International Immunology Advance Access originally published online on October 11, 2006
International Immunology 2006 18(12):1647-1654; doi:10.1093/intimm/dxl098
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Efficiency of peptide presentation by dendritic cells compared with other cell types: implications for cross-priming
1 Department of Dermatology, Charité, University Medicine Berlin, Humboldt University, Schumannstrasse 20/21, D-10117 Berlin, Germany
2 Department of Biology, Technion, Israel Institute of Technology, Haifa, Israel
3 Present address: Department of Immunology, University of Washington, Seattle, USA
Correspondence to: P. Walden; E-mail: peter.walden{at}charite.de
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Abstract |
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Dendritic cells (DCs) play a key role in the induction of cellular immune responses by harvesting antigens from peripheral tissue for cross-priming CD8+ T cells. It has been demonstrated that apoptotic bodies, whole- or degraded-cell-associated or soluble antigens as well as heat shock protein-bound peptides can be taken up, processed and cross-presented by DCs. Since cells are continuously releasing peptides from their surface MHC molecules, DCs in the tissues are exposed to such peptides and might process and present them to T cells as an additional pathway for cross-priming. To investigate this possibility, we compared and characterized the presentation of exogenous peptides by DCs and other cell types employing novel recombinant antibodies with TCR-like specificities for specific peptide–MHC complexes (pMHCs). These analyses reveal that loading of immature and mature DCs with peptide is far less efficient than it is for monocytes, T and B lymphocytes, B-lymphoblastoid, melanoma and TAP-deficient T2 cells. This inefficiency of peptide transfer to the MHC molecules of DCs makes it unlikely that these cells recycle peptides released from the MHC molecules of other cells and may explain why cross-presentation of such peptides has not yet been observed.
Keywords: antigen presentation, cross-priming, dendritic cell, epitope, MHC–peptide complex
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Introduction |
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Cross-presentation of antigens by dendritic cells (DCs) is likely the most important mechanism for inducing CD8 T cells responses (1). A number of studies have been carried out to elucidate both the mechanisms involved in cross-priming and the sources of cross-presented epitopes. It was reported that, in principle, soluble as well as cell-associated antigens could be cross-presented (1). For cell-associated antigens, attempts to identify the precursor molecules that are taken up and processed by DCs have precipitated contradicting findings pointing at chaperone-bound peptides (2), whole proteins (3), proteasome substrates (4) or other intermediates of the antigen-processing pathway (5). In addition to these sources, MHC-bound peptides could be recycled for cross-representation by DCs. Several studies have demonstrated large amounts of degenerated MHC heavy chain-only molecules at cell surfaces, indicating frequent dissociation of the MHC–peptide complexes and, thereby, release of MHC-bound peptides (6–8). It is, thus, likely that DCs in the tissues are exposed to such peptides. However, published data suggest that functional representation of MHC-bound peptides does not occur in vivo (4). To study the possibilities for the transfer of MHC-bound peptides from tissue cells to DCs, we analyzed uptake and loading of free soluble peptides onto the MHC class I molecules of immature dendritic cells (iDCs) and mature dendritic cells (mDCs) in comparison to B lymphocytes, CD4 T lymphocytes, monocytes, SK-mel-24 (SK) melanoma cells, B-lymphoblastoid JY and TAP-deficient T2 cells. Using recently developed recombinant antibodies with TCR-like specificities for defined HLA-A*0201–peptide complexes (9–11), we established the dose–response relationships between the peptide concentration used for pulsing the different cell types and the resulting surface densities of pMHCs and the responses of cognate T cells. Thereby, we observed that the efficiency of loading peptides onto HLA-A*0201 of both iDCs and mDCs is much lower than it is for the other cell types which might explain why functional presentation by DCs of peptides released from other cells has not been observed.
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Methods |
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Cells
For DCs generation (12), PBMC of healthy donors were incubated for 1 h at 37°C in RPMI medium with 50 µM 2-mercaptoethanol (2-ME), 100 U ml–1 penicillin, 100 µg ml–1 streptomycin (all Gibco-Invitrogen, Karlsruhe, Germany) and 1% heat-inactivated human plasma in a tissue culture flask (Nalge Nunc International, Wiesbaden, Germany). Non-adherent cells were washed off with PBS. The adherent cells were cultured in the above medium plus 1000 U ml–1 granulocyte macrophage colony-stimulating factor (Leukomax®, Essex Pharma, München, Germany) and 2000 U ml–1 IL-4 (PromoCell, Heidelberg, Germany). The cytokines were replenished after 48 h. On day 5, non-adherent cells were harvested, washed with PBS and cultured in fresh medium and cytokines as before. After 24 h, tumor necrosis factor
(10 ng ml–1), IL-1ß (10 ng ml–1), IL-6 (25 ng ml–1; all Strathmann Biotech, Hamburg, Germany) and PGE-E2 (0.2 µg ml–1; Minprostin® E2, Pharmacia, Erlangen, Germany) were added to induce maturation. The mDCs were harvested for the experiments between 24 and 30 h later. The iDCs were collected from the day 6 DC cultures before maturation. The DCs were characterized by flow cytometry with biotin-labeled anti-CD80 with streptavidin–rPE as secondary reagent, anti-CD83–FITC, anti-CD86–allophycoerythrin (APC) and anti-HLA-DR–PerCP (all BD-Biosciences, Heidelberg, Germany). T cells, monocytes and B cells were isolated from PBMC by magnetoseperation with the CD4+ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), CD14 Beads (Miltenyi Biotec) or alternatively CD14–FITC mAb (BD-Biosciences) and CD19–FITC (BD-Biosciences) plus anti-FITC magnet beads (Miltenyi Biotec), respectively. T2, JY and SK were cultured in DMEM with 50 µM 2-ME, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin (all Gibco-Invitrogen) and 10% heat-inactivated FCS (Sigma, Taufkirchen, Germany). The medium for SK was supplemented with 2 mM of L-glutamine and 1 mM of sodium pyruvate (both Sigma).
Peptides and peptide pulsing
The peptides gp100154–162 KTWGQYWQV (KTW) and gp100280–288 YLEPGPVTV (YLEP) of the melanoma-associated differentiation antigen gp100, hTERT540–548 ILAKFLHWL (ILA) and hTERT1110–1118 TTLTALEAA (TTL) of the human telomerase reverse transcriptase (hTERT) and TAX11–19 LLFGYPVYV (LLF) of the human T cell leukemia virus type 1 protein TAX were custom-synthesized by EMC microcollection (Tübingen, Germany) and dissolved at 20 mg ml–1 in DMSO (Pierce, Weiterstadt, Germany). For peptide loading, the cells were used directly from the above preparations. Adherent cells were detached with 3 mM EDTA in PBS. All cells were washed once with DMEM/0.5% BSA and, unless stated otherwise, incubated in DMEM/0.5% BSA with the peptides at the indicated concentrations for 90 min at 26°C followed by 90 min at 37°C. After pulsing, the cells were washed to remove free peptide and kept at 4°C until analysis.
Detection of pMHCs
The recombinant Fab 4A9 with specificity for complexes of HLA-A*0201 with ILA (A2/ILA) was used for the analysis of pMHC with biotinylated polyclonal rabbit anti-human Fab IgG antibody (Acris/DPC Biermann, Hiddenhasen, Germany) as secondary and streptavidin–rPE (BD-Biosciences) as tertiary reagents. The biotinylated Fabs G2D12 and T3F2 with specificities for HLA-A*0201 complexes with KTW (A2/KTW) and LLF (A2/LLF), respectively, were tetramerized with streptavidin–rPE or streptavidin–APC (Molecular Probes, Leiden, Netherlands) and then used for cell staining. Where indicated, rPE staining was quantified with QuantiBRITETM PE (BD-Biosciences). Total HLA-A*0201 expression was analyzed with FITC-labeled BB7.2 (BD-Biosciences) and quantified using Quantum Fluorescence Kit for the determination of ‘molecules of equivalent soluble fluorochrome’ units (Sigma).
Generation and analysis of peptide-specific T cells
T cells in freshly isolated PBMC were primed with peptide-loaded DCs from healthy donors. On day 2, 50 U ml–1 IL-2 (Proleukin, Chiron, München, Germany) was added and refreshed every 2–3 days. After 14 days, CD4+ cells were removed by magnetic cell sorting and the CD8+ T cells re-stimulated with peptide-pulsed irradiated PBMC in the presence of IL-2. For the measurement of proliferation, T cells were labeled with 1 µM CSFE (Sigma) for 5 min at room temperature, washed and cultured together with peptide-loaded APC and 50 U ml–1 IL-2. After 2 days, fresh IL-2 was added. After 4 days, the cells were harvested and counterstained with anti-CD8 APC (BD-Biosciences). The CSFE fluorescence signals of CD8+ T cells were analyzed by flow cytometry. FlowJo® analysis software was used to calculate the proliferation index which gives the average number of cell divisions in the proliferating population.
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Results |
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Specific detection of MHC–peptide complexes with recombinant Fabs
A major problem in studying antigen presentation is that pMHCs can be detected only by T cells so that it is neither possible to quantify the amount of peptide presented by an antigen-presenting cell nor possible to separate the antigen presentation from the co-stimulatory aspects of T cell activation. To overcome these limitations, we made use of recently developed recombinant antibodies with TCR-like specificities. The recombinant Fabs employed in this study had been isolated from a large naive human Fab phage display library by repeated panning for clones that specifically bind complexes of soluble recombinant HLA-A*0201 and defined T cell epitopes (9–11). To confirm the specificities of the Fabs, we analyzed their reactivity to the cell types used in this study after pulsing with the cognate epitope for the respective Fabs or control peptides. Staining of T2 cells that, because of their TAP-deficiency, largely lack endogenously processed peptides served as additional background control. With T2, the A2/KTW-specific Fab G2D12 only stains the KTW- but not ILA-pulsed cells (Fig. 1A) and inversely, the A2/ILA-specific Fab 4A9 only ILA-pulsed cells (Fig. 1H). In both cases, the staining for the negative control peptides was the same as for the unpulsed T2 cells proving that the increased cell surface expression of HLA-A*0201 by stabilization of the HLA molecules with the synthetic peptides does not increase nonspecific staining of the cells. Also, the staining of cells with normal TAP and, thereby, HLA expression was dependent on the cognate peptide for the respective Fab. G2D12 (Fig. 1B and G), 4A9 (Fig. 1I and J) and T3F2 (Fig. 1K and L) stained HLA-A*0201-expressing cells only after pulsing with their cognates epitope KTW, ILA and LLF, respectively, but not cells pulsed with other peptides. The cells tested were lymphoblastoid JY cells (Fig. 1B, I and K), SK melanoma cells (Fig. 1C), mDCs (Fig 1D, J and L), monocytes (Fig. 1E), B lymphocytes (Fig. 1F) and T lymphocytes (Fig. 1G). A representative example of the phenotype of the mDCs used in this study is shown in the plots M and N.
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Kinetics of peptide uptake and loading onto the MHC molecules of different cell types
The histograms in Fig. 1 already suggest that different cell types take up and present T cell epitopes with different efficiency. For a more accurate quantification of the efficiency of peptide handling, we pulsed the cells with serial dilutions of the KTW and determined the amount of specific A2/KTW complexes at the surfaces of the cells with the Fab G2D12. For an accurate comparison of the geometric mean fluorescence intensity (gMFI) values measured for the different cells at different peptide concentration used for pulsing, we calibrated the gMFI values with standard PE latex beads included in the same measurements. The results of these analyses are shown in Fig. 2(A and B) as numbers of PE molecules per cell. Since the valency of the binding of the tetramerized G2D12 to A2/KTW at the different cell surface densities of the complexes is not known, we could not exactly calculate the numbers of pMHCs per cell. Nonetheless, the comparison of the loading rates of the different cell types shows strong differences. DCs require about 100-fold higher peptide concentrations than JY cells for a comparable number of A2/KTW complexes at their surfaces. B and T cells as well as monocytes, on the other hand, take up and present less of KTW than DCs. The high numbers of A2/KTW complexes detected at the surfaces of T2 cells even at relatively low peptide concentrations are due to the largely reduced numbers of endogenous epitopes available for loading into the HLA molecules. In fact, the initial phase of the titration curve is paralleled by the increase of total surface HLA-A*0201 expression levels (Fig. 2A). The increase of pMHCs at high peptide concentrations probably reflects replacement of TAP-independent endogenous peptides by KTW.
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The different cell types differed greatly in the levels of HLA expression. To assess the fraction of the HLA-A*0201 molecules occupied by KTW, we quantified the amount of the HLA molecules by flow cytometry, calibrated the gMFI for HLA-A*0201 expression with calibration beads and calculated the ratio of calibrated gMFI values for the specific A2/KTW complexes and total HLA-A*0201 (Fig. 2C). This results in a measure for the fractional occupancy of the HLA molecules with the specific peptide. In the cases of the tumor cell lines JY and SK as well as the primary monocytes and CD4 T cells, the pulsing concentrations of KTW translate in similar degree of occupancy of their surface HLA molecules with the specific peptide. DCs, however, require far higher peptide concentration than the other cells for a comparable occupancy of their HLA molecules. B cells are less efficient in incorporating KTW into their HLA molecules than the other cell types but still far more efficient than DCs. A2/KTW complexes become detectable at the surface of DCs only at an ~100 times higher peptide pulsing concentration than in the cases of the other cell types. These observations are supported by time-course experiments (Fig. 2D), which show that the numbers of specific A2/KTW complexes on DCs saturate rapidly whereas they steadily increase on JY cells.
The results obtained with the KTW are reproduced with the ILA and LLF as shown for a comparison of the loading efficiencies for JY cells and DCs in Fig. 3(A and C). Of particular interest is a comparison of peptide handling by mDCs and iDCs. The iDCs appear to be even less efficient in peptide uptake and presentation than mDCs (Fig. 3D). The large difference in peptide loading, however, does not linearly translate into a corresponding difference in the fractional occupancy of the HLA molecules with KTW. Still mDCs are more efficient than iDCs, but only by 2-fold with respect to the peptide concentration needed to get a similar fractional occupancy (Fig. 3E). Among the cell types tested for the efficiency of peptide uptake and presentation, DCs are the least efficient.
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Possible causes of the low efficiency of peptide presentation by DCs
In search of causes for the low efficiency of the processing of external peptide by DCs, we tested four possibilities. First, DCs might, more than other cells, degrade peptides. To test whether such effect could be responsible for the low rate of peptide binding by DCs, we incubated DCs and JY cells with KTW at concentrations of 160 or 5 µg ml–1, then collected the supernatants, incubated them with T2 cells and quantified the resulting A2/KTW complexes with the Fab G2D12 as a measure for the peptide concentration in the supernatants (Fig. 4A). The T2 cells had been cultured overnight at 26°C to allow peptide-free HLA molecules to accumulate on these cells and, thus, increase sensitivity of detecting the specific peptide. As control, KTW was incubated in culture medium without cells. At the lower peptide concentration, significant reduction of KTW in the supernatant of JY cells and complete elimination of the peptide in the supernatant of DCs were found. At high peptide concentration, however, there was no significant difference between these cell types. The degradation of peptides by DCs reported by Amoscato et al. (13) is, thus, only detectable at low peptide concentration and cannot explain the large shift of the titration curves for peptide loading when DCs and other cell types are compared. Under the limiting peptide concentrations expected in vivo, however, different degrees of peptide degradation in the vicinity of different cell types may be important.
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Second, we considered the possibility that DCs differ from the other cells in the synthesis and turnover rates of the MHC molecules or, third, that loading of exogenous peptides onto MHC molecules in different cell types occurs differently in different subcellular compartments. These two possibilities would depend on active transport of the MHC molecules to the cell surface or active recycling of these molecules. Therefore, we incubated JY cells and mDCs with peptide in the presence of either sodium azide to inhibit all energy-dependent processes including energy-dependent membrane and, with that, MHC turnover or brefeldin A to block export of newly synthesized HLA molecules and thereby decouple peptide loading from conventional MHC class I-specific antigen processing. With both reagents and both cell types, the efficiency of peptide loading is somewhat reduced compared with the controls without inhibitors (Fig. 4B). However, DCs are still less efficient than JY cells in peptide uptake and presentation. The relative efficient loading of the HLA with exogenous peptide in the presence of sodium azide suggests that a large fraction of the complexes are formed at the cell surface by exchange against peptides bound to the HLA molecules. The reduction of specific complexes in the presence of azide is more pronounced for the DCs than for JY cells, suggesting that a larger fraction of the complexes on DCs are formed intracellularly.
The fourth possibility we tested is that HLA molecules of DCs compared with other cells might be occupied by better-fitting peptides, forming more stable HLA–peptide complexes that are less prone to release their peptides. To test whether the stability of different MHC–peptide complexes can impact the efficiency of peptide loading onto the HLA molecules of different cells, T2 cells were pre-incubated with either YLEP or TTL of which the former is a strong and the latter a weak binder for HLA-A*0201. The different binding properties of the peptides were confirmed by the differential increase of total cell surface HLA molecules upon incubation with the peptides which was lower in the case of TTL than in the case of YLEP. After the removal of unbound peptides, the T2 cells were then incubated with serial dilutions of KTW and analyzed with the G2D12 for the resulting A2/KTW complexes. T2 cells either not pre-incubated with peptide or pre-incubated with TTL displayed similar amounts of A2/KTW. In contrast, after pre-incubation with YLEP, a pronounced reduction of specific A2/KTW complexes was found (Fig. 4C). These results suggest that a large fraction of the HLA complexes with exogenous peptides may be formed by replacement of loosely bound endogenous peptides, which implies that the efficiency of external peptide loading is dependent on the stability of the pre-existing HLA–peptide complexes at the surfaces of the cells. We have recently reported that complexes of the same MHC molecules with the same peptide on DCs are more stable than those on other cell types. Thus, in addition to different peptide compositions of the HLA peptidomes of different cells, DCs seem to have mechanisms to produce more stable complexes that other cells lack (11). In summary, while indeed more exogenous peptides are degraded in the presence of DCs than JY cells, neither this difference nor different rates of MHC synthesis and turnover can explain the different efficiencies of external peptide presentation. Rather, a higher stability of the pre-existing HLA–peptide complexes on DCs could be the cause.
pMHC density and T cell stimulation
So far, the capacity of different cell types to stimulate T cells was assessed by correlating the magnitude of the T cell responses with the concentration of the cognate peptide in the assay cultures. Having antibodies with TCR-like specificity makes it possible to determine the surface densities of the cognate HLA–peptide complexes and to base the analysis of the efficiency of T cell stimulation by different cell types on the actual density of the TCR ligands. We compared JY cells and mDCs pulsed with serial dilutions of KTW for their efficiency in stimulation proliferation of HLA-A*0201-restricted, KTW-specific T cells. The T cells were labeled with CSFE and the proliferation determined by flow cytometry as a function of the reduction of fluorescence signal on the cells. Figure 5(A) shows the correlation of the proliferation of the T cells with the peptide concentration in the culture, Fig. 5(B) with the densities of the specific A2/KTW complexes. At low peptide concentration in the cultures, JY seems to be superior. However, looking at the surface densities of the cognate MHC–peptide complexes, DCs are clearly more efficient (~20-fold) in inducing T cell proliferation. This holds true for the dose–response relationship and the magnitude of maximal proliferation.
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Discussion |
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DCs, compared with other antigen-presenting cells, are described as superior in processing and cross-presenting antigens from other cells for cross-priming of CD8 T cells (1,14–17). In addition to whole or partially degraded antigens, peptides released from MHC molecules are potential antigen sources for cross-priming. To test this possibility, we investigated the extent of peptide uptake and presentation by DCs in comparison to various other cell types. These analyses were done with recently developed recombinant antibodies with TCR-like specificities for defined MHC–peptide complexes that enabled us to detect and quantify the complexes directly and independent of T cell responses that, usually used for such analyses, are problematic as they reflect inseparably the effects of both antigen presentation and co-stimulation. Surprisingly, the studies revealed that for mDCs, far higher peptide concentrations are required for detectable pMHCs at the cell surfaces than for other cell types such as primary monocytes, B and T cells or tumor cells. Normalization of the densities of pMHCs with respect to the MHC expression levels of these cells further emphasizes the differences: except for DCs and TAP-deficient T2 cells, a given peptide concentration results in similar specific occupancies of the MHC molecules of all tumor and primary cell types analyzed. For DCs, 10 to 100 times higher peptide concentrations are required for the same cell surface densities of specific complexes.
The inefficiency in peptide handling by DCs could explain published observations that peptides released from MHC molecules do not cross-prime T cells (4). The differences in peptide handling by different cell types may be explained by differences in the availability of the peptides in their vicinity. We had found that supernatants from DCs incubated with peptide contain less peptide than supernatants from B-lymphoblastoid cells incubated with the same amount of the peptide. This reduction of the peptide concentration might be explained with a higher endocytotic activity of DCs with intracellular retention or intracellular degradation of the peptide. However, mDCs have a slower membrane turnover than other cell types, which exclude a major contribution of endocytosis to the removal of peptide from the cultures (11). Moreover, the peptide pulsing was done in a large volume of 1 ml that by far exceeds the volume of the cells and the volume that possibly can be processed through the cells in the time given. A more likely explanation would be that DCs, more than other cells, produce proteases that degrade peptide extracellularly. Such DCs-associated proteases have been described by Amoscato et al. (13, 18). However, we found detectable elimination of peptide from the supernatants of the cells only at low peptide concentrations. At higher concentrations, no difference in the peptide concentrations was seen; yet, the differences in peptide loading remained. While neither of the above two hypotheses can explain the differences in peptide loading in our experiments, at the likely low concentrations of MHC-released peptide in the local environments of tissues, the observed reduction of available peptide for reloading MHC molecules might be significant and could indeed account, at least in parts, for the failures to demonstrate cross-priming by MHC-released peptides.
Because of the above considerations, we believe that the different efficiencies of peptide presentation are due to differences in the generation and turnover of pMHC by the different cells. The total amount of pMHC at the surfaces of cells is, in general, determined by the rate of generation of these complexes and the rate of their elimination from the cell surface by decay or internalization of membrane. We had shown before that pMHC of mDCs are longer-lived than the same complexes on other cell types including iDCs (11). In fact, besides a fast initial decay aspect of specific complexes on macrophages, which is followed by a second phase with a very slow kinetic, pMHC on iDCs are the most short-lived of the cells analyzed. The different apparent stabilities of the pMHC on the different cells are in part explained by the different rates of membrane turnover and in part with a high intrinsic stability of the pMHC on mDCs. The half-life times of the cell surface pMHC of the other tested cell types are significantly lower than the half-life of the pMHC of mDCs but still higher than those of iDCs. With this background from our earlier studies (11), the apparent low efficiency of peptide loading onto the MHC molecules of iDCs seems to be due to a combination of both the instability of their pMHC and the high membrane turnover which prevents any accumulation of pMHC at their cell surfaces. As a consequence, iDCs appear to be inefficient in peptide–MHC loading although the actual initial loading rate might not be low. For mDCs, on the other hand, the high intrinsic stabilities of the pMHC make peptide exchange and, thereby, loading of external peptide onto their MHC molecules less likely. The low membrane turnover, however, allows the complexes that are formed to remain and accumulate at the cell surfaces. The half-life times of the pMHC and the membrane turnover rates of other cell types are somewhere between those of iDCs and mDCs. The biological consequences of these differences in peptide handling would be that iDCs would constantly exchange the peptides presented by their MHC molecules. Once induced to mature, for example, by inflammatory cytokines in their environment or by TLR agonists, the cells would preserve the antigenicity of this environment. The resulting mDCs would retain and carry this information to the draining lymph nodes to induce T cells with specificity for the antigens in the inflamed tissue.
The importance of discriminating the antigen presentation and the co-stimulatory aspects of T cell induction is underlined by the comparison of the antigen dose–T cell response relationships for mDCs and JY cells as antigen-presenting cells. At low peptide concentrations, JY cells appear to be more efficiently stimulating T cells. At higher peptide concentrations this relationship is inverse. Only when the pMHCs are quantified at the surfaces of the cells, and related to the T cell response, is the superior stimulatory capacity of the DCs obvious in the shift of the dose–response curve to lower pMHC cell surface densities by about one order of magnitude.
The findings reported herein have important implications for the development of DC-based vaccines. A number of protocols use DCs specifically loaded with tumor-associated T cell epitopes as therapeutic vaccines for the treatment of cancer. Given the relatively low efficiency of peptide loading onto mDCs, some of these protocols may need revision. As it is done for tumor lysates containing unknown peptides at low concentrations, it should be considered to pulse monocytes or iDCs instead of mDCs with the vaccine peptides and only then induce maturation of the DCs.
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Acknowledgements |
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This work was supported by the Deutsche Forschungsgemeinschaft grants WA 1139/8-1 and FOR 299/2-1,2. We wish to thank Arthur O'Connor for his excellent technical support and Patricia Zambon for her assistance in preparing this manuscript.
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Abbreviations |
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| APC, allophycoerythrin |
| DC, dendritic cell |
| gMFI, geometric mean fluorescence intensity |
| hTERT, human telomerase reverse transcriptase |
| iDC, immature dendritic cell |
| ILA, peptide ILAKFLHWL |
| KTW, peptide KTWGQYWQV |
| LLF, peptide LLFGYPVYV |
| mDC, mature dendritic cell |
| 2-ME, 2-mercaptoethanol |
| pMHC, specific peptide–MHC complex |
| SK, SK-mel-24 |
| TTL, peptide TTLTALEAA |
| YLEP, peptide YLEPGPVTV |
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Notes |
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Transmitting editor: K. Inaba
Received 14 February 2006, accepted 7 September 2006.
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