International Immunology, Vol. 12, No. 5, pp. 731–735
© 2000 The Japanese Society for Immunology
DEC-205 as a marker of dendritic cells with
regulatory effects on CD8 T cell responses
Vadim Kronin, Li Wu, Schiaoching Gong1, Michel C Nussenzweig1 and
Ken Shortman
The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Melbourne,
Victoria 3050, Australia
1The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10021, USA
Keywords: CD8, DEC-205, IL-2 production, regulatory dendritic cells
Abstract
We have previously reported that a population of lymphoid-related CD8α⍣ DEC-205⍣ dendritic cells
(DC) from mouse spleen have ‘regulatory’ effects on the T cells they activate. CD8 T cells produce
IL-2 and give a sustained proliferative response to allogeneic CD8α– DEC-205– splenic DC, but
produce little IL-2 and give a limited response to allogeneic CD8⍣ DEC-205⍣ splenic DC. Although
CD8α and DEC-205 correlate closely among splenic DC, lymph nodes (LN) include a large
population of CD8αlow DEC-205high DC. By i.v. transfer of purified thymic early lymphoid precursors
into irradiated recipient mice we now demonstrate that these CD8αlow but DEC-205high LN DC can
be the progeny of a lymphoid precursor population, apparently corresponding to the CD8αhigh
DEC-205high DC progeny of the same precursors in spleen and thymus. By culture of the separated,
purified DC with allogeneic CD8 T cells we demonstrate that the CD8αlow DEC-205high DC of LN are
also functionally equivalent to the CD8αhigh DEC-205high DC of spleen. Therefore, DEC-205 but not
CD8α serves to segregate functionally distinct DC types in LN. However, DC isolated from the
spleens of genetically manipulated DEC-205null mice and separated on the basis of CD8α
expression have a similar capacity to stimulate CD8 T cells as their heterozygous littermate
controls, with the CD8α⍣ but now DEC-205null DC still giving restricted responses. In conclusion,
high expression of DEC-205 appears to be a good marker of the lymphoid-related regulatory type
of DC, but DEC-205 itself is not responsible for transmitting negative signals to the T cells.
We have previously reported that certain antigen-presenting
dendritic cells (DC) have a capacity to regulate the response
of the T cells they activate (1–3). In mouse spleen two distinct
DC populations may be separated on the basis of CD8α
expression, the CD8α⫹ DC being apparently of lymphoid
origin and the CD8α– DC being apparently of myeloid origin
(1,4–6). Although these two DC types appear equivalent in
their ability to present antigen and activate T cells into cell
cycle, they differ in the subsequent events which determine
the extent and duration of activated T cell proliferation, the
CD8α⫹ DC having distinct negative effects (2,3). In particular,
CD8 T cells when stimulated by CD8α⫹ DC produce very
little IL-2, which restricts their subsequent expansion, whereas
the same CD8 T cells when stimulated by CD8α– DC produce
substantial free IL-2 in the culture supernatant, sufficient to
support extended cell expansion (3). This difference in
induced cytokine output by CD8 T cells is determined within
the first day of culture with the appropriate DC (7). This
‘regulatory’ effect could not be attributed to differences
between the DC in signals from co-stimulator molecules such
as B7-1 or B7-2 which are equivalent on the two populations
nor to differences in soluble factors such as IL-12—some
new signaling system appears to be involved (7).
The possibility that CD8α itself on the DC might transmit
negative signals to the T cells was considered (8); however,
by using CD8 ‘null’ mice and surrogate markers for the
separation of two DC populations, it was demonstrated that
the regulatory effects were independent of DC CD8 expression
(9). This focussed attention on DEC-205 whose surface
expression correlates closely with CD8α on mouse spleen
DC, as well as on mouse thymus DC which are predominantly
CD8α⫹ DEC-205⫹ (5). DEC-205 served as the surrogate
marker for CD8α in the above experiments. DEC-205, a
characteristic marker of interdigitating DC in the T cell areas
Correspondence to: K. Shortman
Transmitting editor: M. Feldmann
Received 7 June 1999, accepted 3 February 2000
732 DEC-205 and DC function
Fig. 1. The relationship between DEC-205 and CD8α expression in
DC isolated from spleen and LN. DC were first extracted and enriched
as described elsewhere (3,4). Briefly, tissues were cut into pieces,
digested at 22°C for 25 min with collagenase DNase, EDTA treated
to break up DC–T cell complexes and then the lightest 3–5% of the
cells selected using Nycodenz density centrifugation. DC were further
enriched by incubating with a cocktail of mAb against cells other
than DC followed by immunomagnetic bead depletion. In the present
studies, in order to ensure macrophage depletion this cocktail
included low levels of anti-Mac-1 and anti-FcRII to deplete only cells
expressing high levels of these surface antigens. We have since
found this leads to some DC loss, particularly of CD8α– DC. However,
subsequent experiments omitting these depletion reagents and using
only F/480 to eliminate macrophages have given similar results to
those in this figure, except for an increase in the relative level of
DEC-205– CD8α– DC. The final DC preparation, 70–90% pure, was
stained in three fluorescent colors with anti-CD11c or anti-class II
MHC to distinguish DC, with NLDC-145 to stain DEC-205 (10) and
with anti-CD8α, using directly conjugated or biotinylated mAb as
described elsewhere (3,5); propidium iodide was added to the wash
solution to stain dead cells. The DC were analyzed on a FACStar Plus
instrument (Becton Dickinson, San Jose, CA), gating for CD11cbright or
class II MHCbright cells with the high forward and side light scatter
characteristics of DC, and gating against propidium iodide-staining
dead cells. The resultant fluorescent distribution of DEC-205 versus
CD8α staining is given. For sorting of DC fractions a similar approach
was taken, generally with only two-color staining for CD11c versus
either CD8α or DEC-205.
of lymphoid organs, is recognized by the mAb NLDC-145
(10). Structurally DEC-205 contains 10 distinct lectin-like
domains and it may be implicated in the uptake of carbohydrate-containing molecules into the DC antigen-presentation
pathway (11). There is nothing so far to suggest DEC-205 is
involved in transmitting signals to T cells and the NLDC-145
mAb has no obvious blocking effects (9). However, this
possible role of DEC-205 had not been critically examined.
Accordingly, in the present study we first assessed the
value of DEC-205 as a marker of those DC which induce only
restricted CD8 T cell responses, extending the study from
spleen to lymph nodes (LN) where the tight correlation
between CD8α and DEC-205 expression on DC breaks down
(5). We then directly tested the role of DEC-205 in transmitting
signals regulating CD8 T cell proliferation, by using DC
isolated from DEC-205null mice, produced in the Rockefeller
Institute laboratory (12).
When DC were isolated from spleen and analyzed by
immunofluorescent staining and flow cytometry there was a
tight correlation between CD8α expression and DEC-205
expression, resulting in two predominant populations,
CD8α⫹ DEC-205⫹ and CD8– DEC-205– (Fig. 1). This con-
firmed our previous studies (5). As would be expected from
this correlation either marker could be used to segregate the
two functionally distinct DC populations. Thus, as shown in
Fig. 2, the DC isolated according to low expression of either
CD8α or DEC-205 induced better proliferation of allogeneic
CD8 T cells than did the DC isolated according to high
expression of either CD8α or DEC-205. This applied over a
wide DC dose range. These cultures were pulsed at day 3,
the time when IL-2 availability begins to limit proliferation in
cultures stimulated by CD8α⫹ DC; the difference is more
pronounced at later time points (3).
However, as shown in Fig. 1, the correlation between CD8α
and DEC-205 expression is not as close amongst the DC
isolated from LN, confirming our previous analyses (5). As
well as the CD8α⫹ DEC-205⫹ and CD8α– DEC-205– DC
populations an additional group of CD8– to low DC expressing
high levels of DEC-205 is apparent. One possibility is that
some of these were lymphoid-derived or lymphoid-related DC
that had failed to acquire CD8α expression. We had previously
observed that DC which develop in culture from the early
thymic lymphoid precursor population lack surface CD8α
expression (13), although the DC progeny of the same
precursors found in thymus or spleen following i.v. transfer to
irradiated recipient mice all express high levels of CD8α (6).
To test whether the LN DC progeny of a lymphoid precursor
population could include DC lacking CD8α expression, the
early thymic lymphoid precursor population was purified from
Ly 5.2 mice and transferred i.v. into irradiated Ly 5.1 recipient
mice, following our earlier procedures (6) except that the Ly
5.2⫹ DC progeny in the LN, as well as in the thymus and
spleen, were analyzed. As shown in Fig. 3, the DC progeny
of the thymic lymphoid precursor population in the LN were
predominantly CD8– to low, although they were all DEC-205high.
In confirmation of our previous studies, the progeny of these
precursors in the thymus and spleen of the same mice all
expressed high levels of both CD8α and DEC-205 (data
not shown).
To determine if these distinct subgroups of DC in LN
showed differential effects on CD8 T cell proliferation, as
found for splenic DC, LN DC were isolated and separated on
the basis of CD8α expression, then cultured with allogeneic
CD8 T cells. As shown in Fig. 4, in contrast to splenic DC,
there was little difference in the extent of induced T cell
proliferation between the CD8α– and CD8⫹ LN fractions. Both
types of LN DC induced a lower level of proliferation compared
to splenic CD8α– DC, especially at the crucial later time
points when IL-2 became limiting. However, when the LN DC
were separated on the basis of DEC-205 expression, the
results were similar to those seen when splenic DC were
separated on the basis of DEC-205 expression or CD8α
expression; the responses to both DC fractions were similar
up to day 2.5 of culture, but after this time the response of
the DEC-205⫹ DC stimulated cultures fell off rapidly (Fig. 4).
It seemed that in LN DEC-205, rather than CD8α, was
marking the ‘regulatory’ DC population which stimulated the
CD8 T cells into cycle but induced only limited IL-2 production
and hence restricted proliferation. Based on the results of
Figs 2 and 3, it could be deduced that the CD8α– fraction
from LN would include the more stimulatory CD8α– DEC-205–
DC of the type found in spleen, together with some ‘regulatory’,
DEC-205 and DC function 733
Fig. 2. Cell dose–response of the capacity of different splenic DC
fractions to induce sustained proliferation in allogeneic CD8 T cells.
Splenic DC were isolated from C57BL/6 mice, and sorted into CD8α⫹
and CD8α– populations, or into DEC-205⫹ and DEC-205– populations,
as illustrated in Fig. 1. Various numbers of the DC were cultured with
20,000 pure CBA CD8 T cells isolated from LN as described previously
(3). Cultures were in 200 µl modified RPMI 1640–10% FCS medium
in V-bottom wells of 96-well culture trays, for 3 days at 37°C in a
humidified 10% CO2-in-air incubator, as described previously (3).
The cultures were then pulsed with 1 µCi of [3H]thymidine for 6
h, the cells harvested on glass fiber filters then the radioactivity
incorporated into cellular DNA determined using a gas scintillation
β-counter. Results represent the mean ⫾ SEM of triplicate cultures,
from an experiment typical of two (DEC-205) or five (CD8α) such
DC dose–response assays. The differences obtained were more
pronounced at later culture times.
lymphoid-related DC that were DEC-205⫹ but had failed to
express CD8α, unlike their splenic counterparts. The excess
of these CD8α– DEC-205⫹ DC seemed likely to account for
the reduced responses to LN CD8α– DC. To check this,
CD8α– DC were isolated from LN, segregated by DEC-205
expression then cultured with allogeneic CD8 T cells. As
shown in Fig. 4, this view was confirmed, the CD8α– DEC205⫹ LN DC giving the curtailed proliferative response
resembling that of the splenic CD8α⫹ DEC-205⫹ DC.
The conclusion from these experiments was that DEC-205
served as a better marker of the DC population giving curtailed
CD8 T cell responses than did CD8α. This fitted with our
previous findings that CD8α itself did not give the negative
signals restricting CD8 T cell cytokine production, but posed
the question of whether DEC-205 was serving to transmit
regulatory signals. To test this possibility, we isolated DC from
DEC-205null mice and compared their CD8 T cell stimulatory
ability with that of DC from littermate DEC-205-expressing
heterozygotes. In these experiments only splenic DC were
studied, since only in spleen could CD8α be used as a
surrogate marker for DEC-205, to segregate the two functional
Fig. 3. The expression of CD8α and DEC-205 on the LN DC progeny
of thymic lymphoid precursors. The early thymic precursors (the ‘low
CD4’ precursors) were purified from 4- to 6-week-old C57BL/6 (Ly
5.2) mice and 30,000 were transferred i.v. into irradiated 7- to
8-week-old C57BL/6 Ly 5.1-Pep3b (Ly 5.1) mice, as described in
detail elsewhere (6). After 4 weeks, the LN (mesentery, aortic, axillary)
of recipient mice were pooled, the DC enriched, and then stained in
four fluorescent colors and analyzed for expression of class II MHC,
of Ly 5.2, and of DEC-205 and CD8α, as described elsewhere (6).
The distribution of DEC-205 and CD8α is presented for cells gated
for high class II MHC and Ly 5.2 expression, with the high forward and
side light scatter of DC. The broken line represents the background
fluorescence omitting only the relevant mAb. The results are typical
of four separate experiments sampling the LN DC progeny from 2 to
6 weeks after precursor cell transfer.
types of DC. DC were found to be present at near normal
total numbers in the spleens of DEC-205null mice, although
the numbers of CD8α⫹ DC recovered was a little higher
(49,000 ⫾ 9000 per spleen compared to 39,000 ⫾ 2000 for
the littermates) and the number of CD8– DC recovered was
lower (7000 ⫾ 3000 per spleen compared to 12,000 ⫾ 5000
for the littermates). The functional tests on these isolated DC
are shown in Fig. 5.
The ability of the two DC fractions from the spleens of DEC205null mice to stimulate allogeneic CD8 T cells was similar
to that of normal mice. In both DEC-205null and normal mice,
the CD8α– DC induced a more extended and higher CD8
T cell proliferative response, while the response to the CD8α⫹
DC was curtailed. It was concluded that the absence of DEC205 had little effect on the ability of the DC to interact with
allogeneic CD8 T cells in culture, and that DEC-205 itself was
not responsible for negative signalling leading to reduced
cytokine output and reduced proliferation by CD8 T cells. The
one caveat on this conclusion is that the role of DEC-205
could be redundant, other related molecules taking over the
function when DEC-205 is absent.
Overall it is clear that DEC-205 can serve as a useful marker
of the type of DC inducing in CD8 T cells only restricted
cytokine production and a curtailed proliferative response (9);
734 DEC-205 and DC function
Fig. 5. Kinetics of the response of CD8 T cells to allogeneic splenic
DC purified from DEC-205 ‘null’ mice or from heterozygous littermate
control mice. Conditions were similar to Fig. 4. The results are means
⫾ SEM from a single experiment, representative of three experiments
performed.
Fig. 4. Kinetics of the response of CD8 T cells to different DC fractions
from allogeneic spleen or LN. DC were purified from C57BL/6 mouse
spleen or LN and sorted into various fractions as in Fig. 1. CD8
T cells were purified from CBA mouse LN as in Fig. 2. The DC (500/
well) were cultured with CD8 T cells (20,000/well) as in Fig. 2. The
cultures were pulsed for 6 h with [3H]thymidine at the times indicated
and incorporation of radioactivity into cellular DNA measured as in
Fig. 2. Results are the means ⫾ SEM of pooled data from two to six
individual experiments, each experiment involving three cultures per
time point. Cultures with T cells alone gave very low counts (see
Fig. 2), stimulation indices at the peak being always in excess of 100.
influence on the earlier steps of antigen uptake and processing
remains to be determined, since in this study we have only
considered responses to endogenous alloantigens.
Surface DEC-205 expression is clearly not an absolute
marker of regulatory DC, since even the population we have
designated DEC-205– includes some DC with low surface
expression and we have found all DC stain positive for
intracellular DEC-205 if permeabilized (5). In addition, the
surface expression of DEC-205 increases on all DC if they
are cultured for a short period (5). However, even under these
circumstances when all DC are clearly surface positive, the
level of DEC-205 expression still serves to distinguish the
populations (5). The fact that the ‘regulatory’ or less stimulatory
type of DC expresses the highest levels of DEC-205 now
presents a paradox, since these are the DC believed to be
concentrated in the T cell areas of spleen and LN (10), and
these might have been expected to be the most stimulatory
DC type.
Acknowledgements
in many circumstances it may be a better marker for this
population than CD8α. This type of ‘regulatory’ DC is likely to
be of lymphoid origin, since similar DC can be produced
artificially by transfer of the thymic lymphoid precursor population (Fig. 3) (6). However, it is clear that DEC-205 itself does
not govern the nature of DC interaction with the T cells. Its
This work was supported by the Cooperative Research Centre for
Vaccine Technology, Queensland Institute for Medical Research, by
a Human Frontier Science Program Grant RG-237-97, and by the
National Health and Medical Research Council, Australia. We thank
David Vremec for the surface phenotype analysis of normal DC
populations, and Dora Kaminaris, Jennie Parker and Frank Battye for
assistance with flow cytometry.
DEC-205 and DC function 735
Abbreviations
DC
LN
dendritic cell
lymph node
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