By
From the Centenary Institute of Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia, 2042
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Abstract |
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Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8 homodimers. The functions of the two subsets remain
controversial, although it has been suggested that the lymphoid-derived (CD8
+) subset induces tolerance, whereas the myeloid-derived (CD8
) subset has been shown to prime naive
T cells and to generate memory responses. To study their capacity to prime or tolerize naive
CD4+ T cells in vivo, purified CD8
+ or CD8
dendritic cells were injected subcutaneously
into normal mice. In contrast to CD8
dendritic cells, the CD8
+ fraction failed to traffic to
the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8
+ dendritic
cells could be detected. Such responses may have been initiated via transfer of peptide-major
histocompatibility complex complexes to migratory host CD8
dendritic cells after injection.
These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8
+ and CD8
dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the
response to immunogenic CD8
dendritic cells by CD8
+ dendritic cells.
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Introduction |
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Appropriate induction of tolerance and immunity (memory) is a crucial function of the normal immune system. The discovery of a novel subset of dendritic cells (DCs) derived from a common lymphoid precursor (1) and located in the thymic medulla and the T cell zones of secondary lymphoid tissue (2), together with preliminary in vitro functional data (3, 4), has led to the suggestion that such lymphoid DCs (LDCs) may be responsible for generating tolerance in naive T cell populations (5, 6). We have recently proposed an evolutionary model of self/non-self discrimination (7) in which LDCs are postulated to induce deletional tolerance to self-antigens by means of preferential internalization and presentation of self-antigen via receptors recognizing the characteristic chemical structures generated by self-biosynthetic enzymes. In contrast, myeloid DCs (MDCs) are already known to express a number of "pattern recognition receptors" (8) that recognize the biosynthetic footprints of foreign organisms, and to stimulate naive T cells in such a way as to generate T cell memory (9).
As an experimental test of whether LDCs and MDCs induce distinct in vivo responses in naive T cells, splenic DCs
were fractionated on the basis of CD8 expression, which
has been defined as a marker capable of distinguishing
splenic LDCs and MDCs in the mouse (2). A novel approach (6) was used in an attempt to ensure that only the
injected DCs were capable of presenting the test antigen, a
peptide of moth cytochrome c which is known to bind to
IE but not IA molecules. By injecting IE+ DCs into a host
that expressed IE only in the thymus, peptide presentation
was restricted to the adoptively transferred APCs, to which
the host T cells were nonetheless tolerant as a result of negative selection to IE in the thymus. Adoptive transfer of a
cohort of purified moth cytochrome c (MCC)-specific naive T cells provided a sensitive detection system for presentation of MCC peptide in vivo.
Surprisingly, we found no evidence that CD8+ DCs
migrated into the draining lymph nodes (DLNs) after subcutaneous injection. Nonetheless, peptide-pulsed, sorted
CD8
+ DCs were able to stimulate a significant T cell response. As expected, donor-derived CD8
DCs were
found in the DLNs and also stimulated T cell division. These data suggest an alternative interpretation of recent
experiments in which subcutaneous injection of antigen-pulsed LDCs was shown to induce Th1 priming, whereas
MDCs biased the response towards Th2 unless IL-12 was
coinjected (12, 13).
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Materials and Methods |
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Experimental Animals.
Transgenic (Tg) mouse lines were bred and housed under specific pathogen-free conditions at the Centenary Institute Animal Facility. Approval for all animal experimentation was obtained from the Institutional Ethics Committee at the University of Sydney. 107-1 and 36-2 lines of IET Cell Purification, Labeling, and Injection.
Pooled inguinal, axillary, subscapular, cervical, and paraaortic LNs of naive (TCR × 107-1) mice served as the source of MCC-specific T cells. Purified T cells were prepared from single cell suspensions and labeled with 5-carboxy fluorescein succinimidyl ester (CFSE) as described previously (18). 107 T cells were injected into the lateral tail vein of unirradiated mice 2 d before injection of DCs.DC Purification, Labeling, and Injection.
A modification of the protocol of Vremec and Shortman (19) was used to purify splenic DCs. Digestion with collagenase/EDTA and density centrifugation (Flow Cytometry.
Five color antibody staining was performed as described previously for analysis of CFSE-labeled T cells (18). For detection of CMFDA-labeled DCs in DLNs, individual popliteal LNs were digested in collagenase/EDTA, as for spleen. A combination of anti-Ly5.1 (biotinylated A20.1 [provided by E.A. Boyse, Memorial Sloan-Kettering Cancer Center, New York] plus allophycocyanin-conjugated streptavidin), anti-CD11c (N418 supernatant plus anti-hamster Texas red [Caltag]), and CMFDA fluorescence was used to distinguish donor-derived DCs. Expression of CD8 ![]() |
Results and Discussion |
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The experimental protocol, designed
to limit antigen presentation to the DC subset of interest,
made use of a pair of MHC Tg lines in which IEd was expressed on the H-2b background, allowing it to pair with
endogenous IE
b to form a functional IE molecule capable
of presenting MCC87-103 peptide to naive Tg T cells expressing the 5C.C7 TCR. Mice from the 36-2 line (14, 15),
which expresses IE
d only in the thymus, were used as hosts
of adoptively transferred purified responder T cells and of
subcutaneously injected DCs. Mice from the 107-1 line (14,
15), in which IE
d is expressed with a wild-type distribution, served both as direct DC donors in the functional
experiments and as parents of (107-1 × B6.SJLPtprca)F1
donors of Ly5.1+ DCs and (TCR Tg × 107-1)F1 donors of
T cells.
To test the capacity of subcutaneously injected DCs to
migrate to the DLNs and act as APCs presenting intravenously administered peptide to naive T cells, CD8 and
CD8
+ DCs were purified from the spleens of 107-1 Tg
donors by two-step positive magnetic bead selection, first
for expression of CD11c and then for CD8
. This yielded
two fractions, a CD8
fraction containing <1% CD8
+
contaminants, and a "CD8
+" fraction containing 50%
CD8
cells and 50% CD8
cells. An aliquot of each purified fraction was pulsed with peptide in vitro. The DCs
were then injected into hind footpads of unirradiated 36-2 Tg mice reconstituted with CFSE-labeled T cells 2 d before. 12 h after DC injection, MCC87-103 peptide was administered intravenously to the animals that had received
unpulsed DCs, and antigen-specific T cell responses were
determined 3 d later in the popliteal LNs. The marked increase in effectiveness of in vitro versus in vivo peptide
loading was apparent from comparison of responses in the
two groups (Fig. 1, A vs. B). We have previously noted a
difference of similar magnitude between in vitro and in vivo peptide loading of naive B cells (6). The size of the response to intravenous peptide correlated with the number
of CD8
DCs in the inoculum, being barely detectable in
the group that received the "CD8
+" DCs consisting of a
mixture of CD8
and CD8
+ DCs, and highest in the
animals receiving a high dose of pure CD8
DCs (Fig.
1 A). The response to both fractions of peptide-pulsed DCs
was high. Analysis of the CFSE division profiles indicated that the number of cells recruited into division was again
proportional to the number of CD8
cells in the inoculum (Fig. 1 B, bottom panels). As expected, all the T cell
responses were localized to the DLNs, as indicated by the
lack of response in the contralateral popliteal node.
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Because of difficulties in achieving sufficient DC purity
in the experiment described above, CD11c+ DCs obtained
by positive bead selection were sorted on the basis of CD8
expression, yielding a CD8
fraction with <0.01% contaminating CD8
+ cells, and a CD8
+ fraction contaminated with 1.0% CD8
cells. After peptide pulsing, the
cells were injected into the hind footpads of 36-2 mice.
Once again, the response to CD8
cells was marginally
higher, as indicated by stimulation of one more T cell division than was seen in the response to CD8
+ cells (Fig.
2 A). As expected, expression of the activation markers CD69 and CD44 was increased before the first cell division
(Fig. 2 B), and the pattern of CD69 downregulation was
consistent with previous data derived from direct peptide
administration to TCR Tg mice (Smith, A.L., and B. Fazekas de St. Groth, manuscript in preparation).
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Tracking CMFDA-labeled DCs purified using the two
methods described above indicated a very striking result,
namely that the donor DCs isolated from the DLNs 22 h
after inoculation were uniformly CD8 (Fig. 3). The absence of donor-derived CD8
+ DCs in the DLNs could
have resulted either from downregulation of CD8
by the
inoculated CD8
+ DCs, or from failure of the CD8
+
DCs to migrate to the DLNs. The correlation between the
number of CD8
DCs in the inoculum and in the DLNs
indicated that the latter was the more likely explanation.
However, the disparity between the substantial T cell response to peptide-pulsed, sorted CD8
+ DCs (Fig. 2) and
the tiny number of donor-derived DCs isolated from the
DLNs (Fig. 3 B) also suggested that antigen may have been
transferred from donor to host cells after injection. Since simple peptide transfer from donor to host cells was excluded by the experimental protocol (Fig. 1), any antigen
transfer from donor cells must have involved transfer of
both peptide and MHC, presumably as a complex formed
during in vitro pulsing. This type of peptide/MHC transfer
between DCs has recently been documented in vitro (21),
and may account for previous reports in which thymic DCs
presented endogenous self-antigen-MHC complexes synthesized by epithelial cells (22). It could also account for
the phenomenon of cross-presentation of MHC class I
molecules in the DLNs (23), a mechanism that is essential to ensure that viruses do not escape immune surveillance merely by failing to infect the APCs required for
priming cytotoxic T cells.
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These data suggest an alternative interpretation of recent experiments
characterizing the response of naive T cells to antigen-pulsed purified DC subpopulations (12, 13). These experiments have been interpreted to indicate that lymphoid
DCs, purified on the basis either of CD8 expression (12)
or of failure to express CD11b at high levels (13), migrate
to DLNs after subcutaneous administration and generate
preferential priming of a Th1 phenotype by means of their
high levels of IL-12 production. In contrast, MDCs (CD8
,
CD11bhi) were suggested to prime for a Th2 response.
However, in light of our finding that no CD8
+ DCs of
donor phenotype could be found in the DLNs after subcutaneous injection, we suggest that priming for both Th1
and Th2 memory results from direct stimulation of naive T
cells by MDCs that have migrated to the DLNs, with the
differential outcomes conditional upon the cytokine environment and/or the amount of available antigen, which
will differ depending on the constituents of the original inoculum. This interpretation is supported by the data of
Maldonado et al. (12), demonstrating that injection of
parenteral IL-12 stimulates T cell production of IFN-
rather than IL-4 in response to subcutaneous injection of
MDCs. Thus, when LDCs are injected subcutaneously, it is
possible that IL-12 production at the site of injection may
influence the ability of MDCs to induce Th1 development in the DLNs, without the LDCs playing any direct role in
antigen presentation to naive T cells in the node. Shift in
the Th1/Th2 balance of the response to soluble subcutaneous antigen after administration of Flt3 ligand (which preferentially expands the LDC population in vivo) versus
GM-CSF (which increases only MDC numbers) (13), is
also consistent with this interpretation. In addition, if peptide-MHC is transferred from LDCs to MDCs (either donor- or host-derived) before their migration to the DLNs,
the concentration of antigen to which T cells are exposed
could change dramatically, which has previously been
demonstrated to influence the Th1/Th2 balance after in
vitro priming (26).
These experiments do not directly address the physiological role of LDCs resident in the T cell zones of secondary
lymphoid tissue. However, they are consistent with a
model in which LDCs are generated in situ from precursors
within the thymus (1) and secondary lymphoid tissue (27)
and comprise a sedentary population which, in contrast to
MDCs, relies on mechanisms other than migration from
the periphery to capture antigen for presentation to T cells
within the T cell zone (7). It has been suggested that LDCs
induce deletional tolerance (3, 5, 6, 7). Our unpublished
data, derived from comparison of the deletional response of
naive peripheral CD4+ T cells to either intravenous peptide or a transgenic neoself-antigen, suggest that induction
of deletional tolerance is accompanied by very early production of Th1 (IL-2, IL-3, IFN-) but not Th2 cytokines
(IL-4 or IL-10), consistent with the high level of IL-12 production by stimulated LDCs (28). Whether Th1 immunogenic (memory) responses to foreign antigen are induced
by direct presentation to T cells by a combination of LDCs
and MDCs (29), or whether LDCs play only an indirect
role in induction of memory responses, namely as cytokine
producers but not APCs, remains to be elucidated.
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Footnotes |
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Address correspondence to Barbara Fazekas de St. Groth, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW, Australia, 2042. Phone: 61-2-9565-6137; Fax: 61-2-9565-6103; E-mail: b.fazekas{at}centenary.usyd.edu.au
Received for publication 23 October 1998 and in revised form 24 November 1998.
The authors wish to thank Karen Knight and her staff for providing expert animal husbandry, and Kate Scott for screening the transgenic mice. A.L. Smith was supported by an Australian Postgraduate Award. B. Fazekas de St. Groth is a Wellcome Trust Senior Research Fellow.
This work was supported by the National Health and Medical Research Council, the Wellcome Trust, and the Medical Foundation of the University of Sydney.
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