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Endogenous Dendritic Cells Are Required for Amplification of T Cell Responses Induced by Dendritic Cell Vaccines In Vivo ¹

Institute for Immunology, Ludwig-Maximilians-Universität München, Munich, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs) loaded in vitro with Ag are used as cellular vaccines to induce Ag-specific immunity. These cells are thought to be responsible for direct stimulation of Ag-specific T cells, which may subsequently mediate immunity. In this study, in transgenic mouse models with targeted MHC class II expression specifically on DCs, we show that the DC vaccine is responsible only for partial CD4⁺ T cell activation, but to obtain optimal expansion of T cells in vivo, participation of endogenous (resident) DCs, but not endogenous B cells, is crucial. Transfer of Ag to endogenous DCs seems not to be mediated by simple peptide diffusion, but rather by DC-DC interaction in lymph nodes as demonstrated by histological analysis. In contrast, injection of apoptotic or necrotic DC vaccines does not induce T cell responses, but rather represents an immunological null event, which argues that viability of DC vaccines can be crucial for initial triggering of T cells. We propose that viable DCs from the DC vaccine must migrate to the draining lymph nodes and initiate a T cell response, which thereafter requires endogenous DCs that present transferred Ag in order induce optimal T cell expansion. These results are of specific importance with regard to the applicability of DC vaccinations in tumor patients, where the function of endogenous DCs is suppressed by either tumors or chemotherapy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)³ are specialized APCs of the immune system that, as immature cells, reside within nonlymphoid tissues and continuously collect foreign Ag. After receiving a maturation signal, DCs migrate to lymphoid tissues and are highly effective at presenting foreign Ag to naive T cells, thereby inducing Ag-specific immune responses (1).

Because they are such potent mediators of immunity, DCs are considered prime candidates for an active cancer immunotherapy (2). In experimental animal models as well as in clinical trials, DCs have been cultured in vitro, loaded with tumor-derived Ag, and injected as vaccines. Despite the fact that DC vaccination has been shown to be a very promising strategy for antitumor vaccination, mixed success has been reported from clinical trials so far (2). Whereas DC vaccines showed high efficacy of eradication of animal tumors early after transplantation, several possible mechanisms for the partial failure of DC vaccines in the setting of well-established tumors have been discussed. It has been shown that, during continued growth, tumors might exert several direct immunosuppressive mechanisms that negatively influence the immunostimulatory capacities of endogenous DCs. For example, DCs isolated from metastases induced a state of Ag-specific anergy in T cells. This effect was caused by IL-10 production within the tumors (3). Also vascular endothelial growth factor-producing tumors increase the percentage of endogenous immature DCs and directly suppress immunostimulation by mature DCs (4, 5, 6). Furthermore, immature DCs have been found to reside preferentially within the tumor tissue, where they might suppress tumor-specific T cells (7). Recently, DCs from patients with myeloid leukemia were shown to display functional impairment (8).

These reports suggest that endogenously growing tumors in a clinical setting might negatively influence APC of the tumor patient. Previous reports described that Ag can be transferred between live DCs in vitro (9) and that DCs can take up Ag from apoptotic or necrotic material (10, 11, 12). We explored whether such a mechanism, in which Ag is transferred from DC vaccines to endogenous DCs, appears probable in vivo, and investigated its role for T cell priming.

Previous studies have shown that DCs injected as DC vaccines leave the site of injection and migrate to draining lymph nodes (13), where they can be found in very small numbers (<1%) (14). There, short-lived migratory DCs can act as a source of Ag (e.g., the MHC II IE polypeptide) to be processed by resident lymph node DCs (15). Recently, it has been reported that specifically CD8⁺ DCs are able to process and re-present material from dying tumor, infected, or allogenic cells in vitro and in vivo (16). However, it remains unclear whether such an Ag-recycling mechanism, which has so far been described for proteins expressed in DCs (15, 16), is also relevant for preprocessed peptides such as on peptide-pulsed DCs. It has further not been shown whether live functional peptide-pulsed DCs are needed for such a cross-presentation, or whether apoptotic/necrotic DCs are sufficient. Whether such a process could play an amplifying role during DC vaccination in vivo has not yet been investigated.

To explore whether and how endogenous DCs take part in induction or amplification of T cell immunity during DC vaccination, we developed a model system that allows us to study this question in vivo. Using mice with targeted expression of transgenic MHC molecules on DCs or B cells, we demonstrate that the participation of endogenous DCs, but not B cells, enhances Ag-specific T cell proliferation and effector function induced by DC vaccination in vivo severalfold. This observation argues that functional endogenous DCs are important for the efficacy of DC vaccines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

T cells from AD-10 mice are transgenic for the TCR specific for moth cytochrome c (MCC) positions 88–103 (MCC_88–103) in the context of MHC class II IE and are positively selected by MHC class II IA^b (17). AD-10 transgenic mice were backcrossed from B10BR for >10 generations to the C57BL/6 background. DC-IE⁺ (18, 19) and B-IE⁺ (20) mice have been described previously. They express transgenic MHC class II IE^b driven by either the CD11c promoter specifically in DC (DC-IE⁺) or the human CD19 promoter specifically in B cells (B-IE⁺). All mice were bred and maintained at Institute for Immunology, Ludwig-Maximilians-Universität, and were used between 6 and 10 wk of age.

Adoptive transfer

CD4⁺ cells from lymph nodes and spleen were prepared as single-cell suspensions. Splenic RBC were removed using ACK buffer (0.15 M NH₄Cl, 1 mM KHCO₃, and 0.1 mM Na₂EDTA (pH 7.4)) for 4 min at room temperature. The percentage of AD-10 TCR-transgenic T cells among CD4⁺ T cells was controlled via FACS analysis with anti-TCR V3- and V11-specific mAbs. Before injection, cells were washed in PBS and filtered through nylon mesh (51-µm pore size; Reichelt Chemie Technick, Heidelberg, Germany). For adoptive T cell transfer, 5 x 10⁶ AD-10 T cells were injected i.v. into the lateral tail vein of recipient mice. In all transfer experiments, donor and recipient mice were sex-matched.

Dendritic cells

DCs were generated from bone marrow progenitors using GM-CSF as described previously (21). Briefly, 5 x 10⁶ total bone marrow cells were resuspended in complete medium (IMDM supplemented with 5% FCS, 50 mM 2-ME, and 1% penicillin/streptomycin) and incubated at 37°C and 5% CO₂. Fresh medium was added on day 3 of culture and additionally later if necessary. On day 8 of culture, DCs were tested for their purity by staining with anti-CD11c and anti-MHC II. Purity of DCs was usually >80%. DCs were pulsed for 3–6 h with 100 µg/ml MCC_88–103 peptide (Neosystem, Strasbourg, France) in complete medium. The cells were then washed extensively in PBS, and 1 x 10⁷ DCs were injected s.c. into the lower hind leg of each mouse.

5-Bromo-2'-deoxyuridine (BrdU) labeling studies

Mice received an injection of 100 mg of BrdU (Sigma-Aldrich, St. Louis, MO) 1 day before T cell transfer and were subsequently given 1 mg/ml BrdU in their drinking water until the day of sacrifice. BrdU incorporation was detected by intranuclear staining using the BrdU flow kit (BD PharMingen, San Diego, CA). Briefly, cells were surface stained with anti-V3 and anti-V11, and fixed for 20 min in Cytofix/Cytoperm (BD PharMingen); then nuclei were opened by incubation with Cytoperm Plus (BD PharMingen) for 30 min, and cells were refixed with Cytofix/Cytoperm solution. Fixed cells were incubated with DNase I solution (300 µg/ml) for 1 h at 37°C. Cells were then stained with FITC anti-BrdU (BD PharMingen) in perm/wash buffer (BD PharMingen) for 20 min at room temperature.

Immunostaining and flow cytometry

Draining popliteal lymph nodes were removed, and cell suspensions were prepared. The mAbs used were anti-V3-PE (KJ25), anti-V11-FITC (RR8-1), anti-CD4-biotin (RPA-T4), PE-conjugated anti-IL-2 (JES6-5H4), anti BrdU-FITC, and streptavidin-APC. To stimulate IL-2 production, cells were incubated for 6 h in the presence of 20 ng/ml PMA, 1 mM ionomycin, and 10 mg/ml brefeldin A (all from Sigma-Aldrich). Intracellular staining for IL-2 was performed using an intracellular staining kit according to the manufacturer’s instructions. All Abs were purchased from BD PharMingen.

A FACSCalibur flow cytometer and CellQuest software (BD Biosciences, Mountain View, CA) were used to collect and analyze the data. Nonviable cells were excluded using forward- and side-scatter electronic gating and by staining with propidium iodide (Sigma-Aldrich).

Proliferation assay

DCs (day 8 of bone marrow culture) were loaded with MCC_88–103 peptide or left unpulsed. T cells from AD-10 TCR-transgenic mice were isolated by negative selection using T cell enrichment columns (R&D Systems, Minneapolis, MN). Varying numbers of DCs were cocultured for 48 h with 1 x 10⁵ T cells in 96-well U-bottom plates (Nunc, Wiesbaden, Germany). [³H]Thymidine (1 µCi; Amersham Pharmacia, Buckinghamshire, U.K.) was added for the last 8 h of incubation. To induce apoptosis, DCs were subjected to UV irradiation (2 mJ/cm²/s). Necrosis was induced by incubating the cell suspension at 56°C for 10 min. Effective induction of apoptosis/necrosis was controlled by flow cytometry using annexin V (BD PharMingen) and propidium iodide (Sigma-Aldrich). In some experiments, Transwell inserts were used to separate different cell populations (Nunc).

Histology

Fresh lymph nodes were frozen in Tissue Tek (Sakura, Zoeterwoude, The Netherlands), and 5-mm sections were cut with a cryostat (Leica, Deerfield, IL). Sections were fixed for 20 min at -20°C in acetone, dried, and frozen at -20°C until use. Before staining, slides were equilibrated to room temperature and rehydrated for 15 min in PBS/0.25% BSA/0.01% NaN₃. Unspecific binding was decreased by incubating sections for 15 min with PBS/0.25% BSA/0.01% NaN₃ and 10% normal mouse serum before staining for 30 min at room temperature in the dark. mAbs used are anti CD11c-biotin (BD PharMingen) and streptavidin Cy-5 (Caltag, Burlingame, CA). Sections were washed with PBS/0.25% BSA/0.01% NaN₃ between incubations, mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL), and visualized with a fluorescence microscope (Leica).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Resident host DCs enhance Ag-specific CD4 T cell expansion induced by a DC vaccine

To investigate Ag transfer from DC vaccines to endogenous DCs, we used a T cell transfer system, where CD4⁺ MCC_88–103-specific transgenic AD-10 T cells were transferred into recipient mice expressing the restriction element MHC class II IE transgenically under the control of the mouse CD11c promoter selectively on DCs (DC-IE⁺). DC-IE⁺ mice were made in the C57BL/6 background, which lacks functional expression of IE due to a mutation in the IE locus. As we have shown previously, transgenic expression of IE cDNA under the control of the DC-specific CD11c promoter leads to expression of functional IE molecules in these mice selectively on DCs (18, 19). These mice and their nontransgenic littermates (IE^-) were immunized with Ag-loaded IE⁺ DCs, and Ag-specific T cell expansion in draining and nondraining lymph nodes was monitored by flow cytometry (Fig. 1a). In DC-IE⁺ animals, in addition to the IE⁺ DC vaccine, endogenous DCs, but not other cells, express the correct restriction element MHC class II IE for Ag presentation to AD-10 T cells. In IE-negative littermates (IE^-), only the IE⁺ DC vaccine can induce AD-10 expansion. T cell expansion in DC-IE⁺ mice was 3-fold higher (Student’s t test, p = 0.0193) than that detected in IE^- recipient mice (Fig. 1b, DC vaccine IE⁺, peptide⁺), which suggests that, in addition to the DC vaccine, endogenous IE⁺ DC further amplify the T cell expansion. In mice that received peptide-pulsed IE^- DCs, substantially lower T cell expansion was observed (Fig. 1b, DC vaccine IE^-, peptide⁺), but approximately two times more T cells were found in draining lymph nodes of DC-IE⁺ mice as compared with IE^- recipients. In fact, this T cell expansion (Fig. 1b, DC vaccine IE^-, peptide⁺) was comparable to that of IE^- recipients that received a peptide-pulsed IE⁺ DC vaccine (Fig. 1b, DC vaccine IE⁺, peptide⁺) and can be explained by endogenous IE⁺ DCs taking up Ag from the IE^- DC vaccine for presentation. Mice immunized with nonpulsed IE⁺ DCs (Fig. 1b, DC vaccine IE⁺, peptide^-) induced weak T cell expansion as compared with nondraining lymph nodes with no significant difference (p > 0.5) in both recipient groups. In general, no T cell expansion was observed in nondraining lymph nodes (Fig. 1c). These data indicate that, in the recipient of a DC vaccine, endogenous DCs are able to amplify T cell responses and must therefore re-present transferred Ag.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. MHC expression by endogenous DCs critically enhances CD4 T cell expansion induced by DC vaccines. CD4+ AD-10 T cells (5 x 106) were adoptively transferred into DC-IE+ () or IE- () mice. One day later, mice were vaccinated s.c. with 1 x 107 IE+ or IE- DCs, either loaded with MCC88–103 peptide or not. Transgenic AD-10 cells in the draining lymph nodes were identified on day 5 post-DC vaccination by flow cytometry with CD4- (not shown), TCR V- and V-specific mAbs as shown in a. The total number of AD-10 transgenic T cells in the draining (b) or nondraining (c) popliteal lymph nodes was determined. Group size of this experiment was three (n = 3), and error bars indicate SEM. The experiment shown is representative of five performed, with similar results. Asterisks indicate significant differences between DC-IE+ and IE- mice as calculated with the Student’s t test (*, p = 0.0193).

We next asked whether the Ag-specific T cells triggered by DC vaccines had different characteristics in the various recipients, and analyzed lymph node cells for the activation marker CD62L, incorporation of BrdU, and IL-2 production by flow cytometry (Fig. 2a). After vaccination with IE⁺ DCs, three times more AD-10 cells showed down-regulation of CD62L as a sign of T cell activation in DC-IE⁺ recipients as compared with IE^- recipients (Fig. 2b). Furthermore, significantly more (60%; Student’s t test, p = 0.049) AD-10 T cells had incorporated BrdU in DC-IE⁺ mice as compared with IE^- recipients (Fig. 2b). The ex vivo analysis of IL-2 production in the same T cells revealed a similar picture. Approximately 70% more IL-2-producing AD-10 T cells were detected in lymph nodes of DC-IE⁺ recipients as compared with IE^- recipients (Fig. 2c). In mice vaccinated with peptide-pulsed IE^- DCs incapable of presenting the antigenic peptide (data not shown), we found low but significant differences in BrdU incorporation, but no significant differences in IL-2 production (data not shown). Taken together, endogenous DCs that express the correct restriction elements appear capable of enhancing the efficiency of DC vaccines, because they increase the numbers of proliferating Ag-specific CD4 T cells with effector functions.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Participation of endogenous DCs in Ag presentation leads to enhanced activation, proliferation, and IL-2 production by T cells. Draining popliteal lymph node cells of DC-vaccinated mice were stained for V11 and V3 (a, dot plot) followed by intranuclear staining for BrdU incorporation, intracellular staining for IL-2 expression, or surface staining for CD62L expression (a, histograms, solid lines). Endogenous CD4+ T cells from the same lymph nodes are shown as controls (a, histograms, dotted lines). Numbers of CD62L- (b), BrdU- (c), or IL-2-positive (d) AD-10 T cells were determined on day 5 postvaccination. Each group contained three mice (n = 3). Asterisks indicate significant differences between DC-IE+ and IE- mice as calculated with Student’s t test (*, p < 0.05)

We next wanted to investigate whether the re-presentation by endogenous cells following DC vaccination is a special property of DCs or whether B cells are also capable of enhancing the proliferation of AD-10 T cells. For this purpose, we took advantage of transgenic mice expressing MHC class II IE^b molecules under the control of the B cell-specific CD19 promoter selectively in B cells (20). When the draining lymph nodes of vaccinated animals were analyzed, in contrast to DC-IE⁺ recipients, the number of AD-10 T cells found in B-IE⁺ mice was not significantly different from that detected in IE^- animals (Fig. 3; Student’s t test, p = 0.09). Because in B-IE⁺ mice, in contrast to IE^- mice, T lymphocytes have been negatively selected against IE, these data argue against an alloreactive influence on the DC vaccine. We investigated this possibility further and injected CFSE-labeled IE⁺ DCs as a vaccine s.c. into DC-IE⁺ or IE^- recipients and subsequently analyzed the draining lymph node at different time points for the presence of labeled DCs. As reported by others (14, 22), we found that numbers of transferred DCs peaked after 2 days postinjection and decreased subsequently. Identical numbers of DCs were detected in both recipients at all time points after injection, indicating that survival times of the DC vaccine was equivalent in both hosts (data not shown). This finding is in accordance with a previous report (15), which showed that no alloreactive effect could be observed when IE⁺ DCs were injected into IE^- mice. Therefore, we conclude that the amplification of the T cell response induced by a DC vaccine is a special property of endogenous DCs, while B cells are less able to process Ag from the vaccine to present it efficiently.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Endogenous B cells do not enhance T cell proliferation induced by a DC vaccine. DC-IE+, B-IE+, or IE- mice were immunized with peptide-pulsed DCs s.c., and the total number of Ag-specific adoptively transferred AD-10 T cells in draining lymph nodes was determined on day 5 postimmunization by FACS analysis with TCR V- and V-specific mAbs. Each group contained three to five mice (n = 3–5). Asterisks indicate significant differences between DC-IE+ and B-IE+ mice as calculated with Student’s t test (**, p < 0.005), whereas differences between B-IE+ and IE- recipients were not significant.

Only a small fraction of DCs (<1%) from the injected DC vaccine seems to arrive in the draining lymphoid organs (data not shown and Ref.14), suggesting that death of DCs occurs at the injection site. One consequence of DC death might be the release of antigenic material and its active uptake by or passive transfer to endogenous DCs. To control for such a possibility, we first analyzed Ag transfer independent of cell-cell contact using a Transwell assay. Viable, necrotic, or apoptotic peptide-pulsed DCs were cultured in the top chamber of a Transwell plate, while AD-10 T cells and peptide-free IE⁺ DCs were cultured in the lower compartment. Effective induction of apoptosis or necrosis of the Ag-pulsed DCs in the top chamber was controlled by flow cytometry using annexin V and propidium iodide (data not shown). As expected, T cells proliferated when soluble peptide was added to the upper compartment of the Transwell and could diffuse freely (Fig. 4a). In contrast, when viable peptide-pulsed DCs were cultured in the upper Transwell, only a background level of proliferation, not different from the medium control, was detected, indicating that the peptide did not reach the lower compartment. Therefore, it seems unlikely that the peptide is released from the live DC vaccines used in previous experiments. DCs that were rendered either apoptotic or necrotic were also unable to release antigenic peptide in a form capable of reaching the lower compartment and therefore did not stimulate T cells (Fig. 4a).

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Dead peptide-pulsed DCs induce weak T cell proliferation in vitro but not in vivo. a, Proliferation assay where AD-10 T cells (1 x 105) and mature IE+ DCs (2.5 x 104) were cocultured in the lower chamber of a Transwell. This culture was separated by 0.2-µm pore filters from the indicated MCC88–103 peptide-pulsed (Ag+) DCs or controls in the upper chamber. T cell proliferation was measured, and SEM is shown for an average of three wells. b, Graded numbers of MCC88–103 peptide-loaded necrotic DCs were cultured in the same chamber with 1 x 105 AD-10 T cells in the presence of no additional DCs (), 1 x 105 nonpulsed IE- DCs (), nonpulsed IE+ DCs (), or peptide-pulsed IE+ DCs (rhomb). T cell proliferation was measured in triplicate and error bars represent SEM. c and d, DC-IE+ mice were vaccinated with viable (c, ) or necrotic (c, ), or viable (d, ) or apoptotic (d, ) MCC88–103 peptide-pulsed IE+ DC vaccines. As controls, peptide-pulsed IE- DCs () were used. Analysis of AD-10 T cells was performed as shown for Fig. 1. The group size was three to five (n = 3–5). Differences between injection of viable or necrotic DCs (c) and viable or apoptotic DCs (d) are both significant (*, p < 0.05; **, p < 0.005) as calculated with Student’s t test.

Apoptotic or necrotic DCs cannot induce T cell proliferation in vivo

As mentioned before, only a small percentage of injected DCs reach the draining lymph nodes (14, 22). We wondered whether 1) these few surviving and migratory DCs from the DC vaccine actually reached the lymph nodes or 2) the majority of DCs dying at the injection site would be the source for Ag. To discriminate between these two possibilities, we injected either IE⁺, peptide-pulsed, necrotic or apoptotic DCs into DC-IE⁺ recipients. Whereas the dead DCs were able to prime naive T cells to some extent in vitro (Fig. 4b), we could not detect statistically significant proliferation in the transferred T cell pool, when necrotic (Fig. 4c) or apoptotic (Fig. 4d) MCC peptide-loaded DC vaccines were injected. We concluded from these findings that, at the conditions we used, either transfer of Ag does not sufficiently take place at the site of injection or dead DCs cannot induce T cell proliferation in vivo.

Injected viable DCs activate endogenous DCs before they die rapidly in the draining lymph node

To follow the fate of DCs after vaccination more closely, we vaccinated DC-IE⁺ mice with a viable DC vaccine that was labeled with the life dye CFSE. At different time points after injection, draining lymph nodes were isolated and analyzed for viability of the DC vaccine. As shown in Fig. 5a, already at day 2 after injection, 25% CFSE-positive DCs in the draining lymph nodes showed signs of secondary necrosis as judged from strong labeling with both annexin V and propidium iodide (Fig. 5a). In contrast, as control, only 2.5% necrotic, resident, CFSE^- DCs could be detected in the same lymph node (Fig. 5a). Comparison of endogenous DCs in injected vs noninjected mice revealed activation of some endogenous DCs by the DC vaccine as detected by their up-regulation of CD86 (Fig. 5b). Although in a nonvaccinated mouse, 34% of DCs expressed high levels of CD86, this percentage went up to 50% in DC-vaccinated animals (Fig. 5b).

View larger version (35K): [in this window] [in a new window]   FIGURE 5. DCs from the vaccine die in the lymph node after interaction with and activation of endogenous DCs. CFSE-labeled DCs (1 x 107) were injected s.c. into DC-IE mice. Two days after injection, draining lymph nodes were taken. a, Flow cytometry analysis of draining lymph nodes. The left dot plot shows representative annexin V/propidium iodide stainings either gated on CFSE+ DCs from the vaccine or gated on endogenous CFSE- DCs. The bar graph on the right side shows a comparison of mean percentages of annexin V+propidium iodide+ cells from either the CFSE+ or the CFSE- DC cell populations. b, Flow cytometry analysis of CD86 up-regulation on endogenous DCs. The left histograms show representative analysis of CD86 on DCs in a mouse injected with a DC vaccine or in noninjected mice. Histograms are gated on CFSE-CD11c+ cells. The bar graph on the right shows a comparison of the mean percentages of CD86 DCs in vaccinated vs nonvaccinated mice. c, Histological analysis of draining lymph nodes. CFSE+ DCs from the vaccine are shown in green, and endogenous CD11c-stained DCs are shown in red. Arrows indicate sites of interaction between injected and endogenous DCs, and arrowheads show CFSE+ membrane patches from injected DCs (green) on endogenous DCs (red).

Injection of apoptotic or necrotic DC vaccines has no immunological effect on Ag-specific T cells

We then controlled to determine whether injection of apoptotic or necrotic DC would interfere with subsequent T cell activation by inducing T cell tolerance, as postulated before (15, 23, 24). To this end, we first immunized mice with Ag-pulsed necrotic or apoptotic DC vaccines and then challenged them with a viable DC vaccine (Fig. 6). Both groups of animals showed strong Ag-specific T cell expansion as compared with control mice (Fig. 6). These data argue against an induction of T cell tolerance by dead DC vaccines, but rather suggest that injection of apoptotic DCs fails to have an immunological effect in our experimental system.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Injection of necrotic or apoptotic DC vaccines does not lead to T cell unresponsiveness. DC-IE+ mice were injected s.c. on day 0 with either 107 apoptotic (), necrotic (), or viable () peptide-pulsed DCs. Eight days later, the same mice were challenged with viable peptide-pulsed IE+ DCs ( and ) to measure T cell responses or were not immunized () to determine background T cell expansion. On day 5 after the second injection, draining lymph nodes were prepared and cell suspensions were stained for the percentage of AD-10 T cells as described in Fig. 1. The group size was three to four (n = 3–4). Differences of T cell expansion in mice injected with apoptotic/viable DCs () vs necrotic/viable DCs () were insignificant (p = 0.24), whereas differences observed after treatments with necrotic/viable DCs () and viable/no DCs () were significant (p = 0.041) as calculated with Student’s t test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several studies have demonstrated transfer of Ag from living cells to DCs in vitro (9, 15) and in vivo (15). In addition, DCs are able to present phagocytosed material from necrotic or apoptotic cells (10, 11, 12). Although presentation of cellular material from dead cells resulted in efficient T cell stimulation in vitro (10, 11, 12), the relevance of such a mechanism in vivo remains hypothetical (24). We have addressed this question using a novel in vivo system that allows us to focus on endogenous DCs or B cells (as control) and their participation in the immune response following DC vaccination. We found that endogenous DCs play an important role during DC vaccination and enhance Ag-specific activation, expansion/proliferation, and effector function of CD4⁺ T cells in vivo (Figs. 1 and 2). In contrast, participation of endogenous B cells had no impact on the efficiency of the DC vaccine (Fig. 3). Using this experimental system, we show that Ag transfer requires cell-cell contact (Fig. 4) and enables endogenous DCs to enhance T cell responses.

However, it remains unclear how Ag transfer takes place. Inaba et al. (15) proposed that transferred MHC class II molecules are endocytosed and the resulting antigenic peptide is subsequently re-presented in the context of endogenous MHC class II molecules. More recent work showed that entire membrane fragments containing MHC class II/peptide complexes may be transferred to endogenous DCs (9), allowing T cell recognition without further peptide processing. This could occur in the shape of exosomes or membrane vesicles. We found that even the presence of viable IE^- DCs unable to express the restricting MHC class II element enhances T cell stimulation induced by necrotic or apoptotic MHC IE⁺ DCs in vitro (Fig. 4). This effect might well be explained as a bystander phenomenon; in this scenario, T cells might recognize MHC class II/peptide on necrotic/apoptotic IE⁺ DCs, with live IE^- DCs simply providing an efficient costimulus. However, the addition of live DCs with the correct MHC class II enhanced T cell proliferation further. This led us to believe that DCs play an active role in the presentation of transferred Ag. This hypothesis is further supported by in vivo experiments, where T cell responses in hosts with endogenous IE⁺ DCs were very strong, as compared with the responses in IE^- hosts (Figs. 1 and 2). Taken together, our in vitro and in vivo experiments support both inactive (bystander phenomenon) and active roles for endogenous DCs in their enhancement of DC vaccinations, but the re-presentation of antigenic peptide on endogenous MHC class II seems to be the more efficient pathway. This interpretation is further supported by a report showing in vitro that a blockade of endocytic proteolysis inhibits re-presentation of transferred MHC class II molecules (15). Our findings show the in vivo consequence of this scenario and indicate that re-presentation via endocytosis accounts not only for peptides derived from the backbone of MHC molecules, as shown previously (15, 16), but also for (MCC_88–103) peptides which are already bound to the peptide-binding groove of the endocytosed MHC molecule. Therefore, we propose that such a pathway of re-presentation, which contains many potential degradation mechanisms for preprocessed small peptides with very short half-lives, is nevertheless very efficiently recycling them for re-presentation in the MHC context.

The next question we investigated was whether the transfer of Ag to endogenous DCs occurs in the periphery or in the lymph node. It is possible that the few DCs that reach the lymph node following DC vaccination might serve as sources for Ag transferred to resident lymph node DCs. Alternatively, the majority of injected DCs that die in situ at the site of injection might provide Ag to migratory DCs from peripheral tissues (e.g., skin). The latter would take up Ag, migrate to the draining lymph node, and present Ag. To discriminate between these two possibilities, we rendered the Ag-loaded DC vaccines either necrotic or apoptotic before injection. In neither case could we find a significant induction of T cell immunity (Fig. 4, c and d). These data suggest that efficient T cell stimulation requires the migration of viable Ag-loaded DCs to the draining lymph nodes, a conclusion that is in accordance with the current working hypothesis for Ag presentation by immature DCs in the steady state (24, 25): resident lymph node DCs present Ag derived from migratory DCs and induce in this case T cell tolerance. In contrast to this, we hypothesize that, in our in vivo model, the DC vaccine activates Ag-specific T cells first and then dies, and Ag is passed over to endogenous immature DCs. This is supported by data showing that DCs from the injected vaccine are in close contact with endogenous DCs in the lymph node (Fig. 5c) but die soon after their arrival there (Fig. 5a). Preactivated T cells can then receive Ag-specific immunostimulatory signals from immature endogenous DCs, a phenomenon described previously in vitro (26). This model might explain why resident lymph node DCs, which are most likely immature DC, are able to enhance the T cell response. In addition, DC vaccination induces activation of a fraction of the endogenous DC as measured by CD86 up-regulation (Fig. 5b). The latter also could be immunostimulatory for naive Ag-specific T cells.

DC vaccination leads to the activation of both CD4⁺ and CD8⁺ T cells. Our data show that endogenous DC significantly enhance the response of MHC class II-restricted CD4⁺ T cells following DC vaccination, but it is unclear whether a similar role for endogenous DCs exists for the stimulation of CD8⁺ T cells. In the latter case, transferred exogenous Ag would have to be endocytosed and re-presented (cross-presented) in the context of endogenous MHC class I.

Cross-presentation has recently been shown to exist in vivo (27, 28, 29), and it will be interesting to determine whether it has a role in the generation of CTL responses following DC vaccination. The findings reported in this study suggest that both DCs injected as vaccine and endogenous DCs from the recipient are necessary for the induction of an optimal CD4⁺ T cell response. Because CD4⁺ T cells play a central role in antitumor responses (for review, see Ref.30), it may be critical to consider the immunological status of endogenous DCs before therapeutic DC vaccinations.

	Acknowledgments

 
We thank K. Kerksiek and G. Riethmueller for critically reading the manuscript, and A. Bol and W. Mertl for expert animal facility maintenance.

	Footnotes

 
¹ This work was supported by the Deutsche Forschungsgemeinschaft (Grant Br 1889/2), Sonderforschungsbereich 456 and a collaborative grant from Jenner Institute for Vaccine Research.

² Address correspondence and reprint requests to Dr. Thomas Brocker, Institute for Immunology, Ludwig-Maximilians-Universität München, Goethestrasse 31, 80331 Munich, Germany. E-mail address: Thomas.Brocker{at}ifi.med.uni-muenchen.de

³ Abbreviations used in this paper: DC, dendritic cell; MCC, moth cytochrome c; BrdU, 5-bromo-2'-deoxyuridine.

Received for publication April 22, 2002. Accepted for publication December 30, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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