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Induction of CTL and Nonpolarized Th Cell Responses by CD8⁺ and CD8^- Dendritic Cells¹

Unité de Biologie des Régulations Immunitaires, Institut Pasteur, Paris, France

	Abstract

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Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8 expression and their localization in lymphoid organs. Several reports suggest that CD8⁺ and CD8^- DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4⁺ and CD8⁺ T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4⁺ T cell priming. Second, we showed that in vivo activation of CD4⁺ T cells by CD8⁺ or CD8^- DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8^- subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8⁺ subset. However, IL-5 was produced by CD4⁺ T cells activated by both DC subsets. When both CD4⁺ and CD8⁺ T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8⁺ T cells, while Th2 cytokines were produced by CD4⁺ T cells. Thus, this study clearly shows that CD4⁺ T cell responses do not influence the development of specific CD8⁺ T cell cytotoxic responses induced either by CD8⁺ or CD8^- DC subsets.

	Introduction

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It is now generally accepted that fully mature dendritic cells (DC)³ are the main professional APCs with the unique ability to induce primary T cell immune response. Immature DC are characterized by their high capacity to capture Ag and a low efficiency to stimulate T cells. After activation by microbial or inflammatory signals such as LPS or TNF-, DC mature and acquire the ability to stimulate naive T cells. Mature DC are characterized by high cell surface expression of MHC class I/II molecules and costimulatory molecules such as CD40, CD80, and CD86 as compared with immature DC (1). Two distinct pathways of DC development have been identified in mice, and DC are usually classified as myeloid and lymphoid. Myeloid DC can be generated from a bone marrow myeloid precursor under the influence of GM-CSF (2), while lymphoid DC seem to develop from early T cell precursor populations in the thymus (3, 4, 5). Both DC subsets express high levels of CD11c, but, until recently, were characterized by the expression of different cell surface markers. Myeloid DC highly express the myeloid marker CD11b and lack CD8 and DEC-205, whereas lymphoid DC are CD11b^low CD8⁺ and DEC-205⁺ (6). However, some recent papers indicate that the expression of the CD8 homodimer at the DC surface is not indicative of lymphoid origin. Indeed, it was recently shown that CD8⁺ DC can develop from a myeloid progenitor (7) as well as from myeloid Langerhans cells after their migration in the draining lymph nodes (8). Furthermore, both CD8⁺ and CD8^- DC could be generated from lymphoid-committed precursors in both thymus and spleen (9). So, CD8 expression on DC could reflect a state of activation, maturation, and/or mobilization rather than ontogeny.

However, even if the expression of the CD8 homodimer does not characterize a specific lineage, important localization and functional distinctions were described between CD8⁺ and CD8^- subsets. In the spleen, the CD8⁺ DC subset resides in the T cell zone, while the CD8^- DC subset resides in the marginal zone (10, 11). Since it was shown that CD8⁺ DC can restrain T cell proliferation by limiting cytokine production by CD8⁺ T cells (12) and by killing CD4⁺ T cells (13), it was suggested that CD8⁺ DC could play a role in peripheral tolerance. By contrast, CD8^- DC induce strong activation of naive T cells (14). However, it was also shown that both populations have the ability to prime CD4⁺ T cells, indicating no inherent tolerizing function for CD8⁺ DC. The differences between the two subsets could rather reside in the type of response they induced, since CD8^- DC were shown to stimulate Th2 responses, whereas CD8⁺ DC induced Th1 responses (15, 16). It was also recently shown that only the CD8⁺ DC subset has the ability to cross-prime CD8⁺ T cell response in vivo (17), but it was demonstrated that both DC subsets induce in vivo strong viral protective cytotoxic T cell response (18). However, in this study, the responses induced by these two DC subsets were analyzed independently of specific CD4⁺ T cell responses since a class I-restricted peptide was used. As DC subsets may affect the polarization of Th cells, it was important to evaluate whether CD4⁺ T cell responses could regulate the induction of cytotoxic response by these two DC subsets. This prompted us to analyze the ability of both DC subsets to induce Th and CTL responses and to determine whether Th responses induced under these conditions could influence the CTL activity. For this study, a panel of class I-, class II-, or class I/II-restricted peptides was used to analyze the Th and CTL responses induced by purified CD8⁺ and CD8^- DC. We showed that both DC subsets are able to induce strong CTL responses after i.v. injection independently of the Th response simultaneously generated. Furthermore, while CD8^- DC seem to be more efficient than CD8⁺ DC to induce Th2 cytokine production, both DC subsets have the ability to generate a nonpolarized Th cytokine response with the production of both Th1 and Th2 cytokines when injected i.v. Finally, we also show that the route of injection influences the polarization of the induced T cell responses.

	Materials and Methods

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Mice

Female BALB/c (H-2^d) and C57BL/6 (H-2^b) mice (6–10 wk old) were used in all experiments and were purchased from CER Janvier (Le Genest St. Isle, France).

Peptides

The synthetic peptide RPQASGVYM carrying the 118–126 sequence from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein corresponding to a class I and class II H-2^d-restricted CTL epitope (19, 20), the synthetic peptide SIINFEKL carrying the 257–264 sequence from OVA corresponding to a H-2K^b-restricted CTL epitope (21), and the synthetic peptide KLFAVWKITYKDTV carrying the 103–116 sequence from the poliovirus I envelope protein VP1 corresponding to a I-E^d-restricted Th epitope (22) were purchased from Neosystem (Strasbourg, France). All peptides and their restriction elements are described in Table I. These peptides were tested for the presence of endotoxin using a chromogenic test (Limulus amebocyte lysate test; BioWhittaker, Fontenay-sous-Bois, France), and no endotoxin was detected.

Complete medium (CM) consisted of RPMI 1640 containing L-alanyl-L-glutamine dipeptide supplemented with 10% FCS, 5 x 10^-5 M 2-ME, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). B3Z T cell hybridoma was maintained by adding 1 mg/ml G418 and 400 µg/ml hygromycin B to the CM (23).

Cell lines

The MV1H7 T cell hybridoma specific of the H-2^d-restricted 118–126 LCMV epitope was generated in the laboratory, as previously described (24). Briefly, female BALB/c mice were immunized s.c. at the base of the tail with 100 µg LCMV peptide p_118–126 in IFA. On the 2 following days, mice were depleted of CD4⁺ T cells with 300 µg CD4-specific rat anti-mouse mAb (GK 1-5). One week later, mice were killed and the inguinal lymph nodes and spleens were removed aseptically. A single cell suspension was prepared in CM and cultured in the presence of an equal number of irradiated syngeneic splenocytes and 10 µg/ml immunizing peptide. Four days later, viable lymphocytes were isolated by fractionation with Lympholyte (Cedarlane Laboratories, Ontario, Canada) and fused with CD8-transfected BW5147 myeloma (kindly provided by M. Viguier, Institut Cochin, Paris, France) in a ratio of 1:1 by using polyethylene glycol (PEG 1500; Boehringer Mannheim, Mannheim, Germany). The cell suspension was brought to a final volume of 40 ml with RPMI 1640 supplemented with 20% FCS, 50 mM 2-ME, 2 mM glutamine, and antibiotics. After incubating for 4 h at 37°C, feeder hypoxanthine/aminopterin/thymidine-sensitive A20 cells were added to a final concentration of 1 x 10⁵ cells/ml. Cells were then plated onto 96-well flat-bottom microtiter plates with 100 µl/well. Sixteen hours later, 20 µl 6x hypoxanthine/aminopterin/thymidine (Boehringer Mannheim) was added to each well. Hybridoma appeared 7 to 15 days after fusion, and were assayed for peptide-specific reactivity, with 1 µg/ml of the immunizing peptide and 5 x 10⁴ P815 as APC. Twenty-four-hour culture supernatants were analyzed for IL-2 content. From over 20 hybridoma specific for the LCMV p_118–126 peptide, MV1H7, a L^d-restricted T cell hybridoma, was selected.

The I-E^d-restricted hybridoma 45G10 specific for the 103–116 sequence of VP1 protein from PV1 was generated in the laboratory, as previously described (25). B3Z (23), a CD8⁺ T cell hybridoma specific for the K^b-restricted OVA 257–264 peptide, was a generous gift from N. Shastri (University of California, Berkeley, CA).

DC purification

DC were purified from spleen by positive selection according to CD11c expression using Automacs (Miltenyi Biotec, Paris, France), following manufacturer’s procedure, except that cells were labeled with PE-conjugated anti-CD11c and FITC-conjugated anti-CD8 mAbs (HL3 and 53-6.7 clones, respectively; BD PharMingen, San Diego, CA) during the incubation with the anti-CD11c microbeads (N418). The recovered cells were routinely >96% CD11c⁺ and consisted of 70% CD8^- and 30% CD8⁺ cells. Then, CD11c⁺ cells were further sorted by flow cytometry on MoFlo (BD Biosciences, Mountain View, CA), according to their CD8 expression. The purity of the two fractions was checked by flow cytometry on FACSCalibur or FACScan, and was usually >97%.

Mouse immunization

CD8^- and CD8⁺ dendritic cells were incubated 90 min with or without 50 µM of the various peptides in Sfem Stem Span medium (StemCell, Meylan, France). After extensive washing, DC were injected into syngeneic mice either i.v. (1 x 10⁵ cells) or s.c. (1 x 10⁵, 1.7 x 10⁵, or 2.5 x 10⁵ cells were injected into hind footpads, as indicated in Fig. 5 legend).

View larger version (13K): [in this window] [in a new window]   FIGURE 5. Ag-specific IL-5 and IFN- production by splenocytes after s.c. injection of DC subsets. The production of IL-5 and IFN- by splenocytes from BALB/c mice injected into the footpads with CD8- () or CD8+ () DC loaded with p118–126 was determined after specific restimulation with this peptide. A total of 1 x 105 DC (A), 1.7 x 105 DC (B), and 2.5 x 105 DC (C) was injected into BALB/c mouse hind footpads. After 1 wk, mice were sacrificed and splenocytes were restimulated in vitro in the presence or absence of the priming peptide, in duplicate. Cytokine level was assessed by ELISA after 72 h of restimulation. Three independent experiments are shown. Results are expressed as Ag-specific cytokine levels corrected for background level.

The stimulation of T cell hybridoma was monitored by IL-2 release in the culture supernatants in the presence of CD8^- or CD8⁺ DC and of various concentrations of the respective peptide. A total of 5 x 10⁴ T cell hybridomas was cocultured in 96-well culture plates in the presence of 2 x 10⁴ DC. After 18 h, culture supernatants were frozen for at least 2 h at -80°C. Then, 1 x 10⁴ cells/well of the IL-2-dependent CTL-L cell line were cultured with 100 µl of these supernatants. After 48 h, [³H]thymidine (50 µCi/ml; ICN, Orsay, France) was added in the wells, and the cells were harvested 6 h later with an automated cell harvester (Skatron, Lier, Norway). Incorporated thymidine was detected by scintillation counting. In all experiments, each point was done in duplicate. Results are expressed as cpm (cpm in the presence of peptide - cpm in the absence of peptide).

In vitro cytotoxicity assay

Splenocytes from immunized mice were isolated 7 days after DC injection and restimulated in vitro for 5 days with p_257–264 (1 µg/ml) or p_118–126 (0.1 µg/ml) in the presence of syngeneic irradiated naive spleen cells. The cytotoxic activity was determined in a 5-h in vitro ⁵¹Cr release assay, as previously described (26). Briefly, P815 (H-2^d) or EL4 (H-2^b) tumor cells loaded with 50 µM concentration of the respective peptide were used as target cells for H-2^d and H-2^b effector cells. Various E:T ratios were used, and all assays were done in duplicate. ⁵¹Cr release in each well was counted using a MicroBeta Trilux liquid scintillation counter (Wallac, Turku, Finland). Percentage of specific lysis was calculated as 100 x [(experimental release - spontaneous release)/(maximal release - spontaneous release)]. Maximum release was obtained by adding 10% Triton X-100 to target cells, and spontaneous release was determined with target cells incubated without effector cells.

IL-2 dosage

Splenocytes from immunized mice were restimulated in vitro in the presence or absence of 1 µg/ml (p_118–126) or 0.1 µg/ml (p_103–116, p_257–264) of the corresponding peptide, and the supernatants were harvested and frozen after 24 h. In some cases, CD4⁺ or CD8⁺ T cells were in vitro depleted by negative selection on Automacs (Miltenyi Biotec). After this depletion, we verified by flow cytometry that the splenocyte suspension contained less than 0.5% of CD4⁺ or CD8⁺ T cells. IL-2 concentrations in the culture supernatants were determined by CTL-L assay and are expressed in pg/ml. Ag-specific IL-2 production was determined by correcting the total production for background level that was about 30% of the specific production.

Cytokine ELISA assay

Splenocytes from immunized mice were restimulated in vitro in the presence or absence of 1 µg/ml (p_118–126) or 0.1 µg/ml (p_103–116, p_257–264) of the corresponding peptide, and the culture supernatants were harvested after 72 h. In some cases, CD4⁺ or CD8⁺ T cells were depleted by negative selection on an Automacs (Miltenyi Biotec). IL-4, IL-5, IL-10, and IFN- concentrations were then measured in these supernatants by a standard sandwich ELISA. Maxisorp plates (Nunc, Roskilde, Denmark) were coated with unconjugated anti-IL-4, anti-IL-5, anti-IL-10, or anti-IFN- capture Abs (BVD4-1D11, TRFK5, JES5-2A5, R4-6A2 clones, respectively; BD PharMingen), and detection was done using corresponding biotinylated mAb (BVD6-24G2, TRFK4, SXC-1, XMG1.2 clones; BD PharMingen). The plates were developed using streptavidin-HRP (BD PharMingen) and o-phenylenediamine as substrate. All dosages were performed in duplicate. The assays were standardized with recombinant murine cytokines (BD PharMingen), and results are expressed in picograms per milliliter. Ag-specific cytokine production was corrected for background production. Background level was about 25% to 30% of the specific production for IFN-, 45% for IL-4, 35% for IL-5, and 40% for IL-10.

Flow cytometry analysis

Cells were washed in PBS supplemented with 1% BSA and 0.1% NaN₃, incubated with anti-CD32/CD16 (2.4G2 clone; BD PharMingen) to avoid nonspecific staining, and labeled 30 min at 4°C with the following Abs purchased from PharMingen: PE-conjugated anti-CD11c mAb (HL3 clone), FITC-conjugated anti-CD8 mAb (53-6.7 clone), APC-conjugated anti-CD4 mAb (L3T4), APC-conjugated anti-CD40 mAb (3/23), APC-conjugated anti-CD80 mAb (16-10A1), APC-conjugated anti-CD86 mAb (GL1), biotin-conjugated anti-I-A^d mAb (AMS-32.1), biotin-conjugated anti-I-A^b mAb (25-9-17), biotin-conjugated anti-K^d mAb (SF1-1.1), biotin-conjugated anti-K^b mAb (AF6-88.5). APC-conjugated streptavidin was used as second Ab to reveal labeling with biotin-conjugated mAb. As negative control, cells were stained with corresponding isotype-matched control mAbs. Acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences), and analysis was done with CellQuest (BD Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotypic and in vitro functional analysis of purified CD8⁺ and CD8^- DC

DC were isolated by positive selection on MACS from spleen of BALB/c or C57BL/6 mice based on the cell surface expression of the CD11c molecule. They were then sorted by FACS to separate CD8⁺ and CD8^- DC. Purity of CD8⁺ and CD8^- DC subsets was superior to 97% for both BALB/c and C57BL/6 mice.

Expression of various surface molecules was assessed on both CD8^- and CD8⁺ DC before and after purification (Table II). Both DC subsets from C57BL/6 expressed similar level of costimulatory molecules, such as CD40, CD80, and CD86, and MHC class I and class II molecules before and after purification. However, all surface molecules tested were expressed at higher levels on the CD8⁺ DC subset, as previously described (18). After 90 min of incubation of purified DC with the peptide p_257–264, a similar pattern of expression was obtained with a slight increase of CD86 and I-A^b expression by CD8⁺ DC. Furthermore, comparable up-regulation of all markers tested (except MHC class I molecules) was observed after activation with LPS for both purified DC subsets (Table II). Similar results were obtained with DC isolated from BALB/C mice (data not shown). These data indicate that purified DC subsets were in the same maturation state as in vivo and efficiently mature after LPS stimulation.

View this table: [in this window] [in a new window]   Table II. Phenotype of CD11c+CD8- and CD11c+CD8+ subpopulations before and after isolation from spleen of C57BL/6 mice1

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Efficient presentation of different peptides to specific T cell hybridoma by both DC subpopulations. CD8- () and CD8+ () DC purified from BALB/c (A and B) or C57BL/6 (C) mice were incubated in the presence of various concentrations of p103–116 (A), p118–126 (B), or p257–264 (C) peptides. These cells were used as APC to stimulate, respectively, 45G10 (A), MV1H7 (B), or B3Z (C) T cell hybridoma. IL-2 secretion by the stimulated hybridoma was determined by the CTL-L proliferation assay. Data represent means of duplicates, and results are expressed in cpm of incorporated [3H]thymidine (cpm in the presence - cpm in the absence of peptide). SD were always <10% and are not represented. Results are representative of three experiments.

In vivo induction of CTL responses by CD8⁺ and CD8^- DC subpopulations

Since it was previously shown that both DC populations reach the spleen after i.v. injection (18), induction of CTL responses by the two DC subsets was assessed in the spleen following i.v. immunization with CD8⁺ or CD8^- peptide-pulsed DC. Freshly purified CD8⁺ and CD8^- DC from BALB/c or C57BL/6 mouse spleens were respectively pulsed with the peptide p_118–126 or p_257–264. Then, 1 x 10⁵ DC were injected i.v. to syngeneic mice. This DC number was previously demonstrated to induce good and reproducible CTL responses for both DC subsets (18 and data not shown). Unloaded CD8⁺ or CD8^- DC were also injected to control mice to assess the specificity of the CTL responses. Seven days after the injection, splenocytes from immunized mice were stimulated in vitro with the peptide used for immunization. Six days later, CTL activity was assessed by ⁵¹Cr release assay using H-2^d P815 or H-2^b EL4 target cells pulsed or not with the respective peptide. As shown in Fig. 2, both DC subsets induced high peptide-specific CTL responses in BALB/c and C57BL/6 mice. These CTL responses were Ag specific since only peptide-sensitized target cells were killed. Furthermore, no significant CTL responses were observed in mice injected with unloaded CD8⁺ and CD8^- DC, showing the specificity of these responses (Fig. 2, A and B). However, CD8⁺ DC appeared to be less efficient in inducing such CTL responses, both in BALB/c and C57BL/6, since significantly higher lysis was observed with all E:T ratios tested with splenocytes of mice immunized with the CD8^- DC subset.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Induction of CTL responses by CD8+ and CD8- DC subpopulations. DC subpopulations were isolated from BALB/c or C57BL/6 mice, pulsed with relevant peptide, and transferred into syngeneic mice. After 1 wk, mice were sacrificed and splenocytes were restimulated in vitro with the priming peptide and irradiated syngeneic splenocytes. CTL responses induced by injection of CD8+ DC () or CD8- DC () pulsed either with p118–126 (H-2d) (A and B) or p257–264 (H-2b) peptide (C and D) into syngeneic mice were tested on 51Cr-labeled P815 (A) or EL4 (C) target cells pulsed with the corresponding peptide. Results on unpulsed target P815 (B) or EL4 (D) are also shown. As control, CTL responses obtained after injection of unpulsed CD8+ (X) or CD8- DC (*) were determined. Cytotoxic activity was tested 5 days later. Each curve corresponds to a single mouse. Results are representative of three experiments.

Induction of cytokine production by CD8⁺ and CD8^- DC subsets

To determine whether a CD4⁺ Th response was induced upon immunization with DC subsets loaded with the various peptides, cytokine production by spleen cells from mice primed with peptide-pulsed DC subsets was assessed. BALB/c and C57BL/6 mice were immunized as for CTL analysis, and splenocytes were stimulated in vitro with the corresponding peptides. Cytokine production was measured by determining the concentration of IL-4, IL-5, IL-10, IFN-, and IL-2 released in culture supernatants.

As shown in Fig. 3, there was no significant difference in the cytokines produced by splenocytes from mice primed in vivo with CD8⁺ or CD8^- DC pulsed with the class I-restricted p_257–264 peptide. Only production of IFN- and of low amount of IL-2 was observed without any production of Th2 cytokines, such as IL-4, IL-5, and IL-10. CD8⁺ or CD4⁺ T cell depletion experiments demonstrated that CD8⁺ T cells, as expected, were responsible for IFN- production (Fig. 4A). IL-2 production was too low to determine the T cell subset that produced it. Therefore, our results strongly suggest that CD8⁺ T cell responses obtained after immunization with CD8⁺ and CD8^- DC pulsed with p_257–264 are CD4⁺ T cell independent. By contrast, in mice immunized with DC loaded with either p_103–116 (class II-restricted peptide) or p_118–126 (class I/II-restricted peptide) peptides, both Th1 and Th2 cytokines were produced (Fig. 3). The CD8^- DC subset induced high level of Th1 and Th2 cytokines both for p_118–126- and p_103–116-immunized mice. The CD8⁺ DC induced good levels of Th1 cytokines and low, but detectable levels of IL-4 and IL-10. Interestingly, IL-5, a Th2 cytokine, was clearly induced by both DC subsets. Selective CD8⁺ or CD4⁺ T cell depletion experiments demonstrated that following p_103–116-pulsed DC priming, all detectable cytokines were produced by CD4⁺ T cells since their production was abolished after CD4⁺ T cell depletion. This indicates that only CD4⁺ T cells were primed under these conditions (Fig. 4B). In mice primed with p_118–126-pulsed DC, IL-4 and IL-5 were produced by CD4⁺ T cells, whereas IFN- was mainly produced by CD8⁺ T cells and IL-2 was produced by both CD4⁺ and CD8⁺ T cells (Fig. 4C). These results indicate that both CD4⁺ and CD8⁺ T cells were stimulated by p_118–126-pulsed DC. In these experiments, the production of IL-10 was too low to determine its origin.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Ag-specific production of a set of cytokines after transfer of CD8+ or CD8- DC. The Ag-specific production of a set of cytokines after transfer into syngeneic mice of CD8+ () or CD8- DC () loaded with p257–264 (H-2b), p103–116 (H-2d), or p118–126 (H-2d) peptides was determined after specific restimulation with the relevant peptide. DC subpopulations were isolated from BALB/c or C57BL/6 mice, pulsed with peptide, and injected into syngeneic mice. After 1 wk, mice were sacrificed and splenocytes were restimulated in vitro in the presence or in the absence of the priming peptide, in duplicate. After 24 h (IL-2) or 72 h (other cytokines) of restimulation, cytokine levels were determined in culture supernatants by CTL-L assay for IL-2 and by ELISA for the other cytokines. Results are expressed as Ag-specific cytokine levels (picograms per milliliter) corrected for background level in the absence of Ag. Data represent mean values of three or four different mice tested in the same experiment ± SD. Results are representative of four experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Inhibition of cytokine production by CD4+ or CD8+ T cell depletion. Percentage of inhibition of specific cytokine production after CD4+ () or CD8+ () T cell depletion of splenocytes is shown. Mice were immunized with either CD8- or CD8+ DC pulsed with p257–264 (A), p103–116 (B), or p118–126 (C), and splenocytes were harvested 7 days later. Each spleen population was divided in three samples, one was left untreated, and the two others were either CD4 or CD8 depleted by negative selection on an Automacs. These three samples were stimulated in vitro in the presence or in the absence of the priming peptide, and cytokine concentration in culture supernatants was determined by CTL-L assay for IL-2 and by ELISA assay for other cytokines. Results are expressed as percentage of inhibition of cytokine production after CD4+ or CD8+ T cell depletion as compared with nondepleted splenocytes. Results are from one representative mouse of two to five mice.

Influence of the immunization route on the polarization of T cell responses

Our results indicate that i.v. injection of CD8⁺ or CD8^- DC subsets loaded with class II- or class I/II-restricted peptides does not induce a clear polarization of the CD4⁺ T cell response. This conclusion contrasts with previous reports (15, 16) demonstrating that CD8^- DC induced Th2 responses, whereas CD8⁺ DC induced Th1 responses. The difference observed between our results and these studies could be due to the route of immunization, since the authors of these studies injected DC subsets into footpads and analyzed lymph node T cell responses. To assess the role of immunization route on the polarization of T cell responses after DC injection, purified DC subsets pulsed with p_118–126 peptide were injected into mouse footpads. After 1 wk, Ag-specific production of IL-5 and IFN- by splenocytes was determined following in vitro peptide stimulation. As shown in Fig. 5, CD8^- DC induced a high production of IFN- and IL-5, whereas CD8⁺ DC induced only IFN- production. Noteworthy, very similar results were obtained in the draining lymph nodes (data not shown). Thus, these results show that the route of injection can influence the polarization of the responses obtained after DC subset injection. In particular, CD8⁺ DC injected s.c. lost their ability to induce IL-5 production by CD4⁺ T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The efficiency of DC to induce strong primary immune responses makes them optimal candidates for Ag-specific immunotherapy protocols both in cancer and infectious diseases. In this work, we compared the ability of CD8⁺ and CD8^- DC subsets to induce in vivo specific CTL and Th responses. CD8⁺ and CD8^- DC were positively selected from spleen according to their CD11c expression, and then CD8⁺ and CD8^- subsets were purified by cell sorting. Under these conditions, purified DC expressed similar costimulatory and MHC molecules than before purification, even after peptide loading, and showed the ability to further mature following LPS as in vivo spleen DC. Since it was previously reported that both subsets reached the spleen after i.v. injection (18), freshly purified CD8⁺ and CD8^- DC were loaded with antigenic peptides and injected to syngeneic mice by the i.v. route.

Using an H-2^b CD8⁺ T cell immunodominant OVA epitope, we show in the present study that peptide-pulsed CD8⁺ and CD8^- DC subsets are both able to induce strong CTL responses, although the CD8^- DC appear more efficient, thus confirming the results of Ruedl et al. (18). Interestingly, analysis of cytokines produced by these CD8⁺ T cells showed a high level of IFN- and a weak production of IL-2. No difference in terms of cytokine production was observed after i.v. immunization with either CD8⁺ or CD8^- DC. These data indicate that both DC subsets are able to generate a type 1 CTL response. Furthermore, we clearly demonstrated that CD4⁺ Th activity was not required for this specific CTL induction. This suggests that the stimulation of CD8⁺ T cell responses by DC loaded with the OVA peptide is CD4 independent, confirming our previous results obtained with optimal peptides (26).

However, Th cells are generally required to activate efficient CTL activity, and it is well established that CD4⁺ T cell responses are strictly required to induce effective antiviral (27, 28) and antitumoral responses (29, 30), even after DC immunization (31). As the two different DC subsets may induce different cytokine production by Th cells (15, 16), it was important to analyze the cytotoxic response induced in the context of a CD4⁺ T cell response triggered by one or the other subset. Using a class I/II H-2^d-restricted epitope to load DC, we show in this study that both DC subsets are able to induce CTL. No difference was observed as compared with the CTL response generated by DC loaded with a class I peptide. However, as expected, CD4⁺ T cell responses were observed after i.v. injection of DC loaded with this class I/II-restricted peptide. These results clearly show that generation of CTL response by peptide-pulsed DC is neither positively nor negatively influenced by the Th responses induced by the DC subsets.

By analyzing the cytokine profiles obtained after i.v. immunization with CD8⁺ or CD8^- DC subsets, we observed that both DC subsets loaded with the p_118–126 (class I/II-restricted) or the p_103–116 (class II-restricted) peptide were able to induce Th1 and Th2 cytokines. CD8^- DC induced high levels of Th1 and Th2 cytokines, whereas CD8⁺ DC induced preferentially Th1 cytokines, except for IL-5, which was efficiently induced by both DC subsets. So, despite minor differences, similar cytokine profiles were obtained after i.v. immunization with DC subsets loaded with either class II- or class I/II-restricted peptides. Interestingly, IL-4 and IL-5 were produced by CD4⁺ T cells, whatever the peptide used for DC loading. In contrast, IFN- was essentially produced by CD8⁺ T cells after stimulation with a class I- or class I/II-restricted peptide, while it was produced by CD4⁺ T cells after immunization with DC loaded with a class II-restricted peptide. IL-2 was produced by both CD4⁺ and CD8⁺ T cells.

These data confirm the results of Pulendran et al. (16), showing that the CD8^- DC subset induced much greater levels of IL-4 and IL-10 production, while no significant difference was observed in IFN- and IL-2 production by T cells primed by either DC subsets. They concluded that CD8^- DC induce preferentially a Th2 response. However, we show in this study that IL-5, a Th2 cytokine, is produced after injection of both CD8^- and CD8⁺ DC. These results are also in contrast with the study of Maldonado-Lopez (15), showing that CD8⁺ DC induce a Th1 response, while CD8^- DC induce a Th2 response. The difference observed between our results and these studies could be due to the site of injection, since they injected DC subsets into footpads and analyzed lymph node T cell responses, whereas we used the i.v. route and studied spleen cell responses. Indeed, when injected into mouse footpads, both DC subsets induced IFN- production, but only the CD8^- DC subset was able to generate a type 2 T cell response both in the spleen and in the popliteal lymph nodes. These results are in agreement with those reported by Pulendran et al. (16). So, the absence of type 2 cytokine production after s.c. immunization with CD8⁺ DC could reflect the influence of the injection route rather than the intrinsic capacity of this subset to prime such responses. As it has previously been established, CD8⁺ DC injected into footpads do not reach lymph nodes. Thus, the T cell response induced under these conditions may not result from direct T cell activation by CD8⁺ DC (32), but rather from cross-priming. In contrast, it was demonstrated that both CD8⁺ and CD8^- DC subsets injected i.v. reached the spleen and can directly stimulate spleen T cells (18).

Unlike Maldonado-Lopez et al. (15), we observed a high IFN- production after injection of CD8^- DC into mouse footpads. This difference could be due to the form of the Ag, as they used a full protein that required processing by DC, while peptides used in our study and in Pulendran’s report (16) do not need to be processed. The differences between our results and this study (15) could also be due to the maturation state of DC used for in vivo immunization. Indeed, Maldonado-Lopez et al. (15) used splenic DC cultured 18 h in the presence of GM-CSF, which could modify the properties of the DC. In our study, the DC used for immunization were similar in terms of maturation to unpurified spleen DC and were able to mature following stimulation, suggesting that these purified DC are physiologically close to splenic DC. However, it is not known whether these DC mature in vivo after injection. Furthermore, these DC were loaded with high concentration of optimal-length peptides that could provide strong signal to specific T cells, even if DC were not fully mature. Indeed, we previously showed that CTL responses induced in vivo by optimal-length peptides do not require CD4⁺ T cell help (26).

So, although CD8⁺ DC induce preferentially Th1 response with a low production of IL-4 and IL-10, presumably due to their capacity to produce high amount of IL-12 (15), we clearly show in this work that both CD8⁺ and CD8^- DC have the potential to prime Th1 and Th2 T cells, at least after i.v. injection. Recently, it was reported that human myeloid DC could alternatively induce Th1 effector/memory cells and Th2 responses/unpolarized central memory, depending on the time point of their activation kinetics (33). Similarly, murine CD8^- DC generated in vitro are able to activate Th0, Th1, or Th2 T cell, depending on cytokines used during their preparation (34). Thus, the Th responses generated by DC could be under control of multiple parameters, such as the nature of DC-activating stimuli, the cytokine microenvironment, the maturation state of the DC, and not essentially by their ontogeny.

In conclusion, in the present study, we show that both DC subsets are able to induce type 1 CTL responses independently of the induction of CD4⁺ Th responses, although the CD8^- DC seem more efficient. While CD4⁺ Th response could be influenced by the DC subset used for immunization, no polarization of Th cells was observed after i.v. immunization, since in both cases Th1 and Th2 cytokines were produced by CD4⁺ T cells. However, the CD4⁺ T cell response could be influenced by the route of immunization. Taken together, these data indicate that both CD8⁺ and CD8^- DC subsets could be efficient in vaccination protocols against infections or tumors.

	Acknowledgments

 
We thank Anne Louise for the sorting of DC subpopulations, and Marika Sarfati, Mohamed El-Azami El-Idrissi, and Hossein Motieian Najar for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by the Agence Nationale de Recherche sur le SIDA.

² Address correspondence and reprint requests to Dr. Gilles Dadaglio, Biologie des Régulations Immunitaires, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France. E-mail address: gdadag{at}pasteur.fr

³ Abbreviations used in this paper: DC, dendritic cell; CM, complete medium; LCMV, lymphocytic choriomeningitis virus.

Received for publication April 5, 2001. Accepted for publication August 9, 2001.

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