International Immunology, Vol. 13, No. 4, 465-473,
April 2001
© 2001 Japanese Society for Immunology
Differential effect of CD8+ and CD8– dendritic cells in the stimulation of secondary CD4+ T cells
Cooperative Research Center for Vaccine Technology, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia
1 Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria 3050, Australia
Correspondence to: L. Brown
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Abstract |
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Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4+ and primary CD8+ T cells; the myeloid-related CD8
– DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8 despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4+ T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8– DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8+ DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8+ DC, but was related to the levels of IL-2 induced. The deficiency in the CD8+ DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8+ splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4+ T cells to sustain their proliferation.
Keywords: CD4 T cells, dendritic cell, dendritic cell subpopulation, T cell activation
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Introduction |
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Dendritic cells (DC) (1) are present in many peripheral tissues where they sample antigens from the environment. In this way, any foreign antigen that is encountered can become rapidly internalized and processed. In response to certain stimuli, such as lipopolysaccharide, tumor necrosis factor-
and IL-1 (2,3), the DC undergo a switch in chemokine receptor expression (4) and migrate from the site of antigen encounter via the afferent lymph or blood to the T-dependent areas of lymph nodes and spleen respectively (1). Here they complete their maturation, a process that results in up-regulation of adhesion molecules, down-regulation of endocytic activity, and accumulation of MHC class II complexes (5,6). The DC take up residence in the path of recirculating T cells, facilitating their interaction with those bearing TCR capable of recognizing the processed foreign antigen complexed with MHC molecules on the DC surface. The interaction of CD4+ T cells with DC has been shown to be a crucial step in the initiation of both humoral and cellular responses, and can result in a change in the functional state of both the CD4+ T cell and the DC. A naive CD4+ T cell becomes primed to participate in cognate interaction with B cells for the initiation of T-dependent antibody responses (7) and the dendritic cell becomes more efficient at activating naive CD8+ T cells by cross-priming (8,9).
In recent years it has become apparent that there are different subclasses of DC whose interactions with T cells have different outcomes (reviewed in 10). Freshly isolated mature DC from mouse spleen exist as two distinct populations: CD8+DEC-205+CD11b– DC (also found in the thymus) and CD8–DEC-205–CD11b+ DC (11,12). In addition to differences in surface markers, the CD8+ and CD8– DC populate different regions of the spleen, and are thought to be derived from lymphoid and myeloid lineages respectively (10,11). The CD8– DC population are efficient antigen-presenting cells (APC) capable of activating naive T cells to proliferate and produce cytokines, while the CD8+ DC population are less potent despite their mature phenotype and equivalent expression of MHC and co-stimulator molecules (11,12). The CD8+ DC in fact induce the Fas-mediated death of many of the naive CD4+ T cells they activate (13). In addition, the CD8+ DC are much less potent inducers of the production of IL-2 and other cytokines by naive CD4+ and especially CD8+ T cells, and this limits subsequent T cell expansion (14–16).
In the present study, we further examine the interaction of these different populations of DC with CD4+ T cells, and ask whether the distinct functional outcomes observed when naive T cells interact with either CD8+ and CD8– DC are also evident with primed T cells which might be expected to exhibit less stringent requirements for activation. We show that the CD8– DC population is dramatically more efficient than the CD8+ population at stimulating both CD4+ T cell clones and populations of antigen-specific splenic T cells.
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Methods |
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Animals
Mice were obtained from the animal facilities of either the Department of Microbiology and Immunology at the University of Melbourne or the Walter and Eliza Hall Institute of Medical Research (Melbourne, Australia). The BALB/c mouse strain was used throughout except for measurement of primary allogeneic T cell responses, where the strain combination used was CBA T cells with BALB/c DC.
Synthetic peptides
The peptides used in this study were based on sequences representing T and B cell determinants from the influenza hemagglutinin (17–19). The B cell determinant TLKLATG (B) represents residues 313–319 of the heavy chain (HA1) of H3 subtype hemagglutinin. The T1 determinant PKYVKQNTLKLA overlaps the B determinant in the native sequence while the T2 determinant is located on the HA2 chain of the molecule and is quite distinct from B. Both T cell determinants are I-Ed restricted, and are processed and presented by splenic APC when intact virus is used as the antigen source. Branched synthetic constructs containing a single copy of either the T1 or T2 T cell determinant with two lysine residues at the C-terminus and two copies of the B cell determinant, synthesized from the 
amino groups of each lysine, were assembled as previously described (20) using KR100 resin to which two Fmoc lysine-(Dde) residues had been attached. These constructs are referred to as T1-KK-BB and T2-KK-BB respectively. Simple linear peptides with the sequences PKYVKQNTLKLATGMRNVPEKQT (parent peptide, residues 306–328) and PKYVKQNTLKLATG (T1-B), which contains B and T1, were assembled by conventional solid-phase methodology in a Milligen 9050 Plus automatic peptide synthesizer using Fmoc chemistry throughout (20).
Virus
The influenza virus used was the recombinant A/Memphis/1/71xA/Bellamy/42 (H3N1). Virus was grown for 2 days in the allantoic cavity of 10-day embryonated hen's eggs. Allantoic fluid containing virus was stored in aliquots at –70°C. A hemagglutination assay (21) was used to quantitate virus and titers are expressed in HAU/ml.
Culture medium
Cells were maintained and assays were performed in RPMI 1640 medium (CSL, Victoria, Australia) supplemented with 10% heat-inactivated (56°C, 30 min) FCS, 2 mM glutamine, 2 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 30 µg/ml gentamicin, 100 IU/ml penicillin and 100 µg/ml streptomycin.
Cell lines
The cell lines HT2 (22) and CT4S (23) are cytokine dependent, and were used to assess the production of IL-2 and IL-4 respectively in culture supernatants. HT2 cells were maintained in the presence of supernatant from concanavalin A-stimulated splenocytes as a source of IL-2 and CT4S cells were maintained with recombinant murine IL-4.
CD4+ T cell clones
T cell clone 4.51, which recognizes the T1 determinant, was derived by limit dilution of cultured lines generated from the spleens of peptide 305–328-immunized DBA/2 (H-2d) mice (17). T cell clone 12V1, which recognizes the T2 determinant (16), was isolated from the lymph nodes of BALB/c (H-2d) mice primed to the HA2 chain isolated from the influenza virus hemagglutinin. T cell clones were maintained as previously described (19) by alternate passage with IL-2 followed by influenza virus plus irradiated spleen cells as a source of APC. Clones were used in proliferation assays 6–7 days after passage in IL-2-containing medium and were depleted of dead cells by centrifugation over Isopaque-Ficoll (24).
Isolation of DC
The procedure for isolation of DC subpopulations has been described in detail previously (11,15). Briefly, spleens from BALB/c mice were digested with collagenase and DNase, the DC–T cell complexes dissociated with EDTA, then a density-cut procedure (25) using Nycodenz (Nyegaard Diagnostics, Oslo, Norway) was performed to separate a low-density population of cells. T cells, B cells, macrophages, granulocytes and erythrocytes were then removed from this population using immunomagnetic bead depletion. The remaining cells were sorted for CD11chigh cells, defining the DC population, and for CD8+ and CD8– subpopulations using a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA).
Isolation of primed T cells
Mice received four i.p. inoculations at 3-weekly intervals with 15 nmol of the parent peptide 306–328 conjugated to Pam3Cys (tripalmitoyl-S-glyceryl cysteine) (26,27) as a built-in adjuvant moiety. Four days after the last inoculation, spleens were removed, single-cell suspensions prepared by passage through a wire sieve and erythrocytes lysed by incubation in Tris–ammonium chloride buffer. T cells were enriched by passage over nylon wool columns (28) and used as a source of primed T cells in proliferation assays.
T cell proliferation assays
Assays using antigen-primed T cells were performed in duplicate or triplicate in flat-bottom 96-well microtiter trays (Nunc, Roskilde, Denmark) in a total volume of 250 µl. Cultures contained either nylon wool-enriched secondary T cell preparations (3x105 cells/well) or T cell clones (1x104 cells/well), purified DC (at the indicated number) and serial dilutions of peptide antigen. Cultures were incubated for 4 days, unless stated otherwise, at 37°C in an atmosphere of 5% CO2. Proliferation of T cells was determined by addition of 1 µCi/well [3H]thymidine 18 h prior to termination of culture. Cells were harvested onto glass fiber filters and incorporation of radioactive label was measured on a Packard Matrix 9600 direct ß-counter.
Studies with primary CD4 or CD8 T cells were performed in cultures established in V-bottom 96-well microtiter trays (Nunc, Denmark) as described previously (15). Cultures contained purified lymph node primary CD4 or CD8 T cells from CBA mice together with purified BALB/c CD8– or CD8+ DC. Proliferation of T cells was measured by the incorporation of [3H]thymidine after a 6-h pulse on day 4 of culture. Radioactivity was detected by liquid scintillation counting.
Cytokine assays
T cells and DC were established in culture as described above with a single dose of antigen. At different times, the contents of replicate cultures were pooled and the cells removed by centrifugation. The supernatant was diluted in fresh tissue culture plates in a volume of 50 µl to which either 4x103 HT2 cells or 5x103 CT4S cells, washed 4 times prior to seeding, were added in 50 µl. The cultures were incubated overnight at 37°C and then 1 µCi/well of [3H]thymidine was added for a further 4 h when using HT2 cells or a further 24 h when using CT4S cells. Cells were then harvested onto glass fiber filters and radioactivity determined as above.
Direct cell counts of DC-stimulated T cells
Cultures of T cells, DC and antigen were established under conditions described above. At different times, 25 µl of 0.1 M EDTA was added to replicate cultures, and the cells resuspended thoroughly to break up DC–T cell rosettes. After re-suspension, the contents of four wells were transferred to an Eppendorf tube, the cells pelleted and then resuspended in 100 µl of culture medium. After staining with an equal volume of 0.2% eosin to distinguish dead cells, viable cell counts were determined in a hemocytometer under phase contrast microscopy.
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Results |
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Ability of DC subpopulations to stimulate proliferation of primary T cells
As a prelude to the studies using secondary T cells, some of our previous findings with primary CD8+ and CD4+ T cells were repeated using BALB/c rather than C57BL/6 DC for purposes of comparison. This served to test if the BALB/c splenic DC used in this study gave results generally in line with our previous studies on C57Bl/6 mice and to test if both the CD8+ DC as well as the CD8– DC could show, under some circumstances, a strong T cell stimulatory capacity. As shown in Fig. 1
, the CD8– DC from BALB/c spleen, just like those from C57BL/6 spleen, showed a greater capacity to stimulate primary allogeneic CD8+ T cells than did the CD8+ DC. However this difference disappeared in the presence of exogenous IL-2, in agreement with previous studies (15). Thus the CD8+ DC were effective stimulators of primary CD8 T cells into cell cycle but were less effective at inducing the IL-2 production required to maintain their proliferation. When the BALB/c CD8– and CD8+ DC were used to stimulate allogeneic CD4 T cells the differences seen were much less, both being effective stimulators (Fig. 1). As demonstrated elsewhere (16), this was largely because primary CD4 T cells produce much higher levels of IL-2 than primary CD8 T cells and although CD8– DC are more potent stimulators of IL-2 production, the availability of IL-2 is much less limiting than with CD8+ T cells. More pronounced differences between the stimulatory capacity of CD8+ and of CD8– DC for primary CD4 T cells were observed in cultures in which T cells from BALB/c hemagglutinin peptide-specific TCR-transgenic mice were tested for proliferation in response to the peptide (13). CD8– DC were considerably more effective stimulators than CD8+ DC in this system and we assume that this is because IL-2 becomes more limiting than in the allogeneic response. Overall, we conclude that CD8– DC from BALB/c mice, like those from C57Bl/6 mice, are more effective at stimulating the proliferation of primary T cells than are the CD8+ DC, but the latter do become effective stimulators provided IL-2 is not limiting primary T cell proliferation.
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Ability of DC subpopulations to stimulate proliferation of antigen-primed T cells
Further to these findings with primary T cells, we wished to establish the capacity of DC subpopulations to provide the necessary signals to induce proliferation of secondary T cells. Primed T cells can interact with other APC types and may not require the same magnitude or spectrum of signals for activation and division as do primary T cells. For this reason it could not be assumed that secondary T cells would be limited in their proliferative response to CD8+ DC. To test this we measured the response of influenza virus-specific T cell clones to antigen-presenting, syngeneic CD8+ and CD8– DC from BALB/c spleen (Fig. 2
). CD8– DC were found to induce substantial proliferation of both T cell clones 12V1 and 4.51 to two different forms of synthetic antigen containing their respective determinant, while the proliferation induced when antigen was presented by CD8+ DC was markedly weaker and, in the case of 12V1, not significant. As shown in Fig. 3, the differential ability of the two subpopulations to stimulate T cell clones was apparent over a range of DC numbers. CD8– DC were found to be highly efficient with as few as 50 cells/well capable of stimulating clone 4.51.
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) and CD8– (•) DC to T cell clones 12V1 and 4.51. Proliferative response of T cell clone 12V1 to the synthetic peptide construct T2-KK-BB (A) or monomeric T2 (B) presented by 100 DC/well. Proliferative response of T cell clone 4.51 to the synthetic peptide construct T1-KK-BB (C) or monomeric T1 (D) presented by 50 DC/well. Radioactivity was measured in this and subsequent experiments by direct ß counting in gas phase. Counts are expressed in (A), (C) and (D) as the mean c.p.m. of triplicate samples ± SEM; in (B), cell yields were sufficient for testing only in duplicate. Peptide dose is given as nmol/250 µl culture.
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Failure to detect stimulation of secondary T cells by CD8+ DC is not due to altered response kinetics
The kinetics of the proliferative response of T cell clones to peptide presented by the two DC populations was investigated using clone 4.51 (Fig. 4
). While similar low levels of label were incorporated in cultures containing CD8+ and CD8– DC during the first time interval (day 1; 24–42 h), the response in cultures containing CD8– DC rapidly increased to reach a maximum at day 3 and persisted at this level through to day 6. In contrast, no increase in thymidine incorporation occurred during this time period with CD8+ DC.
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To ensure that the difference in proliferative capacity as measured by thymidine incorporation was a true representation of the expansion of the T cells, rather than a difference in, for example, the capacity of cells to take up thymidine, we directly measured the number of cells present in the cultures at various time points. Multiple replicates of cultures containing clone 4.51, a constant amount of peptide antigen and either CD8+ or CD8– DC were established, and viable cell counts performed by microscopy at 1 and 24 h intervals out to 7 days. The results (Fig. 5
) show that significant expansion of cells only occurred in cultures containing CD8– DC. The cell numbers initially appeared to decrease slightly but on day 3 showed a 2-fold increase, corresponding well with the thymidine incorporation data. Cells continued to expand until a peak was achieved on day 5.
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A faster death rate or lower viability of CD8+ DC is not responsible for their poor stimulatory capacity
It seemed possible that the CD8+ DC die faster than the CD8– DC when cultured with the T cell clones and that this difference was responsible for the differences in stimulatory capacity. To address this, an experiment was performed in which cultures of T cell clone 4.51 together with peptide antigen and either CD8+ or CD8– DC were supplemented 1 and 2 days after initiation with additional antigen plus the respective DC. In this way the T cells would have continued exposure to viable DC. The cultures were labeled on the third day and thymidine incorporation determined 18 h later. Figure 6
shows that addition of antigen alone to the cultures did not significantly alter the proliferative response of the T cells compared to cultures that received additional medium only. Cultures receiving both antigen and DC supplements had enhanced T cell proliferation but the differential capacity of the DC subpopulations to stimulate T cells was maintained. This result shows that even under conditions where the supply of viable DC to present antigen is not exhausted, T cells responding to CD8+ DC were not able to proliferate to the same extent as those responding to CD8– DC.
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T cells cultured with CD8+ DC do not produce IL-2
In cultures containing very high doses of antigen, T cell clones apparently received signals that resulted in inhibition of their proliferation, as seen in Fig. 2 (A–C)
. Such non-proliferative T cells have nevertheless been shown to be able to secrete cytokines (29,30). This phenomenon was further characterized by Suzuki et al. (31) and attributed to re-occupancy of the TCR with antigen–MHC complexes shortly after initial triggering. It was therefore possible that the signals T cells receive from CD8+ DC could have resulted in cytokine production in the absence of proliferation. To test this, supernatants from cultures of T cell clones, DC and antigen were sampled after 24, 48 and 72 h, and assayed for the presence of IL-2 using proliferation of the IL-2-dependent cell line HT-2 as an indicator. Both clones are known to produce predominantly IL-2 when stimulated with antigen and an unsegregated preparation of splenic APC (18,32). When tested with DC as APC, clone 4.51 (Fig. 7) and clone 12V1 (not shown) produced significant levels of IL-2 in the first 24 h in culture with CD8– DC. Somewhat increased amounts of IL-2 were present in parallel cultures sampled after 48 h and those sampled at 72 h showed an additional increase. The levels of IL-2 detected in cultures containing CD8+ DC were consistently low over all sampling intervals. Very small amounts of IL-4 were also detected in the supernatants of cultures containing CD8– but not CD8+ DC (data not shown). This indicates that deviation of the cytokine profile of the T cell clone from IL-2 to IL-4 production when recognizing antigen presented on CD8+ DC was not a likely explanation for its lack of observed IL-2 secretion and low proliferative capacity.
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Antigen-primed T cell populations also respond differently to antigen presented by CD8+ or CD8– DC
The difference in stimulatory capacity of the two DC subpopulations was observed, not only with long-term T cell clones, but also when the DC were used to present peptide antigen to cultures of T cells isolated directly from the spleens of peptide-primed animals (Fig. 8
). CD8– DC were found to induce strong proliferative responses in the primed T cell population which was, for certain antigen doses, as much as 10-fold higher than that achieved with CD8+ DC.
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Differential T cell stimulation by CD8+ or CD8– DC is also observed with live virus as antigen
The two T cell clones used in this study also recognize live influenza virus when presented by splenic APC (18,19). We could therefore use these clones to determine whether the lack of ability of CD8+ DC to stimulate proliferation of T cells when presenting peptide antigen could be overcome if the same determinants were presented in the form of virus, which can potentially utilize alternate processing pathways and also provide additional signals to the DC. In assays similar to those described above, using live virus instead of peptide antigen in the culture, relatively poor responses were again achieved with the CD8+ population of DC as APC and, although the CD8– DC induced higher levels of stimulation of clone 4.51, proliferation was only observed at DC numbers ~10-fold greater than required to induce a response to the peptide constructs (Fig. 9
).
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Discussion |
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In earlier studies (11,12,14) we have extensively characterized the CD8+ and CD8– DC from spleen, and shown they both can be classified as mature DC, expressing similar surface levels of MHC molecules and co-stimulator molecules such as B7-1, B7-2 and CD40. More recently, the two subpopulations were also shown to express equal levels of the new co-stimulator molecule ICOS-L or B7RPI (I. Caminsche, unpublished data). Although both CD8+ and CD8– DC can be further activated or matured by injection of factors such as bacterial lipopolysaccharide, they up-regulate co-stimulator molecules in parallel rather than one subpopulation maturing to the phenotype of the other (J. Pooley and K. Shortman, in preparation). Both CD8
+ and CD8– DC can induce proliferative responses in both CD8 and CD4 primary T cells but, as shown previously (13–16) and confirmed in Fig. 1, CD8– DC usually produce more extensive proliferation than CD8+ DC. With primary allogenic CD8 T cells the difference is only seen from 3 days of culture onwards and disappears if exogenous IL-2 is added to the cultures (14,15), as confirmed in Fig. 1. The difference in proliferative response has been shown to be due to an enhanced production of IL-2 by the CD8+ T cells when CD8– rather than CD8+ DC are used as stimulators (13,16); the signals from the DC governing IL-2 production are not yet understood but appear to be separate from those given by peptide–MHC or co-stimulator molecules (14). With primary CD4 T cells, which are more efficient IL-2 producers, IL-2 seldom becomes limiting under these culture conditions although the production of IL-2 is still less when CD8+ DC are the stimulators (16). However, when these CD4 T cells interact with CD8+ DC, additional factors, including a Fas-mediated T cell apoptosis, may act to limit their proliferation (13). Overall, both DC types behave as mature DC, and effectively initiate primary CD4 and CD8 T cell division, but then other factors such as a reduced production of IL-2 limit the extent of the subsequent proliferative response. Antigen-primed CD4+ T cells examined in this study also showed a differential ability to undergo sustained proliferation in response to antigen presented by the two different DC subpopulations and the differences observed appeared even more pronounced than for primary T cells. The CD8– DC population was extremely efficient in presentation of synthetic-peptide-based immunogens to two distinct T cell clones, with as few as 50 DC/culture yielding a significant proliferative response. In contrast, the CD8+ DC failed to induce proliferation of one of the clones and supported the expansion of the other to only a very limited extent. The ability of one clone but not the other to proliferate in response to CD8+ DC may reflect their relative sensitivity to IL-2 itself or perhaps to some signal from the DC that triggers IL-2 production.
The differential capacity of CD8+ and CD8– DC to support proliferation of T cells was also very marked when the T cells used were antigen-primed splenic T cells of mixed specificity, indicating that the data derived with clones was representative of the T cell population as a whole.
The low response to the CD8+ DC initially raised the possibility that these DC could be entirely non-functional. However, they were clearly not totally devoid of the capacity to induce proliferation of T cells as seen when greater numbers of DC were present in the cultures, either from initiation (Fig. 3) or when added each day over a 3-day period to negate any effects of differential longevity of the two subpopulations (Fig. 6). Although, in these experiments, the level of proliferation never reached that of equivalent cultures containing CD8– DC, in experiments measuring the primary allogeneic response of CD4 T cells or CD8 T cells in the presence of IL-2 (Fig. 1), BALB/c CD8+ DC stimulated levels of proliferation comparable to that with CD8– DC, indicating that the CD8+ DC population is capable of driving a T cell response under certain conditions.
In the present study the cultures containing secondary T cells were performed in flat-bottomed wells and so could be followed microscopically. At no stage could wells containing CD8+ DC be distinguished from those containing CD8– DC; both types of DC induced clustering of T cells and there were no overt signs of change in relative T cell numbers nor in accumulation of dead cells. When we performed viable counts on the cells from these cultures to confirm that thymidine incorporation by the T cells was indeed a true reflection of proliferation, it was clear that the level of cellular expansion even with CD8– DC was slight. The increase in T cell numbers at the peak of the response only amounted to a doubling of the starting number. Clearly the full proliferative potential of the T cells was not being realized under these culture conditions.
The restraining factor which appears to limit extensive expansion of T cells even in response to the more stimulatory CD8– DC is the relatively low levels of cytokines in the culture supernatants. Though significantly greater than the levels seen in cultures containing CD8+ DC, they are, from our experience with the same T cell clones stimulated with unseparated splenic APC, quite low. However, when the cultures were supplemented with 10 IU/ml recombinant human IL-2 in experiments designed to address this possibility, the T cells displayed high levels of proliferation both in the presence (121,000 ± 4000 c.p.m.) and absence (116,000 ± 33,000 c.p.m.) of antigen. The same effect was observed with lower doses of IL-2. The antigen independence of this proliferation makes it difficult to examine any enhancing effect that cytokines may have on the antigen-specific stimulation by CD8– DC. For the same reason we were also unable to determine directly whether the failure of the CD8+ DC subpopulation to stimulate the secondary T cells as well as did the CD8– DC was entirely a consequence of a reduced IL-2 induction. However, in view of the extreme dependence of the system on IL-2 availability, we consider this the most likely explanation.
The ability of the DC to present intact virus was also examined. Although the processing capacity of classical DC is thought to be greatest prior to their migration to lymphoid organs, DC isolated directly from spleen have been shown to process and present complex antigens within the first 24 h of culture. After this time, only peptide antigens that do not require processing can be presented by the cultured DC (1). We show that the CD8– DC can present whole virus when it is added at the initiation of the cultures but this was less efficient than the presentation of the peptide constructs, requiring ~10-fold more DC to achieve measurable T cell proliferation. Though some of the constructs used in this study are branched structures incorporating B cell determinants as well as T cell determinants and therefore more complex in nature than simple linear peptides, they nevertheless appear to be efficiently handled by the CD8– DC. In fact, in a separate study (33) we report that branched immunogens are more potent stimulators of T cell proliferation when presented by these DC than the equivalent determinants in the form of a linear peptide.
Overall, this study clearly shows that the myeloid-related CD8– DC can activate antigen-primed CD4+ T cells and stimulate proliferation. In contrast, interaction with lymphoid-related CD8+ DC, though able to induce clustering of the T cells, results in only poor and sometimes barely significant levels of both cytokine production and proliferation. Therefore, despite both the increased sensitivity to antigen stimulation and the greater effectiveness of the TCR complexes to transduce signals in secondary versus naive T cells (34), it appears that the CD8+ DC still fail to provide the appropriate stimulus to allow expansion of T cells that are already in the activated or memory state. Since the essential difference between the CD8+ and CD8– DC does not appear to be in obvious factors such as maturation state or expression of certain co-stimulator molecules, we are currently examining the differences in gene expression between the two DC types, in search of the relevant signaling molecules.
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Acknowledgments |
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This work was supported by grants from the Cooperative Research Centre for Vaccine Technology and the National Health and Medical Research Council of Australia (980664 and 960184).
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Abbreviations |
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| DC dendritic cell |
| APC antigen-presenting cell |
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Notes |
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Transmitting editor: J. Schrader
Received 25 February 2000, accepted 20 December 2000.
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