Content uploaded by Jianping Yang
Author content
All content in this area was uploaded by Jianping Yang on Jul 21, 2023
Content may be subject to copyright.
RESEARCH ARTICLE | MARCH 15 2000
CD8
+
T Cell-Dependent Elimination of Dendritic Cells In Vivo Limits the
Induction of Antitumor Immunity
1
Ian F. Hermans; ... et. al
J Immunol (2000) 164 (6): 3095–3101.
https://doi.org/10.4049/jimmunol.164.6.3095
Related Content
Impaired Ability of MHC Class II−/− Dendritic Cells to Provide Tumor Protection is Rescued by CD40 Ligation
J Immunol (July,1999)
Tumor-Specic Tc1, But Not Tc2, Cells Deliver Protective Antitumor Immunity
J Immunol (December,2001)
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
CD8
ⴙ
T Cell-Dependent Elimination of Dendritic Cells In Vivo
Limits the Induction of Antitumor Immunity
1
Ian F. Hermans, David S. Ritchie, Jianping Yang, Joanna M. Roberts, and Franca Ronchese
2
The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the
migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response.
DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers
detectable ⬃40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the
lymph node with kinetics that parallel the known kinetics of activation of CD8
ⴙ
T cells to effector function. In the presence of high
numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection.
The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in “memory” mice
in which memory CD8
ⴙ
cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-
mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8
ⴙ
T cells failed to divide
in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity
by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback
mechanism to limit the activity of DC within the lymph node. The Journal of Immunology, 2000, 164: 3095–3101.
Dendritic cells (DC)
3
are highly specialized APCs that
serve a sentinel function in immunity by internalizing
and processing peripheral Ags for presentation to naive
T cells (1). DC residing in the peripheral tissues are initially of an
“immature” phenotype characterized by a high propensity for Ag
capture but relatively poor ability to stimulate naive T cells (2).
However, upon exposure to cytokines associated with inflamma-
tion or tissue injury such as TNF-
␣
or IL-1

, DC migrate from the
peripheral tissues to regional lymph nodes (3, 4) and undergo a
maturation process characterized by a reduction in Ag capture
function and up-regulation of MHC and costimulatory molecules
(5, 6). Final DC activation is induced by Ag-specific Th cells via
the interaction of CD40-ligand on the Th cell with CD40 on the
DC (7, 8). Activated DC can prime naive CD8
⫹
T cells to CTL
(9–11), which have the capacity to recognize and kill cells that
express specific Ag in the peripheral tissues. The fate of DC after
they have reached the lymph node is unknown. Because no cells
with the morphology of DC can be demonstrated in efferent lymph
(12), it has been proposed that DC die in situ, although the mech-
anism remains unclear.
Previous studies have shown that DC injected s.c. or i.v. have
the capacity to migrate to the regional lymph nodes or the spleen,
respectively (13). Fluorescent-labeled DC injected s.c. have been
shown to migrate to draining lymph nodes (DLN) where they in-
teract with Ag-specific CD4
⫹
T cells to form clusters in the para-
cortex (14). DC that had not been loaded with specific Ag failed to
form such clusters. Surprisingly, it was also observed that the num-
bers of Ag-loaded DC in the DLN had declined by 48 h, whereas
the non-Ag-loaded DC persisted for longer periods of time. This
suggested that the Ag-loaded DC may have been eliminated by an
immune-mediated mechanism.
We wished to examine in more detail the fate of peripherally
administered DC in a model of CTL-mediated immunity and to
establish how the immune response affected the persistence of DC
in the lymph node. Our results suggest that DC clearance occurs in
the presence of previously activated CTL and also during the ac-
tivation of naive Ag-specific CTL precursors. Furthermore, the
rapid clearance of tumor Ag-loaded DC was associated with im-
paired capacity to induce tumor immunity. Therefore, CTL-medi-
ated clearance of Ag-loaded DC may serve as a negative feedback
mechanism to limit the activity of DC within the lymph node.
Materials and Methods
Mice
C57BL/6 mice were from breeding pairs originally obtained from The
Jackson Laboratory (Bar Harbor, ME). Strain 318 mice (15), transgenic for
a TCR specific for H-2 D
b
plus fragment 33–41 of the lymphocytic cho-
riomeningitis virus glycoprotein (LCMV
33–41
),
3
were kindly provided by
Dr. H. Pircher (Institute of Medical Microbiology and Hygiene, University
of Freiburg, Freiburg, Germany). The B6Aa
0
/Aa
0
MHC class II
⫺/⫺
mice
(16) were supplied by Dr. H. Bluethmann (Hoffmann-LaRoche, Basel,
Switzerland). All mice were maintained at the Biomedical Research Unit of
the Wellington School of Medicine by brother ⫻sister mating; in vivo
experimental protocols were approved by the Wellington School of Med-
icine Animal Ethics Committee and were performed according to institu-
tional guidelines.
Tumor cell line
LL-LCMV is a derivative of the Lewis lung carcinoma LLTC (C57BL/6,
H-2
b
) that has been modified to express a minigene encoding LCMV
33–41
under the control of a CMV promoter (17).
Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington,
New Zealand
Received for publication July 6, 1999. Accepted for publication January 13, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Cancer Society of New Zealand, New
Zealand Cancer Institute, and Otago University Research Fund and by a generous
donation by Sir Roy McKenzie. F.R. is the recipient of a Wellington Medical Re-
search Foundation Malaghan Senior Fellowship.
2
Address correspondence and reprint requests to Dr. Franca Ronchese, Malaghan
Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand. E-
mail address: fronchese@malaghan.org.nz
3
Abbreviations used in this paper: DC, dendritic cell; DLN, draining lymph node;
LCMV, lymphocytic choriomeningitis virus; CM, complete medium; CFSE, carboxy-
fluorescein succinimidyl ester.
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
In vitro culture media and reagents
Unless otherwise stated, all cultures were maintained in complete medium
(CM) comprising of IMDM with 2 mM glutamine, 1% penicillin-strepto-
mycin, 5 ⫻10
⫺5
M 2-ME, and 5% FBS (all from Life Technologies,
Auckland, New Zealand). The synthetic peptides LCMV
33–41
(KAVYN
FATM) and OVA
257–264
(SIINFEKL) were from Chiron Mimotopes (Clay-
ton, Australia).
Culture of bone marrow-derived DC
Bone marrow cells from C57BL/6 mice or B6Aa
0
/Aa
0
mice were cultured
at 4 ⫻10
5
cells/ml in CM containing 20 ng/ml IL-4 and 20 ng/ml GM-CSF
as described previously (18). Cultures were provided fresh CM and cyto-
kines every 3 days and incubated at 37°C until the time of assay (6–8
days). Cultures typically contained 70–100% DC as determined by fluo-
rescent staining with the anti-CD11c Ab N418. DC (1 ⫻10
6
cells/ml) were
loaded with peptide Ag by incubation at 37°C in CM containing 10
M
synthetic peptide for 2 h and then were washed three times with IMDM to
remove excess peptide.
DC migration assay
DC were labeled with the fluorescent dye carboxyfluorescein succinimidyl
ester (CFSE; Molecular Probes, Eugene, OR) by incubation in PBS con-
taining 1
M CFSE for 10 min at 37°C before one wash in five volumes
of ice-cold PBS and two washes with IMDM. Unless otherwise stated,
mice were injected with 1 ⫻10
6
CFSE
⫹
DC by s.c. injection in the distal
forelimb (volar aspect). At the indicated times, both axillary and brachial
lymph nodes were removed from immunized mice and incubated in 2.4
mg/ml collagenase type II (Life Technologies) and 1 mg/ml DNase I (Sig-
ma, St. Louis, MO) for 90 min at 37°C. The tissue was then disrupted by
aspiration through an 18-gauge needle. The cell suspension was sieved
through gauze, washed in PBS, and resuspended for flow cytometric anal-
ysis in PBS containing 2% FBS and 0.01% sodium azide.
Lymph node suspensions were analyzed using a FACSort and CellQuest
software (both from Becton Dickinson, Mountain View, CA). The region
containing DC was identified on the basis of forward/side light scatter
profile. No CFSE
⫹
cells were found outside this region. In initial experi-
ments, the total number of DC within the DLN was calculated as follows:
% CFSE
⫹
cells in the DC region ⫻% of DLN cells in the DC region ⫻
number cells in DLN; data are expressed as average number of DC within
DLN for each experimental group. Because it was observed over repeated
experiments that the percentage of CFSE
⫹
cells in the DC region was
always proportional to the absolute number of CFSE
⫹
cells across the
experimental groups, data were expressed as average percentage of CFSE
⫹
cells thereafter. Only events falling within the gated DC region were col-
lected and stored (⬎250,000 events for each DLN suspension).
Frozen sections (8
m) were prepared from the axillary lymph nodes of
animals injected with CFSE
⫹
DC 20 h earlier. The sections were stained
with hematoxylin and eosin and analyzed by standard and fluorescence
microscopy. CD8
⫹
T cells are depleted by one i.v. injection of 500
g
purified 2.43 mAb 24 h before DC immunization.
Ab staining
Ab staining was in PBS containing 2% FBS and 0.01% sodium azide. The
anti-Fc
␥
RII mAb 2.4G2 was used at 10
g/ml to inhibit nonspecific stain-
ing. Anti-MHC class II (3JP) and anti-CD11c (N418) were affinity purified
from culture supernatants using protein G-Sepharose (Pharmacia Biotech,
Uppsala, Sweden), conjugated to biotin as described (19), and revealed
using streptavidin-PE (PharMingen, San Diego, CA). Instrument compen-
sation was set in each experiment using single color-stained samples.
Adoptive transfer and DC immunization
Pooled lymph node cell suspensions were prepared from strain 318 mice,
and the percentage of T cells expressing the transgenic TCR was deter-
mined on one sample of cells by staining with anti-V
␣
2 and anti-V

8.1,
8.2 Abs and by FACS analysis. The cells were then labeled by incubation
in 1.25
M CFSE (Molecular Probes) in PBS for 10 min at room temper-
ature at a cell concentration of 2 ⫻10
7
cells/ml. The cells were pelleted by
centrifugation in the presence of 50% FCS, washed two times in CM, and
then washed again in IMDM. Suspensions containing 5 ⫻10
6
V
␣
2
⫹
V

8
⫹
cells were transferred into C57BL/6 recipients by i.v. injection into the tail
vein. One day after adoptive transfer, the mice were immunized s.c. with
10
5
DC. After 66 h, the draining axillary and brachial lymph nodes were
removed, teased through gauze to prepare single-cell suspensions, and an-
alyzed by FACS.
Tumor immunity assay
Groups of C57BL/6 mice (n⫽5) were immunized by s.c. injection into the
left flank with 10
5
DC. Secondary immunizations were by s.c. injection
into the contralateral flank 7 days later. One week after the last immuni-
zation, all animals were challenged with 10
6
LL-LCMV tumor cells in-
jected s.c. into the left flank. Mice were monitored for tumor growth every
3–4 days, and tumor size for each group was calculated as the mean of the
products of bisecting diameters (⫾SE). Measurements were terminated for
each group when the first animal developed a tumor in excess of 200 mm
2
.
Results
CFSE
⫹
DC injected s.c. are detected in the DLN
We examined the migration of injected DC to the secondary lym-
phoid organs of mice. DC were cultured from bone marrow pre-
cursors in the presence of GM-CSF and IL-4 for 7 days and labeled
with CFSE, an amine-reactive dye that can be retained in live cells
for many generations. DC were administered s.c. into the distal
forelimb of mice without any deliberate preincubation with Ag. As
can be seen in Fig. 1, 20 h after injection CFSE
⫹
cells could be
detected in the DLN by fluorescence microscopy on frozen tissue
sections and were located in the paracortical region of the lymph
node. Analysis of DLN cell preparations by flow cytometry also
revealed the presence of CFSE
⫹
cells in the DLN (Fig. 2). No
CFSE
⫹
cells could be detected in the non-DLN. In addition,
CFSE
⫹
cells could not be detected in the DLN if the DC were heat
killed before injection. These results imply that the CFSE
⫹
cells
had reached the DLN via an active migratory process.
The possibility that the CFSE
⫹
cells found in the DLN were in
fact endogenous cells that had acquired CFSE from the injected
cells was examined by injecting MHC class II
⫺/⫺
DC and then
using MHC class II expression to distinguish the endogenous from
the injected DC. C57BL/6 mice were injected with CFSE
⫹
DC
cultured from either C57BL/6 mice or MHC class II
⫺/⫺
B6Aa
0
/
Aa
0
mice. MHC class II expression was then examined on CFSE
⫹
cells in the DLN. As shown in Fig. 2B, MHC class II expression
could be demonstrated on CFSE
⫹
cells obtained from mice that
had received C57BL/6 DC. In contrast, no expression of MHC
class II was found on CFSE
⫹
cells from mice that received MHC
class II
⫺/⫺
DC. We conclude that the CFSE
⫹
cells detected in the
DLN were the same DC that had been injected peripherally and
that these cells had actively migrated from the site of injection to
the DLN.
The proportion of CFSE
⫹
DC that reached the DLN and their
kinetics of appearance were also examined. As shown in Fig. 3A,
the absolute number of CFSE
⫹
DC found within the DLN repre-
sented only a small proportion (⬃0.1%) of the original number of
DC injected. The injected DC appeared in the DLN within 12 h of
injection, reaching maximum numbers before 48 h. Considerable
numbers of CFSE
⫹
DC could still be demonstrated in the DLN at
172 h postinjection (Fig. 3B), the latest time point examined.
In summary, CFSE
⫹
DC can be demonstrated to actively mi-
grate from the site of s.c. injection to the DLN and to persist in
vivo in excess of 7 days from the time of injection.
DC loaded with MHC class I-binding peptide Ag are cleared
from the DLN
Next, we sought to determine the fate of Ag-loaded DC after s.c.
injection into mice. CFSE
⫹
DC were loaded with LCMV
33–41
,an
MHC class I-binding peptide from the LCMV glycoprotein, and
injected into the forelimbs of C57BL/6 recipients. The numbers of
Ag-loaded DC in the DLN were compared with the numbers in the
DLN of mice that received DC not loaded with Ag. As can be seen
in Fig. 4A, loading with Ag had no effect on the number of CFSE
⫹
DC recovered from the DLN at 16 h after injection. By 72 h, a
small reduction in the number of DC in the DLN was observed in
3096 CTL-MEDIATED ELIMINATION OF DC IN VIVO
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
the experiment shown, but this reduction was not always observed
in repeated experiments. In contrast, when the number of CD8
⫹
T
cells specific for the Ag presented on DC was increased as in TCR
transgenic recipients, a significant and highly reproducible reduc-
tion in the number of Ag-loaded DC in the DLN was observed 72 h
after injection. No reduction was apparent at 16 h. Depletion of
CD8
⫹
T cells from TCR transgenic recipients before DC injection
prevented the reduction in number of Ag-loaded DC in the DLN
FIGURE 3. Quantitation and kinetics of DC migration to the DLN. A,
Groups of mice were given s.c. injections in both distal forelimbs with the
indicated numbers of CFSE
⫹
DC; DLN were removed for FACS analysis
20 h later. Each group contained three animals with each injected forelimb
treated as an independent event. B, Mice were injected with 10
6
CFSE
⫹
DC, and the DLN were removed for FACS analysis at the indicated time
points. Three animals were analyzed at each time point with each injected
forelimb treated independently (n⫽6). Data are presented as the mean
percentage of DC collected in a gate encompassing only large cells (⫾SE),
as outlined in Materials and Methods.
FIGURE 1. DC injected s.c. migrate to the paracortex of the DLN. Mice were injected s.c. with 5 ⫻10
5
CFSE
⫹
DC in the distal forelimb, and axillary
and brachial lymph nodes were removed for histological analysis 20 h after injection. Frozen tissue sections of the DLN were prepared and stained with
hematoxylin and eosin. CFSE
⫹
cells were detected by fluorescence microscopy. A, Low-power magnification with a representative CFSE
⫹
cell indicated
(arrowhead). B, High-power magnification showing three CFSE
⫹
cells.
FIGURE 2. DC that have migrated to the DLN can be detected by
FACS analysis. Mice were injected s.c. with 5 ⫻10
5
CFSE
⫹
DC in the
distal forelimb, and axillary and brachial lymph nodes were removed for
FACS analysis 20 h after injection. Data were collected in a gate encom-
passing only large cells as based on the known size of cultured DC. No
CFSE
⫹
cells were found outside this gate. A, Results are shown for a
noninjected animal, an animal injected with CFSE
⫹
DC (DLN and con-
tralateral nodes), and an animal that was injected with CFSE
⫹
DC that had
previously been heated to 80°C for 10 min. B, DLN from C57BL/6 mice
injected with either C57BL/6 DC (MHC class II
⫹/⫹
DC) or B6Aa
0
/Aa
0
DC
(MHC class II
⫺/⫺
DC). DLN cell suspensions were stained with anti-MHC
class II to determine MHC class II expression on CFSE
⫹
cells.
3097The Journal of Immunology
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
(Fig. 4B). These results suggest that activation of TCR transgenic
CD8
⫹
T cells to cytotoxic effector cells is associated with the
disappearance of DC expressing specific Ag from the DLN. DC
clearance occurs between 16 and 72 h after injection, a period
consistent with the acquisition of effector function by CD8
⫹
T
cells as reported by other investigators (20, 21). The minimal
clearance of Ag-loaded DC demonstrated in the DLN of naive,
non-TCR transgenic mice probably reflects the smaller number of
Ag-specific T cells that become activated in these mice and hence
the lower number of interactions that result in DC clearance.
Clearance of Ag-loaded DC is accelerated in the presence of an
Ag-specific immune response
Next, we next investigated the fate of Ag-loaded DC in the context
of an ongoing immune response. To this end, CFSE
⫹
DC loaded
with the MHC class I-binding peptide LCMV
33–41
were adminis-
tered to mice that had been injected s.c. in the flank 7 days before
with 10
5
DC alone or with 10
5
DC loaded with the same
LCMV
33–41
peptide. The 7-day interval was chosen because our
previous studies have shown that Ag-specific T cell expansion is
maximal at this time (22); also, resistance to challenge with
LCMV
33–41
-expressing tumors can be demonstrated (17). In ad-
dition, “memory” recipients were also used. These animals had
received LCMV
33–41
-loaded DC 6 mo before and had subse-
quently rejected a challenge with tumor cells expressing the
LCMV
33–41
epitope. All mice received the LCMV
33–41
-loaded,
CFSE
⫹
DC by s.c. injection into the anterior forelimb. As can be
seen in Fig. 5, similar numbers of CFSE
⫹
DC were observed in the
DLN of mice that were either previously immunized with DC
without Ag or in those of mice that had received no previous
treatment. In contrast, a large reduction in the number of CFSE
⫹
DC was observed in the DLN of animals immunized with
LCMV
33–41
peptide-loaded DC 7 days earlier. The reduction was
clearly detectable as early as 20 h after injection, suggesting that it
was related to the presence of peptide-specific effector cells in the
recipient mice. Similar results were also obtained with MHC class
II
⫺/⫺
DC (data not shown), indicating that CD4
⫹
T cells were not
involved in DC elimination. A decrease in the number of CFSE
⫹
DC was also observed in the DLN of mice from the “memory”
group. However, in this latter case the decrease in CFSE
⫹
DC was
observed only at 72 h and not at 20 h after in vivo injection. This
result suggests that the reduction in the number of CFSE
⫹
DC was
related to the acquisition of effector activity by pre-existing mem-
ory CD8
⫹
T cells in the recipient mice.
Taken together, the data presented above indicate that the dis-
appearance of CFSE
⫹
DC from the DLN of recipient mice is as-
sociated with the presence or the development of effector CTL. We
suggest that in the course of an immune response, Ag-loaded DC
become targets for CTL activity and are eliminated from the
lymph node.
CTL-mediated clearance of DC from the lymph node impairs
induction of further immune responses
We wished to establish whether elimination of Ag-loaded DC dur-
ing an immune response impacts upon T cell activation initiated by
the same DC. As an in vivo readout of T cell activation, we ex-
amined the proliferation of CFSE-labeled, LCMV
33–41
-specific
CD8
⫹
T cells adoptively transferred into syngeneic recipients.
Proliferation of CFSE
⫹
T cells can be monitored in the DLN of
immunized mice by flow cytometry and is detected as progressive
halving of cellular fluorescence with every cell division completed
(23). Thus, we examined the specific proliferation induced by DC
that were or were not the targets of a pre-existing immune re-
sponse. Mice were preimmunized s.c. in the flank with DC ⫹
OVA
257–264
peptide to elicit an OVA-specific CTL immune re-
sponse or with DC only as a control. One week later, both groups
of mice received an adoptive transfer of CFSE
⫹
LCMV
33–41
-spe-
cific T cells and were immunized s.c. in the forelimb with DC
loaded with both the OVA
257–264
and LCMV
33–41
peptides. These
FIGURE 4. DC loaded with MHC class I-binding peptide Ag are
cleared from the DLN. Groups of C57BL/6 or TCR transgenic mice were
injected s.c. in the forelimb with either 10
6
CFSE
⫹
DC or 10
6
CFSE
⫹
DC
that had been loaded with the LCMV
33–41
peptide recognized by the trans-
genic TCR. A, DLN were removed for FACS analysis 16 or 72 h after
CFSE
⫹
DC injection. B, TCR transgenic recipients were depleted of CD8
⫹
T cells or were left untreated before injection with CFSE
⫹
DC. DLN were
removed for FACS analysis 72 h later. All data are presented as in Fig. 3B.
FIGURE 5. Clearance of Ag-loaded DC is accelerated in the presence
of an Ag-specific immune response. The presence of CFSE
⫹
, LCMV
33–41
peptide-loaded DC was monitored in the DLN of mice that had been im-
munized 1 wk before with 10
5
LCMV
33–41
-loaded DC injected s.c. in the
flank or with 10
5
DC not loaded with Ag or in mice that had received no
previous treatment. A fourth group, termed the “memory” group, was also
assessed. This group had been immunized with 10
5
LCMV
33–41
peptide-
loaded DC 6 mo before and had also successfully rejected a challenge with
10
6
LL-LCMV tumor cells 7 days after immunization. All groups were
injected s.c. with 10
6
CFSE
⫹
, LCMV
33–41
peptide-loaded DC in each fore-
limb. Three animals from each group were analyzed at the indicated time
points after CFSE
⫹
DC administration. Data are presented as in Fig. 3B.
3098 CTL-MEDIATED ELIMINATION OF DC IN VIVO
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
mice were used to examine the proliferation of LCMV
33–41
-spe-
cific T cells. In addition, separate mice that had also been immu-
nized in the flank with DC ⫹OVA
257–264
or with DC only re-
ceived CFSE
⫹
DC that had been loaded with both the OVA
257–264
and LCMV
33–41
peptides by s.c. injection in the forelimb. These
latter mice were used to evaluate the numbers of CFSE
⫹
DC in the
DLN. As shown in Fig. 6, OVA
257–264
⫹LCMV
33–41
-loaded DC
were rapidly eliminated in mice that had previously been immu-
nized with DC ⫹OVA
257–264
, most likely due to the induced
OVA-specific CTL response. In contrast, a considerable propor-
tion of OVA
257–264
⫹LCMV
33–41
-loaded DC could be demon-
strated in the DLN of mice previously immunized with DC only.
Fig. 6 also shows the proliferative response of LCMV
33–41
-spe-
cific T cells in mice that had received the same DC immunizations.
In mice in which DC could be demonstrated in the DLN,
LCMV
33–41
-specific T cells underwent several cycles of cell di-
vision, indicating the onset of an active immune response. In con-
trast, in mice in which DC had disappeared from the DLN, only
limited proliferation of LCMV
33–41
-specific T cells could be dem-
onstrated. We conclude from these results that CTL-mediated
elimination of DC may have a profound impact on T cell immune
responses and may prevent the initiation of immune responses to
other Ags that are simultaneously presented on the same DC.
CTL-mediated clearance of tumor Ag-loaded DC from the lymph
node impairs antitumor immune responses
We wished to extend our findings by examining whether elimina-
tion of Ag-loaded DC during the induction of an antitumor im-
mune response has a significant negative impact on antitumor im-
munity. For these studies we used the tumor cell line LL-LCMV,
a Lewis lung carcinoma expressing the LCMV
33–41
epitope (17).
All mice were preimmunized s.c. in the flank with DC loaded with
OVA
257–264
to induce OVA
257–264
-specific CTL responses. One
week later, mice received a second s.c. immunization in the op-
posite flank with MHC class II
⫺/⫺
DC that were loaded with both
OVA
257–264
and the “tumor Ag” LCMV
33–41
or with MHC class
II
⫺/⫺
DC loaded with the “tumor Ag” LCMV
33–41
only. MHC
class II
⫺/⫺
DC were used in this experiment to rule out any effects
mediated by CD4
⫹
T cells. Similar results were obtained when
MHC class II
⫹/⫹
DC were used (data not shown), because DC are
eliminated by pre-existing CTL responses regardless of MHC class
II expression. DC presenting OVA
257–264
and tumor Ag became
targets of the anti-OVA
257–264
CTL response and were eliminated
(data not shown). In contrast, DC presenting tumor Ag alone re-
mained unaffected. One week after the second DC immunization,
mice were challenged with LL-LCMV, and tumor size was mea-
sured. In Fig. 7, we show that DC presenting both OVA
257–264
and LCMV
33–41
were less effective at inducing antitumor immu-
nity than DC presenting the LCMV
33–41
Ag alone were. This re-
duction in efficacy was most likely due to the rapid clearance of
OVA
257–264
-loaded DC by OVA
257–264
-specific CTL, which pre-
vented the productive initiation of an LCMV
33–41
-specific im-
mune response. However, a limited degree of antitumor immunity
was reproducibly induced by OVA
257–264
-loaded DC in these ex-
periments, suggesting that the DC were able to induce some degree
of T cell activation before being eliminated. Alternatively, DC-
associated Ag had been released and taken up by other APC, al-
lowing a specific immune response to be initiated.
Discussion
The capacity of DC to migrate from peripheral tissues to lymphoid
organs and to initiate immune responses has been the subject of a
number of investigations (reviewed in Ref. 1). In contrast, the sub-
sequent fate of DC has received little attention. DC are not ob-
served in the efferent lymph (12), suggesting that they may not be
FIGURE 6. CTL-mediated clearance of Ag-loaded DC from the DLN
limits the induction of further immune responses. Groups of mice were
immunized s.c. in the flank with either 10
5
OVA
257–264
-loaded DC or with
10
5
DC alone. All mice received a second injection of DC loaded with
OVA
257–264
and LCMV
33–41
s.c. in the forelimb. Survival of the DC ad-
ministered with the latter injection and their induction of a T cell response
were examined. Left panel, CFSE
⫹
DC simultaneously loaded with
OVA
257–264
and LCMV
33–41
are eliminated in OVA-immunized mice.
Data were collected 66 h after injection of CFSE
⫹
DC and are presented as
in Fig. 3B.Right panel, Adoptively transferred CFSE
⫹
TCR transgenic
cells specific for LCMV
33–41
fail to divide in response to specific peptide
presented on DC if the DC are eliminated by an existing CTL response.
Data were collected 66 h after DC immunization; only CFSE
⫹
,V
␣
2
⫹
V

8.1,8.2
⫹
events are shown. FACS profiles shown are of one represen-
tative mouse of the six in each group.
FIGURE 7. CTL-mediated clearance of tumor Ag-loaded DC from the
DLN impairs the generation of antitumor immune responses. Two groups
of mice (n⫽5) were immunized with 10
5
OVA
257–264
peptide-loaded DC
by s.c. injection in the flank, and then 1 wk later, one group was immunized
with LCMV
33–41
peptide-loaded MHC class II
⫺/⫺
DC in the contralateral
flank, and the second group was immunized with MHC class II
⫺/⫺
DC
loaded with both LCMV
33–41
and OVA
257–264
peptides. After 7 days, both
of these groups and an additional control group were challenged with 10
6
LL-LCMV tumor cells presenting the LCMV
33–41
epitope. Mice were
monitored for tumor growth every 3–4 days, and tumor size for each group
was calculated as the mean of the products of bisecting diameters (⫾SE).
Measurements were terminated for each group when the first animal de-
veloped a tumor in excess of 200 mm
2
.
3099The Journal of Immunology
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
capable of leaving the lymph node and may die in situ. It has been
speculated that DC may be eliminated by the very immune re-
sponses they elicit. This would allow the activated T cells to pro-
liferate freely and to migrate away from the lymph node to pe-
ripheral tissues or, in the case of CD4
⫹
T cells, it would allow the
T cells to migrate to the B cell areas of the lymph node to deliver
help for Ab production (14, 24). Furthermore, elimination of DC
could function as a negative feedback mechanism to limit T cell
responses and also could prevent excessive accumulation in the
lymph node of DC that carry Ags to which the immune system has
already been sensitized.
In this paper we have monitored the migration of CFSE
⫹
DC
injected s.c. into mice. The effect of Ag presented on MHC class
I on DC survival in the DLN was also examined. We show that
Ag-loaded DC disappear from the lymph node during the course of
a CD8
⫹
T cell response, with kinetics that parallel the known
kinetics of activation of CD8
⫹
T cells to effector function (20, 25).
Ag-loaded DC were immediately cleared in recently immunized
mice with a reduction in DC numbers observed in the DLN within
20 h of injection, suggesting that some DC may have been elim-
inated before reaching the lymph node. The kinetics of clearance
were somewhat slower in “memory” mice, in which labeled DC
were clearly demonstrated in the DLN at 20 h after injection but
not at 72 h after injection. Memory CD8
⫹
cells presumably would
need to reacquire effector function before mediating elimination of
DC in these mice (21). Lastly, Ag-loaded DC were cleared from
the DLN between 16 h and 72 h after s.c. injection into naive, TCR
transgenic mice. This indicates that Ag-loaded DC may be elim-
inated as a consequence of a primary CD8
⫹
T cell immune re-
sponse, perhaps as a mechanism to allow turnover of DC within
the lymph node. This process may contribute to the restriction of
the number of T cell specificities generated in a given immune
response, a phenomenon that has been reported for viral infections
(26–28). High-avidity T cells that become successfully activated
in response to Ag on a given DC may eliminate that DC, thereby
preventing the full activation of lower-avidity T cells that recog-
nize different antigenic epitopes presented on the same DC.
The close parallel of the kinetics of DC clearance with the de-
velopment of a CTL response suggests that the failure to demon-
strate CFSE
⫹
DC in the DLN is most likely due to active elimi-
nation of labeled DC rather than to their migration to another site
or to selective loss of the CFSE label from otherwise viable DC.
This hypothesis is also supported by the observation that DC elim-
ination was inhibited by anti-CD8 Ab treatment and did not require
MHC class II expression by the DC. Elimination of DC was clearly
a systemic event in that it was observed at sites distant from the
site of original immunization, as would be expected on the basis of
the CTL’s ability to recirculate to different sites. However, the
mechanism of DC clearance, although clearly Ag-specific, is as yet
undefined. We cannot exclude the possibility that there was a con-
stant rapid turnover of DC in the DLN and that the clearance ob-
served represented a failure of DC to migrate to the DLN in the
presence of activated CTL. However, the fact that Ag-loaded DC
were present in the DLN of naive TCR transgenic mice at 16 h and
then were lost by 72 h is highly suggestive of a cytolytic mecha-
nism operating within the node. Furthermore, the kinetics of DC
clearance in naive animals are consistent with the reported time
required for CTL to gain cytolytic function upon activation (20,
25). Preliminary experiments indicated that DC clearance is not
impaired in IFN-
␥
-deficient mice (data not shown), suggesting that
this cytokine is not critical to the clearance process and that other
effector mechanisms, presumably cytotoxicity, have a greater role.
Other authors have also reported a decrease in the number of Ag-
loaded DC in the lymph node during a CD4
⫹
T cell immune re-
sponse (14). That decrease appeared less profound than the one we
describe here, probably reflecting different mechanisms of DC
elimination during CD4
⫹
T cell responses compared with CD8
⫹
T
cell responses. DC elimination has also been demonstrated by his-
tological means in mice exposed to viruses that induce strong CTL
immune responses (29).
We also show here that CTL-mediated clearance of Ag-loaded
DC can have significant consequences on immune responses. Ag-
specific T cell activation and proliferation, and induction of anti-
tumor immunity were severely impaired when the DC were
cleared by an existing CTL response. However, it should be noted
that a limited degree of T cell proliferation and antitumor immu-
nity was reproducibly demonstrated in these experiments, suggest-
ing that some T cell activation was occurring despite the rapid
clearance of DC. This weakened T cell response may have resulted
from suboptimal stimulation by DC in the process of being elim-
inated or may have been induced by a low number of healthy DC
that had escaped elimination.
It has been reported that Ag from short-lived migratory DC can
be processed and presented by recipient DC within the lymph node
(30). If DC are cleared from the DLN by a cytolytic mechanism,
these dying cells may provide a ready source of Ag for lymph
node-resident DC. The recipient DC may be of the “lymphoid”
subclass that has been proposed to serve a regulatory, tolerogenic
function (31, 32). This process could account for the reduced an-
titumor responses observed when the DC were “cleared” by an
existing CTL response. Alternatively, it is possible that transfer of
Ag from migratory DC to lymph node-resident DC, in fact, may be
stimulatory but that this process was inefficient in our experimental
system.
One surprising finding in our study is that a proportion of the
injected DC appeared to persist in the DLN for a long time (in
excess of 7 days from the in vivo transfer). This suggests that DC
may have the ability to “wait” for T cells of the appropriate spec-
ificity to migrate through the lymph node and to recognize Ag on
their surface. The prolonged survival of some DC may be simply
a stochastic process or alternatively may reflect heterogeneity in
the degree of activation of our DC before injection in vivo. It is
also possible that the DC become activated in vivo by host CD4
⫹
T cells to become the “temporal bridge” hypothesized by Ridge et
al. (10), thereby becoming “conditioned” to stimulate further im-
mune responses. Our experiments do not distinguish among these
possibilities. In addition, we cannot exclude the possibility that this
apparent prolonged persistence of DC in the DLN may in fact
represent a slow turnover of DC that are continuously replaced by
fresh DC migrating from the site of injection. We find this possi-
bility less attractive in that it would appear that the lymph node
should offer a more favorable environment for DC survival com-
pared with that of the periphery. However, additional experiments
are required to clarify this issue.
The process of DC clearance has implications for the design of
DC-based immunotherapy regimes. From our results it can be con-
cluded that repeated immunizations at short intervals with DC
loaded with a given MHC class I-binding peptide may not be ef-
fective at enhancing responses to this Ag because the DC used in
the secondary injections would be rapidly cleared by existing CTL.
Indeed, our own experiments have suggested that repeated immu-
nization with LCMV
33–41
-loaded DC does not lead to enhanced
CTL responses against LL-LCMV challenge (data not shown).
However, repeated DC injections may be effective in maintaining
effector function in “memory” CD8
⫹
T cells that may have lost
activity due to suboptimal presentation of Ag in the context of
tumor tissue.
3100 CTL-MEDIATED ELIMINATION OF DC IN VIVO
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
Acknowledgments
We thank the personnel of the Wellington Medical School Biomedical
Research Unit for animal husbandry and Drs. H. Pircher and H. Bluethman
for the modified mouse strains. The expert help of Dr. H. H. Teoh,
A. Richardson, and R. Irlam of the Department of Laboratory Services
(Anatomic Pathology) at Wellington Hospital in the preparation and anal-
ysis of lymph node tissue sections is also gratefully acknowledged.
References
1. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392:245.
2. Romani, N., S. Koide, M. Crowley, M. Witmer-Pack, A. M. Livingstone,
C. G. Fathman, K. Inaba, and R. M. Steinman. 1989. Presentation of exogenous
protein antigens by dendritic cells to T cell clones: intact protein is presented best
by immature, epidermal Langerhans cells. J. Exp. Med. 169:1169.
3. Cumberbatch, M., and I. Kimber. 1992. Dermal tumour necrosis factor-
␣
induces
dendritic cell migration to draining lymph nodes, and possibly provides one stim-
ulus for Langerhans’ cell migration. Immunology 75:257.
4. Cumberbatch, M., and I. Kimber. 1995. Tumour necrosis factor-
␣
is required for
accumulation of dendritic cells in draining lymph nodes and for optimal contact
sensitization. Immunology 84:31.
5. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen
by human dendritic cells is maintained by granulocyte/macrophage colony stim-
ulating factor plus interleukin-4 and downregulated by tumor necrosis factor-
␣
.
J. Exp. Med. 179:1109.
6. Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. Dendritic cells use
macropinocytosis and the mannose receptor to concentrate macromolecules in the
major histocompatibility complex class II compartment: downregulation by cy-
tokines and bacterial products. J. Exp. Med. 182:389.
7. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand,
and J. Banchereau. 1994. Activation of human dendritic cells through CD40
crosslinking. J. Exp. Med. 180:1263.
8. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, and
G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high
levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via
APC activation. J. Exp. Med. 184:747.
9. Bennett, S. R. M., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. A. P. Miller,
and W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40
signalling. Nature 393:478.
10. Ridge, J. P., F. Di Rosa, and P. Matzinger. 1998. A conditioned dendritic cell can
be a temporal bridge between a CD4
⫹
T-helper and a T-killer cell. Nature 393:
474.
11. Schoenberger, S. P., R. E. M. Toes, E. I. H. Vandervoort, R. Offringa, and
C. J. M. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by
CD40-CD40L interactions. Nature 393:480.
12. Pugh, C. W., G. G. MacPherson, and H. W. Steer. 1983. Characterization of
nonlymphoid cells derived from rat peripheral lymph. J. Exp. Med. 157:1758.
13. Kupiec-Weginski, J. W., J. M. Austyn, and P. J. Morris. 1988. Migration patterns
of dendritic cells in the mouse. J. Exp. Med. 167:632.
14. Ingulli, E., A. Mondino, A. Khoruts, and M. K. Jenkins. 1997. In vivo detection
of dendritic cell antigen presentation to CD4
⫹
T cells. J. Exp. Med. 185:2133.
15. Pircher, H., K. Buerki, R. Lang, H. Hengartner, and R. M. Zinkernagel. 1989.
Tolerance induction in double specific T-cell receptor transgenic mice varies with
antigen. Nature 342:559.
16. Ko¨ntgen, F., G. Su¨ss, C. Stewart, M. Steinmetz, and H. Bluethmann. 1993. Tar-
geted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol.
5:957.
17. Hermans, I. F., A. Daish, P. Moroni-Rawson, and F. Ronchese. 1997. Tumor
peptide-pulsed dendritic cells isolated from spleen or cultured in vitro from bone
marrow precursors can provide protection against tumor challenge. Cancer Im-
munol. Immunother. 44:341.
18. Garrigan, K., P. Moroni-Rawson, C. McMurray, I. Hermans, N. Abernethy,
J. Watson, and F. Ronchese. 1996. Functional comparison of spleen dendritic
cells and dendritic cells cultured in vitro from bone marrow precursors. Blood
88:3508.
19. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober.
1991. Current Protocols in Immunology. Wiley, New York.
20. Oehen, S., and K. Brduscha-Riem. 1998. Differentiation of naive CTL to effector
and memory CTL: correlation of effector function with phenotype and cell divi-
sion. J. Immunol. 161:5338.
21. Ehl, S., P. Klenerman, P. Aichele, H. Hengartner, and R. M. Zinkernagel. 1997.
A functional and kinetic comparison of antiviral effector and memory cytotoxic
T lymphocyte populations in vivo and in vitro. Eur. J. Immunol. 27:3404.
22. Hermans, I. F., D. S. Ritchie, A. Daish, J. Yang, M. R. Kehry, and F. Ronchese.
1999. Impaired ability of MHC class II
⫺/⫺
dendritic cells to provide tumor pro-
tection is rescued by CD40 ligation. J. Immunol. 163:77.
23. Lyons, A. B., and C. R. Parish. 1994. Determination of lymphocyte division by
flow cytometry. J. Immunol. Methods 171:131.
24. Garside, P., E. Ingulli, R. R. Merica, J. G. Johnson, R. J. Noelle, and
M. K. Jenkins. 1998. Visualization of specific B and T lymphocyte interactions
in the lymph node. Science 281:96.
25. Zimmermann, C., A. Prevost-Blondel, C. Blaser, and H. Pircher. 1999. Kinetics
of the response of naive and memory CD8 T cell antigen: similarities and dif-
ferences. Eur. J. Immunol. 29:284.
26. Pantaleo, G., J. F. Demarest, H. Soudeyns, C. Graziosi, F. Denis,
J. W. Adelsberger, P. Borrow, M. S. Saag, G. M. Shaw, R. P. Sekaly, and
A. S. Fauci. 1994. Major expansion of CD8
⫹
T cells with a predominant V

usage during the primary immune response to HIV. Nature 370:463.
27. Vitiello, A., L. Yuan, R. W. Chesnut, J. Sidney, S. Southwood, P. Farness,
M. R. Jackson, P. A. Peterson, and A. Sette. 1995. Immunodominance analysis of
CTL responses to influenza PR8 virus reveals two new dominant and subdomi-
nant K
b
-restricted epitopes. J. Immunol. 157:5555.
28. Callan, M. F., N. Steven, P. Krausa, J. D. Wilson, P. A. Moss, G. M. Gillespie,
J. I. Bell, A. B. Rickinson, and A. J. McMichael. 1996. Large clonal expansions
of CD8
⫹
T cells in acute infectious mononucleosis. Nat. Med. 2:906.
29. Odermatt, B., M. Eppler, T. P. Leist, H. Hengertner, and R. M. Zinkernagel.
1991. Virus-triggered acquired immunodeficiency by cytotoxic T-cell-dependent
destruction of antigen-presenting cells and lymph follicle structure. Proc. Natl.
Acad. Sci. USA 88:8252.
30. Inaba, K., S. Turley, F. Yamaide, T. Iyoda, K. Mahnke, M. Inaba, M. Pack,
M. Subklewe, B. Sauter, D. Sheff, et al. 1998. Efficient presentation of phago-
cytosed cellular fragments on the major histocompatibility complex class II prod-
ucts of dendritic cells. J. Exp. Med. 188:2163.
31. Kronin, V., K. Winkel, G. Suss, A. Kelso, W. Heath, J. Kirberg, H. von Boehmer,
and K. Shortman. 1996. A subclass of dendritic cells regulates the response of
naive CD8 T cells by limiting their IL-2 production. J. Immunol. 157:3819.
32. Suss, G., and K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cells
via Fas/Fas-ligand-induced apoptosis. J. Exp. Med. 183:1789.
3101The Journal of Immunology
Downloaded from http://journals.aai.org/jimmunol/article-pdf/164/6/3095/1115875/3095.pdf by guest on 21 July 2023
