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- and CD8+ subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo
* Department of Discovery Research, Immunex Corporation, Seattle, Washington; and
Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles, Gosselies, Belgium
Correspondence: Thibaut De Smedt, Discovery Research Department, Immunex Corporation, 51 University Street, Seattle, WA 98101. E-mail: desmedtt{at}immunex.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression. We have
studied the phenotype and maturation state of mouse splenic
CD8- and CD8+ DCs. Both subsets were
found to reside in the spleen as immature cells and to undergo a
phenotypic maturation upon culture in vitro in
GM-CSF-containing medium or in vivo in response to
lipopolysaccharide. In vitro and in vivo
analyses showed that this maturation process is an absolute requisite
for DCs to acquire their T-cell priming capacity, transforming
CD8- and CD8+ DCs into potent and
equally efficient activators of naïve CD4+ and
CD8+ T cells. Furthermore, these results highlight the
importance that environmental factors may have on the ability of DC
subsets to influence Th responses qualitatively; i.e., the ability to
drive Th1 versus Th2 differentiation may not be fixed immutably for
each DC subset.
Key Words: CD4+/CD8+ • T-cell activation • GM-CSF • lipopolysaccharide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since their discovery more than two decades ago, the idea that
dendritic cells (DCs) play a central role in the development of
acquired immunity has become accepted widely [1
].
Indeed, among APCs, DCs appear to be unique in their capacity to
activate naïve T cells. This property correlates with their
expression of high levels of antigenic peptide/MHC, costimulatory
molecules such as CD80, CD86, and CD40, and their capacity to produce
interleukin (IL)-12 and possibly other cytokines [2
3
4
5
6
].
However, DCs do not display a constitutive costimulatory function
in situ and are immature immunologically; i.e., they do no
act as potent APC for naïve T cells. To acquire this adjuvant
property, DCs must undergo a process termed maturation. Upon pathogen
entry, tissue damage, or danger signals, DCs undergo functional
modifications, which include enhanced expression of MHC and
costimulatory molecules and secretion of stimulatory cytokines such as
IL-12 and tumor necrosis factor (TNF-), and alter their
responsiveness to chemokines such that they are directed to the
T-cell-rich areas of lymphoid organs [1
]. As they
acquire the capacity to prime naïve T cells, DCs also lose the
ability to capture and process antigens [7
]. Factors
inducing DC maturation in vitro include culture on plastic
[3
, 4
], bacterial components such as
lipopolysaccharide (LPS) [8
] and CpG-rich DNA motifs
[9
] or viral compounds such as double-stranded RNA
[10
]. Inflammatory signals such as LPS
[11
, 12
], CpG-rich DNA motifs
[13
, 14
], or Toxoplasma gondii tachyzoite
extracts [12
] induce in vivo maturation of
DCs. Once the activated DCs reach the lymphoid organs, they interact
with T cells and are able to generate primary immune responses.
Recently, improved isolation techniques have led to the identification
of multiple DC subsets. In mice, most investigators distinguish at
least two different subclasses of freshly isolated DCs on the basis of
their relative expression of CD8 or DEC-205 [15
,
16
]. DCs have also been subdivided on the basis of the
relative expression of the myeloid-related marker CD11b
[17
]. The CD8+ fraction of splenic DCs
falls within the CD11bdull population and comprises about
half of that subset [17
, 18
], whereas all
of the cells within the CD11bhigh population are
CD8-. Studies conducted in vitro have
suggested that CD8+ DCs could play a role in the
regulation of immune responses and may be responsible for generating
peripheral tolerance in naïve T cells, whereas
CD8- DCs may be more stimulatory [19
,
20
]. Indeed, some data have shown that only
CD8- DCs are able to activate naïve
CD4+ and CD8+ T cells in vitro and
that CD8+ DCs induce low activation and proliferation of
T cells [19
, 20
]. It has also been proposed
that the low proliferation induced in CD4+ T cells by
CD8+ DCs is a result of induction of apoptosis of the
reactive T cells through a Fas-dependent pathway [19
].
Conversely, other in vivo experiments have shown that either
subset, pulsed in vitro with antigen, can prime T cells but
CD8- DCs skew the T-cell response toward Th2, and
CD8+ DCs skew toward a Th1 response [5
,
6
]. Several studies have shown that CD8+
DCs are the major producers of IL-12. In vitro,
CD8+ DCs but not CD8- DCs are able to
respond to stimulation by secreting IL-12 p70 [5
,
6
]. In vivo, Reis e Sousa et al.
[12
] demonstrated that most of the IL-12, p40-positive
DCs in the spleen of mice exposed to soluble extracts form Toxoplsama
gondii or LPS belong to the CD8+ subset. Interferon
(IFN)-
has also been shown to be produced by the CD8+
DCs [21
]. Thus, these results suggest potential
differences in the function of DC subsets, subjected to change upon
maturation. To our knowledge, no studies have been undertaken to study
the maturation of DC subsets carefully and the role of the maturation
process on the capacity of DC subpopulations to activate naïve
T cells. Therefore, we have examined in vitro and in
vivo the maturation of the different DC subsets and analyzed the
interactions of immature and mature DCs of both subsets with
CD4+ or CD8+ T cells. We have found that
CD8- and CD8+ DC subsets require a
functional maturation to transform them into potent activators of
naïve T cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Injections
Mice were injected i.p. with 10 µg recombinant human FL
for 11 consecutive days to induce DC expansion. LPS from
Escherichia coli (serotype 055:B5) was purchased from Difco
Laboratories (Detroit, MI), and mice were injected i.p. with 5 µg LPS
solubilized in phosphate-buffered saline (PBS). Control animals were
injected with the same volume of PBS.
Cell line
The I-Ad-restricted, OVA-specific, T-cell hybridoma
DO11.10 [24
] (from Dr. P. Marrack, Howard Hugues Medical
Institute, National Jewish Medical and Research Center, Denver, CO) was
used to assess the processing capacity of DCs.
Flow cytometry
Cells were analyzed by flow cytometry with a FACScan cytometer
(Becton Dickinson, Mountain View, CA). The cells were stained in FACS
buffer [PBS containing 2% fetal bovine serum (FBS), 1% normal rat
serum, 1% normal hamster serum, and 10 µg/ml 2.4G2, a rat anti-mouse
Fc receptor monoclonal antibody (mAb)]. All mAbs were from PharMingen
(San Diego, CA) except the KJ1-26 mAb, anti-clonotypic Ab for DO11.10 T
cells, which was produced and labeled at Immunex. The mAbs used were as
follows: phycoerythrin (PE) or fluorescein isothiocyanate (FITC)
anti-mouse CD11c (HL3); FITC, biotin, or PE anti-mouse CD8 (53-6.7);
FITC anti-mouse CD90.1 (Thy-1.1); FITC-hamster immunoglobulin (Ig) or
-rat IgG2a or IgG2b as isotype controls; FITC anti-mouse
I-Ad or I-Ab; FITC anti-mouse CD40 (3/23); FITC
anti-mouse CD80 (B7.1, 16-10A1); FITC anti-mouse CD86 (B7.2, GL1);
biotin anti-mouse DEC-205 (NLDC-145); and PE anti-mouse CD4 (RM4-5).
Biotinylated Abs were revealed by Streptavidin Cy-Chrome. Cells were
gated according to forward- and side-scatter, or PI was added in the
fluorescence-activated, cell-sorter (FACS) buffer to eliminate dead
cells and debris from analysis.
Culture medium
Medium used in all experiments, unless otherwise noted, was
RPMI-1640 and Iscove’s modified Dulbecco’s medium mixed in equal
proportion, supplemented with 5% FBS, penicillin, streptomycin,
nonessential amino acids, sodium pyruvate, 2-mercaptoethanol (2-ME),
and L-glutamine.
Purification of DCs
Splenic DCs were purified from FL-treated animals following a
protocol described previously [6
]. To purify fresh,
immature DCs, low-density cells floating on a 45% Nycodenz gradient
(Nycomed, Oslo, Norway) were stained with FITC-CD11c and PE-CD8 mAbs
and sorted on a FACSVantage cytometer into CD11c+
CD8- or CD11c+ CD8+ DCs.
Activated, mature DCs were obtained by culturing DCs overnight in
complete medium containing 50 ng/ml recombinant
murine-granulocyte-macrophage colony-stimulating factor
(GM-CSF) or by i.p. injection of 5 µg LPS and separated as described
above.
For peptide-pulsing, DCs were incubated in complete culture medium containing 1 µM OVA peptide class II (323-339; DO11p) or OVA peptide class I (OT-Ip) for 30 min at 37°C and then washed twice. The cells were resuspended in PBS, and 3 x 105 DCs were injected subcutaneously into the hind footpads of recipient mice.
Measurement of CD4+ T-cell activation in
vitro
Serial dilutions of fresh or activated DCs were plated in
96-well culture plates with 0.5 µg/ml DO11p and 1 x
105 CD4+ T cells from DO11.10 mice in complete
medium. To enrich CD4 T cells, total lymph node (LN) and splenic cells
were incubated with anti-MHC class II mAbs, anti-B220, and anti-GR-1
for 30 min at 4°C. Ab-coated cells were depleted with anti-Ig
magnetic beads (Dynabeads, Dynal, Oslo). Proliferation was measured by
3H-thymidine incorporation.
Measurement of antigen-processing capacity in vitro
Serial dilutions of fresh or cultured DCs were plated in 96-well
culture plates with 0.5 mg/ml OVA protein (Calbiochem, La Jolla, CA) or
0.5 µg/ml DO11p and 1 x 104 DO11.10 T-cell
hybridoma in culture medium. After 24 h, culture supernatants were
harvested and assessed for IL-2 content using a sandwich enzyme-linked
immunosorbent assay (ELISA; PharMingen).
Measurement of CD4 T-cell expansion in vivo and
restimulation in vitro
BALB/c mice recipients of DO11.10 spleen cells were immunized
with peptide-pulsed DCs in the hind footpads, and 4 days later,
popliteal LN cells were harvested and counted, and T-cell expansion was
measured by flow cytometry using anti-CD4-PE mAb and anti-clonotypic
mAb KJ1.26-FITC. The LN cells were also restimulated in
vitro in triplicate with graded doses of the DO11p in DMEM medium
supplemented with 0.5% heat-inactivated mouse serum and additives.
Triplicate culture supernatants were pooled and assayed by ELISA for
IL-2 after 24 h and for IFN- and IL-4, -5, and -10, after
72 h of incubation. The detection limits were 25 pg/ml for IL-2,
25 pg/ml for IL-4, 25 pg/ml for IL-5, 40 pg/ml for IL-10, and 2 ng/ml
for IFN-.
Measurement of CD8+ T-cell expansion in vivo
and restimulation in vitro
C57BL/6 mice recipients of OT-Ip Thy1.1 spleen cells were
immunized with peptide-pulsed DCs in the hind footpads, and 5 days
later, popliteal LN cells were harvested and counted, and T-cell
expansion was measured by flow cytometry using anti-CD8-PE mAb and
anti-CD90.1-FITC mAb. For restimulation in vitro, twofold
serial dilutions (starting at 2.5x105 cells/well) of LN
cells were cultured in vitro with 2.5 x
105 irradiated, C57BL/6 spleen cells pulsed with 1 µM
OT-Ip in complete medium. The 3-day culture supernatants were assessed
for IFN- production by a two-site ELISA from PharMingen. Cytolytic T
lymphocyte (CTL) activity was measured after 7 days of culture in a
standard 51Cr-release assay. Briefly, C1498 target cells
were pulsed for 1 h at 37°C with or without 1 µM OT-Ip in the
presence of 51Cr in complete medium. Cells were washed
three times, and 1 x 104 cells/well were added to the
culture of LN cells restimulated in vitro with C57BL/6
spleen cells in a total volume of 200 µl. After 6 h of incubation at
37°C, 100 µl supernatant was removed, and radioactivity was counted
in a -counter. Spontaneous and total release was determined by
adding culture medium or detergent to target cells, respectively.
Percent-specific 51Cr release was calculated as 100 x
(experimental release-spontaneous release)/(total release-spontaneous
release).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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+ fraction of DCs (Fig. 1
). Immediately following separation, the expression of MHC and
costimulatory molecules on DC subsets was analyzed by flow-cytometry.
To analyze the expression of the same markers after maturation in
vitro, total low-density cells were left unseparated (because
factors released by bystander cells could play a role in the maturation
of DCs) and cultured overnight in medium containing GM-CSF, a cytokine
that has been shown to act as a DC-activating and -survival factor
[25
]. The cells were stained with CD11c and CD8 and
analyzed by flow cytometry, gating for expression of high levels of
CD11c and lacking CD8 or expressing CD8. Figure 1
shows that
freshly isolated DCs of CD8- and CD8+
subsets express low, but detectable, levels of I-A and the
costimulatory molecules CD80, CD86, and CD40, consistent with previous
studies [17
, 18
]. The expression of these
molecules was increased strongly after overnight culture in
GM-CSF-containing medium. The levels of expression for these molecules
were similar for CD8- and CD8+ DCs,
suggesting that the two subsets were activated similarly by overnight
culture.
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- and CD8+ subsets of fresh and
cultured DCs were used as APC for naïve T cells in
vitro. The activation of naïve, CD4+ T cells
is known to depend on the presentation of the antigen in the context of
MHC class II molecules, as well as the delivery of costimulatory
signals by the same APC. Serial dilution of the DC subsets was cultured
with naïve, CD4+ T cells from DO11.10 OVA-specific,
transgenic mice with the cognate OVA-derived peptide (DO11p). The
capacity of these mouse-DC subsets to induce the proliferation of the
naïve T cells was measured. As presented in Figure 2A
, CD8- and CD8+ cultured DCs are
more potent activators of naïve T cells than their fresh
counterparts, suggesting that maturation induced by culture is
necessary for all DC subpopulations to induce optimal naïve
T-cell activation in vitro. Also, once activated, both
subpopulations are able to activate T cells equally. Another
characteristic of DCs is their capacity to process protein antigen, a
function that is decreased after culture in vitro or after
maturation with LPS in vivo. Fresh and cultured DC
subpopulations from FL-treated mice were purified as described above
and cultured with the OVA-specific, DO11.10 T-cell hybridoma plus OVA
protein or the DO11p peptide. Activation of the hybridoma cells is
independent of costimulation and was measured as IL-2 secretion.
Freshly isolated CD8- DCs were able to process OVA
protein and induce high levels of IL-2 secretion (Fig. 2B)
. Fresh
CD8+ were also capable of processing OVA, although to a
considerably lesser extent than fresh CD8- DCs (Fig. 2B)
. Both subsets exhibited a dramatic decrease in the processing
capacity after maturation induced by overnight culture (Fig. 2B)
.
However, all DC preparations induced T-cell activation when coated with
DO11p, which does not require processing to be presented on MHC class
II (Fig. 2C) . It is interesting that high numbers of the
CD8+ subset of immature DCs induced a lower plateau in
the response compared with fresh CD8- and the activated
DCs, although the response induced at lower cell numbers was equivalent
for all DC preparations (Fig. 2C)
.
|
- or CD8+ DCs were pulsed
in vitro with the relevant peptide
(I-Ad-restricted DO11p or H-2Kb-restricted
OT-Ip) and injected subcutaneously into the hind footpads of the host
animals. T-cell expansion in draining lymph nodes was monitored by FACS
4 or 5 days after immunization for the CD4+ and
CD8+ T cells, respectively, when the expansion of each
T-cell subset was maximal (unpublished results).
Fresh CD8- DCs induced some expansion of
CD4+ T cells in vivo (on average, 20-fold
increase compared with the transferred-only group considered as
background), whereas the effect with fresh CD8+ DCs was
lower (sevenfold increase; Fig. 3A
). Maturation of both subsets of DCs by overnight culture in
GM-CSF-containing media increased their capacity strongly to induce
expansion of naïve, CD4+ T cells in vivo
(90-fold and 50-fold increase induced by cultured CD8-
DCs and CD8+ DCs, respectively). LN cells from these
immunized animals were restimulated in vitro with graded
doses of DO11p, and cytokines were measured in the culture supernatant.
As shown in Figure 3B
, LN cells from mice immunized with fresh DCs did
not secrete IL-2 or IFN- upon restimulation. In contrast, both
subsets of cultured DCs induced activation of T cells that produced
high levels of IL-2 and IFN- upon restimulation in vitro.
Of note, Th2 cytokines (IL-4, -5, and -10) were below the levels of
detection of our ELISAs (unpublished results), suggesting that both
subsets induced a Th1-like activity in this assay.
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- DCs
induced a reproducible expansion of CD8+ T cell in
vivo (on average, 20-fold increase above background), but only
cultured DCs were able to drive a high level of expansion (Fig. 4A
; on average, 100-fold and 220-fold increase induced by cultured
CD8- DCs and CD8+ DCs, respectively). We
also measured the development of effector functions of these expanded
cells. To this end, lymph-node cells from immunized mice were
restimulated in vitro with irradiated spleen cells pulsed
with the OT.Ip peptide. The production of IFN- was assessed from the
3-day culture supernatants (Fig. 4B)
, and the lytic activity was
determined 7 days after restimulation (Fig. 4C)
. In vivo
priming with fresh CD8+ DCs did not induce IFN-
secretion by the LN cells upon in vitro restimulation.
Fresh, CD8- DCs were able to prime for low levels of
IFN- production, but cultured DCs of either subset were able to
drive high production of IFN- (Fig. 4B)
. Also, only the mature DCs
were able to prime the LN CTLs to be active lytically toward
peptide-coated target cells (Fig. 4C) .
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- and
CD11c+ CD8+ DCs isolated from LPS-treated
animals displayed increased levels of MHC class II and CD40, CD80, and
CD86 costimulatory molecules as compared with control animals
(Fig. 5
). To test if the up-regulation of MHC and costimulatory molecules
on both subsets of DCs was associated with a functional maturation of
DCs, splenic CD8- and CD8+ DCs were
purified from naïve or LPS-injected animals and tested
immediately as accessory cells and stimulators for the presentation of
DO11p to naïve, CD4+ T cells from DO11.10
OVA-specific, transgenic mice. Figure 6
shows that both subsets of in vivo, matured DCs were
better activators of CD4+ T cells than DC from control
animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and CD8+ DCs to activate
naïve T cells in vitro and in vivo. We
found that splenic DCs reside in the tissue in an immature state, and
maturation of CD8- and CD8+ DCs is a
prerequisite for their optimal activation of naïve
CD4+ and CD8+ T cells. This correlated with our
finding that overnight culture of low-density spleen cells (enriched
for DCs) in medium containing GM-CSF induced strong up-regulation of
costimulatory and MHC molecules on CD8- and
CD8+ DCs. Furthermore, although we used FL to increase
the number of DC in the spleen to facilitate the analysis, the same
enhancement of costimulatory molecule expression was observed after
overnight culture of DCs purified from untreated mice (unpublished
results). These data differ somewhat from the findings of Vremec
and Shortman [16
] in that they did not observe an
up-regulation of CD86 after overnight culture of DCs. However, this
discrepancy may result from differences in DC preparation and culture
system; i.e., Vremec and Shortman cultured freshly isolated DCs,
obtained by depletion of the non-DC cells in the low-density fraction
of collagenase-treated spleen followed by sorting the different
subpopulations of DCs, whereas we cultured total low-density cells,
which are enriched for DCs but also contain some T cells, B cells, and
macrophages that could release factors contributing to the DC
maturation and were responsible for the more dramatic activation that
we observed. Indeed, although GM-CSF may influence DC maturation and
induces some increase in CD86 expression [3
], the nature
of other factor(s) that regulate CD86 expression on DCs remains to be
elucidated. We also found that in vivo injection of LPS led
to a similar phenotypic maturation of both subsets of DCs.
Antigen-pulsed, immature DCs of CD8- and
CD8+ subsets were able to induce some expansion of TCR
transgenic, antigen-specific CD4+ and CD8+ T
cells. However, this weak expansion does not seem to reflect a full
activation of the T cells because restimulation of these T cells did
not lead to cytokine secretion for CD4+ T cells or
differentiation of the CD8+ T cells into CTLs (Figs. 3 and 4)
. A similar weak expansion and lack of effector function induced by
immature DCs have been described by Jenkins and co-workers
[30
] who showed that injection of soluble antigen, which
does not induce maturation of DCs but is processed by DCs, provokes a
transient accumulation of adoptively transferred CD4+
TCR-transgenic T cells in lymphoid organs. However, these T cells do
not migrate to B-cell follicles, do not provide B help for antibody
production, and do not migrate out of lymphoid organs, as effector
cells do. Moreover, Viney et al. [31
] showed
that increasing the numbers of immature DCs in vivo, by
treating mice with FL, enhanced the induction of oral tolerance. In
contrast, matured DCs of either subset, when loaded in vitro
with the relevant OVA peptide and then injected into the footpads of
syngeneic hosts carrying adoptively transferred, OVA-specific,
transgenic CD4+ T cells, were able to drive the expansion
of the naïve, CD4+ donor cells and their
development into Th1 cells upon restimulation in vitro (Fig. 3)
. This is also in keeping with the results by Jenkins and colleagues
[30
], who found that co-injection of a bacterial
adjuvant such as LPS, which induces maturation of DCs in
vivo ([11
, 12
] and this paper),
enhanced the expansion of antigen-specific T cells, their migration
into B-cell follicles, and their migration into periphery. Moreover,
Williamson et al. [32
] showed that
administration of IL-1 or cholera toxin, which induces maturation of
the DCs, prevented induction of oral tolerance. Our results show that
both subsets of mature DCs induced the development of transferred,
OVA-specific, transgenic CD4+ T lymphocytes into Th1 cells.
This is a contradiction of a previous study by Pulendran et
al. [5
] who showed that in the same transfer
system, the freshly isolated cells of the CD11bhigh subset
induce secretion of Th2 cytokines (IL-4 and IL-10) in addition to Th1
cytokines (IFN- and IL-2). However, the same preparations of mature
CD8- and CD8+ DCs that we used in this
study, when pulsed with keyhole limpet hemocyanin (KLH), direct the
development of distinct Th cells (unpublished results), as previously
described [6
]. The reason for this discrepancy between
our study and the results obtained by Pulendran et al.
[5
] may be related to differences in the purification
procedures, the subpopulations studied (CD11b vs. CD8), the
precursor frequency of antigen-reactive T cells, and/or the maturation
state of the DCs transferred.
We also showed in an antigen-specific system that only matured DCs of
both subsets are able to drive expansion of naïve,
CD8+ T cells in vivo, as shown by Ruedl and
Bachmann [33
]. Also, in that system, immature DCs were
unable to induce the activation of naïve, CD8+ T
cells in vivo, and maturation is a necessary step to
transform DC subsets for priming of CTL immunity. This is in accordance
with a recent study by Schuurhuis and colleagues [34
]
who used a DC cell line (D1) as APC and demonstrated that maturation of
DCs is essential for efficient induction of CTL responses. This
suggests that DCs, independent of their expression of the CD8
marker, need to undergo maturation and then are able to drive
functional T-cell activation in vitro and in
vivo. Our results support the notion that the different potential
outcomes of T-cell activation could depend on the maturation state of
the DCs presenting antigen.
Previously, our group has shown that the CD11bdull and
CD11bhigh DCs differ in their capacity to phagocytose
zymosan particles, with the CD11bhigh subset being more
effective [18
]. Here, we show that splenic
CD8+ DCs have a low capacity to capture and process OVA
protein and subsequently present its peptides in the context of MHC
class II molecules, although they are able to present OVA peptide,
which does not require processing. CD8- DCs, when
pulsed with OVA protein, are able to process this molecule into
peptides and present it efficiently to MHC class II-restricted T cells.
Reis e Sousa and Germain [35
] have shown that upon
injection of hen egg lysozyme into naïve mice, splenic
IDC (which are mostly CD8+) [18
] do not
present detectable levels of HEL peptides complexed with their MHC
class II molecules at early time points after injection. Later, as new
CD8+ DCs are recruited into the T-cell area, these cells
began to display high levels of HEL peptides complexed with MHC class
II molecules, presumably because they captured antigen in the
periphery. Of note, the CD8+ DCs used in the present
study were isolated from the spleen and, accordingly, would no longer
have been able to process native protein. This suggests that the
processing capacity of CD8+ DCs may be regulated over
time and space. They may be highly endocytic or phagocytic in the
periphery and may lose this function upon migration to the lymphoid
organs, although they may be immature phenotypically and
functionally as measured by other parameters. It is also interesting to
note that high numbers of immature CD8+ DCs were not
able to drive the same levels of IL-2 secretion as CD8-
DCs (Fig. 2C)
. This may be related to the observation by Inaba et
al. [36
], which suggested that interdigitating DCs
(a majority of them belonging to the CD8+ subset)
express high levels of self peptides and induce apoptosis in a T-cell
hybridoma specific for this MHC class II self-petide complex. It could
also be related to the proposed regulatory role of CD8+
DCs, whereby immature cells are tolerizing rather than immunogenic
[37
].
Upon exposure to maturation signals, DCs modify their expression of chemokine receptors, leave peripheral tissues, and migrate to draining LNs where they interact with naïve T cells [38 , 39 ]. It is clear that mature DCs are much better APCs for naïve T cells than immature DCs, because we observed a difference between fresh and cultured DCs in the activation of naïve T cells in vitro (Fig. 2A) , where DCs and T cells are in close contact, and migration is not an issue. However, we cannot rule out the possibility that some of the differences we saw when we transferred DCs back into mice were because of trafficking differences between fresh and cultured DCs.
It has been proposed that DCs have a role in promoting or maintaining
peripheral tolerance to self antigens and that CD8+ DCs
belong to a specific lymphoid lineage of DCs related to thymic DCs,
which are involved in induction of T-cell tolerance in vivo
[19
, 37
, 40
]. Our findings
indicate that neither immature CD8- nor
CD8+ DCs are able to induce optimal T-cell activation
in vivo, and maturation is a prerequisite for both subsets
to become potent activators of naïve T cells. Because a
majority of the DCs that are tightly associated with T cells in mouse
lymphoid organs in the absence of an active immune response are
CD8+ DCs ([18
] and our own unpublished
data), and because these cells have the functional characteristics of
immature DCs (our results), these cells would appear to be in a
position to induce self-tolerance. However, inflammation and pathogen
signals would result in the co-localization of T cells and mature DCs
of both subsets capable of inducing strong T-cell responses.
In the unpertubated state, both major subpopulations of splenic DCs are immature, and as such, maturation induced by inflammation or danger signals is required to transform these cells into potent APCs. Given the important role of DCs in the induction of immunity, and potentially tolerance, it is important that we define further the nature and function of different DC subsets in conditions of health and disease.
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ACKNOWLEDGEMENTS |
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Received October 2, 2000; revised December 22, 2000; accepted December 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo J. Exp. Med. 189,587-592 production by CD8+ lymphoid dendritic cells J. Exp. Med. 189,1981-1986This article has been cited by other articles:
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 J. R. Gordon, F. Li, A. Nayyar, J. Xiang, and X. Zhang CD8{alpha}+, but Not CD8{alpha}-, Dendritic Cells Tolerize Th2 Responses via Contact-Dependent and -Independent Mechanisms, and Reverse Airway Hyperresponsiveness, Th2, and Eosinophil Responses in a Mouse Model of Asthma J. Immunol., August 1, 2005; 175(3): 1516 - 1522. [Abstract] [Full Text] [PDF]
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T. Yasumi, K. Katamura, I. Okafuji, T. Yoshioka, T.-a. Meguro, R. Nishikomori, T. Kusunoki, T. Heike, and T. Nakahata Limited Ability of Antigen-Specific Th1 Responses to Inhibit Th2 Cell Development In Vivo J. Immunol., February 1, 2005; 174(3): 1325 - 1331. [Abstract] [Full Text] [PDF]
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S. Marino, S. Pawar, C. L. Fuller, T. A. Reinhart, J. L. Flynn, and D. E. Kirschner Dendritic Cell Trafficking and Antigen Presentation in the Human Immune Response to Mycobacterium tuberculosis J. Immunol., July 1, 2004; 173(1): 494 - 506. [Abstract] [Full Text] [PDF]
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P. Bjorck Dendritic Cells Exposed to Herpes Simplex Virus In Vivo Do Not Produce IFN-{alpha} after Rechallenge with Virus In Vitro and Exhibit Decreased T Cell Alloreactivity J. Immunol., May 1, 2004; 172(9): 5396 - 5404. [Abstract] [Full Text] [PDF]
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T. Yasumi, K. Katamura, T. Yoshioka, T.-a. Meguro, R. Nishikomori, T. Heike, M. Inobe, S. Kon, T. Uede, and T. Nakahata Differential Requirement for the CD40-CD154 Costimulatory Pathway during Th Cell Priming by CD8{alpha}+ and CD8{alpha}− Murine Dendritic Cell Subsets J. Immunol., April 15, 2004; 172(8): 4826 - 4833. [Abstract] [Full Text] [PDF]
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A. D. Edwards, D. Chaussabel, S. Tomlinson, O. Schulz, A. Sher, and C. Reis e Sousa Relationships Among Murine CD11chigh Dendritic Cell Subsets as Revealed by Baseline Gene Expression Patterns J. Immunol., July 1, 2003; 171(1): 47 - 60. [Abstract] [Full Text] [PDF]
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R. K. Veeraswamy, M. Cella, M. Colonna, and E. R. Unanue Dendritic Cells Process and Present Antigens Across A Range of Maturation States J. Immunol., June 1, 2003; 170(11): 5367 - 5372. [Abstract] [Full Text] [PDF]
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C. Vasu, R.-N. E. Dogan, M. J. Holterman, and B. S. Prabhakar Selective Induction of Dendritic Cells Using Granulocyte Macrophage-Colony Stimulating Factor, But Not fms-Like Tyrosine Kinase Receptor 3-Ligand, Activates Thyroglobulin-Specific CD4+/CD25+ T Cells and Suppresses Experimental Autoimmune Thyroiditis J. Immunol., June 1, 2003; 170(11): 5511 - 5522. [Abstract] [Full Text] [PDF]
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A. D. Straw, A. S. MacDonald, E. Y. Denkers, and E. J. Pearce CD154 Plays a Central Role in Regulating Dendritic Cell Activation During Infections That Induce Th1 or Th2 Responses J. Immunol., January 15, 2003; 170(2): 727 - 734. [Abstract] [Full Text] [PDF]
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 G. Schiavoni, F. Mattei, P. Sestili, P. Borghi, M. Venditti, H. C. Morse III, F. Belardelli, and L. Gabriele ICSBP Is Essential for the Development of Mouse Type I Interferon-producing Cells and for the Generation and Activation of CD8{alpha}+ Dendritic Cells J. Exp. Med., December 2, 2002; 196(11): 1415 - 1425. [Abstract] [Full Text] [PDF]
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