic DCs. Splenic DCs were purified
according to Inaba (8) with modifications. Briefly, cell
suspension was prepared from collagenase-perfused
spleens and fractionated by centrifugation on high-den-
sity bovine serum albumin (BSA) solution (100 g BSA
dissolved into 186 ml PBS, ρꢃ1.080). The low density
cells were incubated at 37 C in a plastic petri dish (φ60
mm, 0.5 spleen in 2.5 ml/dish) for 1.5 hr, and after
removing non-adherent cells, adherent cells were further
cultured overnight. Cells detached from the plastic sur-
face during the overnight culture were collected and
treated with a mixture of antibodies (anti-Thy1.2, anti-B-
220) and prescreened rabbit complement (26) to remove
contaminating T and B cells. Dead cells were removed
by centrifugation over Lymphosepar II (IBL, Maebashi,
Japan).
Induction of DCs from bone marrow cells. DCs were
also induced in vitro from bone marrow cells by cultur-
ing the cells in the presence of GM-CSF (granulocyte
macrophage colony stimulating factor) for 9 days (9).
GM-CSF was actually added as culture supernatant of
GM-CSF-transfected plasmacytoma X63-Ag8 (18),
which contained approximately 5,000–10,000 units/ml of
GM-CSF as determined by functional assays (volume:
10%, final concentration: 500–1,000 units/ml). Bone
marrow cells obtained from femurs, tibias and pelvises
were cultured in 6-well plastic culture plates in medium
containing GM-CSF. During the first 7 days, culture
medium was changed every other day to remove floating
cells. On the 8th day of culture, floating cells were
replated into new wells to enrich mature DCs and further
Determination of IFN-γ (interferon-γ) production. T
cells (1ꢂ105/well) were incubated with purified DCs in
a flat-bottomed microtiter plate in triplicate. After 16 hr’s
incubation, concentration of IFN-γ in the supernatant
was determined using an ELISA kit (BioSource Inter-
national, Camarillo, Calif., U.S.A.).
Results
Staining Profiles of DCs
Splenic and bone marrow DCs were prepared as
described in “Materials and Methods.” In order to enrich
mature cells, bone marrow DCs were replated on day 7
and floating cells in the secondary culture were used
on the next day. Figure 1 shows the surface staining pro-
files of murine DCs freshly isolated from spleens or
induced from bone marrow cells in vitro. DCs were

287
FAS-BASED LYSIS OF DC
ing CD11b and did not contain significant numbers of
ꢀ
CD8 lymphoid DCs (panel I). The bone marrow-
derived preparations did not contain lymphoid DCs
either (panel J) as predicted.
Apparent Resistance of DCs to Jo2 Due to the Lack of
FcγR
In order to study the sensitivity of DCs to the Fas-
based induction of apoptosis, bone marrow-derived DCs
were studied for their sensitivity to anti-Fas monoclonal
51
antibody, Jo2. Figure 2A shows the results of Cr
release experiments carried out with a DC preparation
along with a B cell tumor line, A20.2J. Jo2 never caused
detectable specific release of 51Cr from the DC prepara-
tion in three repeated experiments while it was capable of
lysing A20.2J, confirming the observation by Ashany et
al. (1). Since A20.2J expresses FcγRII/III while DCs
do not (Fig. 3), the possible involvement of FcγR in the
Jo2-mediated induction of apoptosis was studied (Fig.
2B). A monoclonal anti-FcγRII/III antibody (2.4G2)
was examined for its effect on Jo2-mediated lysis of
A20.2J. The induction of apoptosis in A20.2J by Jo2
was completely abrogated in the presence of high con-
centrations of 2.4G2, suggesting the involvement of
FcγR.
Therefore, the sensitivity of DCs to Jo2 in the presence
ꢀ
of FcγR by-stander cells was studied. 51Cr-labeled
BALB/c bone marrow-derived DCs were treated with Jo2
in the presence of cold A20.2J. As shown in Fig. 2C,
DCs became sensitive to Jo2 in the presence of A20.2J,
although the level of the lysis was relatively low. Three
experiments consistently showed low but significant 51Cr
release. The induction of apoptosis was completely
inhibited by 2.4G2, confirming the involvement of
FcγRII/III on A20.2J. This phenomenon was not unique
to DCs, since essentially the same result was obtained
with Fas/LY, an FcγR-negative transfectant lymphoma
line expressing transgenic Fas (Figs. 2C and 3). Thus,
Fas/LY was insensitive to Jo2, but gained the sensitivity
in the presence of A20.2J, which was again inhibitable by
2.4G2. The conclusions drawn from these experiments
are that DCs are not intrinsically resistant to Fas-mediated
induction of apoptosis and that their total resistance to
Jo2 merely reflects the lack of expression of FcγR
required for cross-linking Jo2.
Fig. 1. Staining profiles of purified DCs. Splenic DCs purified
from BALB/c mice (A, C, E, G and I) and DCs induced in vitro
from C3H bone marrow cells (B, D, F, H and J) were stained with
antibodies with indicated specificities. Through the induction
and separation procedures described in “Materials and Meth-
ods,” we consistently obtained highly enriched (80–90%) DC
preparations (A and B, cells were stained with PE-anti-CD11c).
Staining profiles in panels C–H (FITC-staining) were obtained
with the cells gated for the expression of CD11c with double
staining. Negative controls were the cells stained with appropri-
ately labeled antibodies with irrelevant specificities. The cells
shown in panels I and J were stained with anti-CD11c (PE),
anti-CD11b (TC) and anti-CD8 (FITC) and gated for positive
staining with anti-CD11c.
ꢀ
defined as CD11c positive cells. Panels A and B show
that the cell preparations were highly enriched (90% or
more) for DCs. In the following experiments, cell prepa-
rations of comparable purity were used. The cells
expressed class II MHC antigens, Fas and B7.2 (C–H).
The splenic DC populations prepared by our hands con-
tained almost exclusively myeloid type DCs express-
Lysis of DCs by Perforin(ꢁ/ꢁ) CD8 CTL
Since the above mentioned experiments have revealed
that the apparent resistance of DCs to Jo2 was attributable
to their lack of FcγR expression rather than their intrin-
sic resistance, the sensitivity of DCs to cell-mediated Fas-
based lysis was examnied. P0K is an allogeneic H-2k-
ꢀ
specific CD8 CTL line with homozygous perforin

288
A. YOKOTA ET AL
Fig. 2. Resistance of DCs and sensitivity of A20.2J to Jo2-treatment. A: BALB/c bone marrow-derived DCs (open circle) and A20.2J
(BALB/c B cell tumor, closed circle) were labeled with 51Cr, incubated with indicated concentrations of Jo2 (anti-Fas antibody) and 8
hr 51Cr release was measured. B: Inhibitory effect of anti-FcγR antibody on the apoptosis induction of A20.2J by Jo2. 51Cr-labeled A20.2J
was incubated in the medium containing 100 ng/ml of Jo2 for 8 hr in the presence of indicated concentrations of anti-FcγR monoclonal
ꢀ
ꢁ
ꢀ
antibody, 2.4G2. C: FcγR by-stander cells enabled Jo2 to lyse FcγR Fas cells. Fas/LY was cultured in the medium containing 100 ng/ml
ꢀ
of Jo2 for 8 hr. FcR cells (A: A20.2J, B: P815) were added to the culture at indicated ratios. Anti-FcγR antibody, 2.4G2, was added at
ꢀ
400 ng/ml. Note that the presence of FcR cells made BALB/c DCs as well as Fas/LY sensitive to the apoptotic effect of Jo2, and the
effect was inhibited by 2.4G2.
Fig. 3. Expression of FcγR on the cells used in the experiments. DCs derived from BALB/c BM, A20.2J and a Fas-transfected lymphoma,
Fas/LY, were stained with FITC-labeled antibodies to Fas (Jo2: upper panels) and FcγR (2.4G2: lower panels).

289
FAS-BASED LYSIS OF DC
knockout genes and has been shown to be capable of per-
forming exclusively Fas-based target cell lysis (14).
The lytic effect of this line on bone marrow-derived
DCs of C3H mice (H-2k) was examined. As shown in
Fig. 4A, P0K killed C3H DCs, albeit the percentage of
the lysed cells was relatively low, whereas it did not
touch C3H-lpr DCs. The induction of apoptosis of the
C3H DCs was confirmed by staining the cells with
ꢀ
annexin-V (Fig. 4B). A significant fraction of CD11c
B7.2high cells became apoptotic. Jo2 was added to the
assay system as a specific blocker of Fas (bottom panel),
taking advantage of the absence of FcγR on cells of
both sides used in this experiment. The addition of Jo2
inhibited the induction of apoptosis, confirming the
dependency of this cell-mediated killing of DCs on Fas
(and FasL). These observations indicate that a fraction of
DC population is sensitive to lysis by FasL-expressing T
cells.
ꢀ
Antigen-Specific Cytolytic Effect of CD4 T Cells on
DCs
ꢀ
Since soluble antigen-specific Th1 type CD4 T cells
are the most likely in vivo FasL-armed attackers of anti-
gen-presenting cells, the effect of antigen-specific recog-
ꢀ
nition of DCs by cytolytic CD4 T cells was studied.
DCs induced from bone marrow of C3H mice along
with Fas-deficient C3H-lpr DCs were studied for their
sensitivity to the antigen-specific lytic activity of 4CK, a
ꢀ
C3H-derived KLH-specific Th1 type bulk CD4 T cell
line. As shown in Fig. 5A, 4CK lysed KLH-pulsed
C3H DCs in an antigen-specific manner. On the contrary,
4CK failed to lyse KLH-pulsed C3H-lpr DCs, indicating
that the lytic activity of 4CK was entirely dependent
on Fas. Figure 5B shows that lpr DCs were fully capa-
ble of presenting KLH to 4CK as judged by IFN-γ pro-
duction. These results indicate that DCs are sensitive to
ꢀ
the Fas-based lytic activity of Th1 type CD4 T cells.
ꢀ
The lytic effect of Th1 type CD4 T cells was also
examined on splenic DCs. DCs isolated from spleens of
BALB/c mice were pulsed with KLH, labeled with 51Cr
and examined for their sensitivity to the lytic activity of
ꢀ
ꢀ
Fig. 4. Fas-dependent lysis of DCs by perforin (ꢁ/ꢁ) CD8
BK1, a Th1 type KLH-specific CD4 T cell clone. BK1
CTL. A. P0K, an H-2k-specific allogeneic CD8 CTL line with
ꢀ
lacks the expression of perforin and its cytolytic activity
has been shown to be entirely dependent on the Fas/FasL
pathway (10, 14). As shown in Fig. 5C, splenic DCs
were also lysed by BK1 in an antigen-specific manner.
The lytic effect of BK1 in this case was comparable to
homozygous disrupted perforin genes (23), was examined for its
lytic activity on bone marrow-derived DCs obtained from C3H
(closed circle) and C3H-lpr (open circle) mice in a 4 hr 51Cr
release assay. Two experiments gave the same results. B. The mix-
ture of P0K and DCs were incubated for 6 hr, stained with FITC-
annexin-V and analyzed by flow cytometry. The mixture of the
cells was gated for the expression of CD11c. The number below
ꢀ
the perforin-based killing by OE4, a CD8 H-2k-spe-
cific allogeneic CTL clone.
ꢀ
the window indicates the percentages of the gated (CD11c )
cells falling within the window (annexin V positive). The data are
representative of two independent experiments.
Sensitivity of Subpopulations of DCs to Fas-Based Apo-
ptosis
In an attempt to distinguish the Fas-sensitive sub-

290
A. YOKOTA ET AL
Fig. 6. Both mature and immature DCs are sensitive to apoptosis
induction. B6 bone marrow DCs replated on day 7 were incubated
in the presence of 1 mg/ml of soluble trimeric FasL for 6 hr. The
treated cells were analyzed by flow cytometry for the expression
of class II MHC antigen (anti-I-Ab) and phosphatidylserine
(annexin V). The staining profiles of the untreated (upper panel)
and FasL-treated cells (lower panel) gated for the expression of
CD11c. The number in each quadrant indicates the percentage of
the cells falling there. In this experiment, cells were not gated for
forward scatter to avoid exclusion of dying cells. Such processing
of the data resulted in inclusion of a large number of nonspecif-
ic background dots that formed a diagonal group in the lower-left
quadrant, making the percentage of the cells falling in this region
illusively higher. Thus the actual ratios of dead/live of I-Ab-low cells
should have been much higher than the ones calculated from
the numbers shown.
Fig. 5. Specific lysis of DCs by T cells. A: A C3H-derived KLH-
specific CD4 Th1 type bulk T cell line, 4CK, was examined for
ꢀ
its lytic activity on bone marrow-derived DCs obtained from
C3H (left panel) and C3H-lpr (right panel) mice. A representative
result of three experiments is shown. Closed circles: KLH-pulsed
DCs, open circles: non-pulsed DCs. B: IFN-γ production by
4CK stimulated with indicated DCs. Note that lpr DCs were
resistant to the lytic effect of 4CK in spite of their ability to
present KLH to the T cell line. This experiment was performed
only once along with the experiment shown in A. Nevertheless,
similar experiments carried out on 4CK with B cells gave the
same results (unpublished observations). C: A KLH-specific
population from the Fas-resistant subpopulation of DCs,
bone marrow-derived DCs replated on day 7 were incu-
bated with soluble FasL. The cells were analyzed for
their expression of class II MHC and phosphatidylserine
as determined by binding of annexin-V. As shown in Fig.
6, significant fractions of both class II MHChigh and class
II MHClow DCs became positive for annexin-V staining 6
hr after the treatment with FasL, although mature DCs
were less sensitive to the treatment than immature DCs.
Thus it was concluded that both mature and immature
DCs were sensitive to the Fas-based induction of apo-
ptosis.
ꢀ
CD4 Th1 type T cell clone, BK1, was examined for its lytic
activity on splenic DCs. BALB/c splenic DCs were incubated with
(black bar) or without (open bars) 100 µg/ml of KLH overnight
and labeled with 51Cr. The labeled cells were incubated with
ꢀ
graded numbers of BK1 for 6 hr. OE4, a CD8 allogeneic CTL
clone (anti-H-2d class I MHC), was used as a positive control for
specific cell lysis. The result was reproduced in 4 independent
experiments.

291
FAS-BASED LYSIS OF DC
cells to soluble Jo2 does not trigger the death signal, and
A20.2J can be lysed by such a treatment owing to their
expression of FcγR. Although the precise role of FcγR in
contributing to anti-Fas-induced cell death is not entire-
ly clear, the most feasible mechanism is the crosslinking
of anti-Fas antibody by FcγR resulting in aggregation of
Fas and the formation of a death-inducing signal complex
(DISK) (11, 13). In the case of cell-mediated cytolysis,
the aggregation of Fas molecules through interaction
with FasL fixed on the cell surface probably fulfils the
condition for triggering the cascade of caspase activation.
Based on these observations, we speculate that DCs
may well be eliminated after presenting antigens to
Discussion
The results shown in the present report indicate that at
least a fraction of DCs is sensitive to the Fas-based cell-
mediated lysis. The lytic activity of BK1 has been
shown to be exclusively dependent on Fas. It expresses
FasL on the surface upon activation, lacks the expression
of perforin and fails to lyse Fas-negative cells (6). Fur-
thermore, its lytic activity is inhibitable by soluble Fas
molecules or with anti-FasL antibody (14 and Yokota
unpublished observation). Likewise, 4CK could lyse
C3H DCs while they failed to kill DCs of the lpr mouse
(Fig. 5A). Thus the observation by Matsue et al. (20) has
been confirmed. Within our observations, however, it has
been noticed that the maximum percentage of 51Cr release
by DCs was generally lower than the values obtained
with other types of sensitive cells (for example, the
experiment shown in Fig. 2C), suggesting their relative
resistance to Fas-mediated apoptosis or the presence of
a resistant subpopulation. Flow cytometric analysis
indicated that both class II MHClow population (immature
DCs) and the class II MHChigh population (mature DCs)
are sensitive to the Fas-based apoptosis induction. Nev-
ertheless, it also suggested that mature DCs are less
sensitive to the apoptosis induction than immature DCs.
There has been a report indicating the resistance of
DCs to Fas-mediated apoptosis in spite of their expres-
sion of Fas (1). Although the reason for the discrepancy
between the conflicting observations is not fully eluci-
dated at present, the above mentioned relative resis-
tance of DCs to the Fas-mediated lysis might partly
explain it. The observed low sensitivity of DCs, partic-
ularly of mature DCs, probably reflects their enhanced
expression of FLIP (17). It appears also possible that the
heterogeneity of DCs has contributed to the difference in
the observations made by different groups. Our prepa-
rations of DCs consisted exclusively of myeloid DCs
expressing CD11b and was enriched for mature (class II
MHChigh) cells (Fig. 1). Thus, our data do not provide
information on other types of DCs. It is not clear
whether or not there have been significant differences
between DCs prepared by us and by others, since stain-
ing profiles of DCs of other groups are not available.
Although it is not unlikely that minor differences in the
procedures for preparing DCs have resulted in drastic dif-
ferences in the prepared DC population, there is no
pressing reason to believe that there have been drastic dif-
ferences in the nature of DCs prepared in different lab-
oratories, as far as the available information is con-
cerned. Another source of the conflict stems from the
FcγR-dependency of the induction of apoptosis by anti-
Fas antibodies. The simple exposure of Fas-positive
ꢀ
cytolytic CD4 T cells. DCs in peripheral tissues active-
ly internalize extracellular antigens and synthesize pep-
tide/class II MHC complexes. Later, they migrate into
local lymph nodes where they stop taking any more
new antigens and just continue to present the antigens
they have collected in the peripheral tissues (37). Con-
sidering such a life cycle of DCs, it appears necessary to
facilitate the turnover of terminally differentiated DCs in
lymph nodes in order to keep the antigenic information
from local tissues updated. Old DCs might be pro-
grammed to undergo spontaneous death after a certain
length of time. Alternatively, they might be actively
eliminated after providing the antigenic information to
ꢀ
the T cell compartment. Cytolytic CD4 T cells appear
ꢀ
to be suited for this purpose. If cytolytic CD4 T cells
interact and kill the specific target APC, they simulta-
neously get stimulated and undergo lymphokine pro-
duction and proliferation. It has been reported that
Langerhans cells labeled in situ appeared to be eliminated
after migrating into lymph nodes (12). The reduction of
the labeled cells was significantly delayed in lpr mice,
indicating the involvement of the Fas-mediated elimi-
nation of DCs. Such a regulatory system would prevent
unwanted long persistence of old antigenic information
and may well facilitate updating antigenic information by
making room for DCs newly coming in from local
peripheral tissues. It is reported that excessive respons-
es to antigenic stimulations are observed in lpr mice
(20). Further in vivo studies on the interaction between
ꢀ
cytolytic CD4 T cells and DCs from this point of view
are necessary.
This work has been supported by the Integrative Research
Program of the Graduate School of Medical Science, Kitasato
University (Yokota), and by The Hitech Research Center Grant
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
The authors cordially thank Dr. Takashi Saito of Chiba Uni-
versity for supporting this work. We are also thankful to Dr.
Uchiyama of the animal facility, Kitasato University School of
Allied Health Sciences.

292
A. YOKOTA ET AL
hara, S., and Takayama, H. 1994. Two distinct pathways of
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