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Immunology |
Cancer Immunotherapy and Gene Therapy Program, Istituto Scientifico H San Raffaele, 20132 Milan, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCs that are induced in vitro from CD34+ precursors (5 , 6) can be loaded with the antigen of choice and can be re-infused into the donor’s tissues as potent anticancer vaccines (2) .
Nevertheless, the issue of the state of activation of the DC to be used as vaccine is still under debate (2
, 7)
. Indeed, DCs exposed to maturation stimuli appear to be more potent APCs than immature DCs (2
, 7)
. It is likely however, that later during the development of an immune response, DCs are limited in their APC function to avoid the risks of excessive proliferation of selected lymphocyte clones, and/or development of autoimmune reactions. Kinetics studies have shown that, whereas recently activated DCs express cell surface molecules at high levels and produce relevant amounts of soluble factors, all required for T cell activation, DCs exposed for long periods to promaturation stimuli undergo functional "paralysis" and/or "exhaustion" (8, 9, 10)
. Those functional and phenotypic changes directly affect the type of immune response induced. Langenkamp et al. (10)
, in particular, showed that, after in vitro activation by LPS, as well as by poly(I)-poly(C), and TNF-
plus IL-1ß, DCs produce IL-12, a cytokine directly involved in the generation of CTL responses (11)
, only transiently, with a peak between 5 and 8 h, and complete extinction after 18 h, the time at which they become refractory to further stimulation by CD40L. Similar kinetics has been found, using microarray techniques (12
, 13)
. Even more interestingly, recently activated DCs (8hDC) preferentially induced Th1 responses, whereas exhausted DCs (48hDC) primed Th2 and nonpolarized T cells (10)
.
We investigated whether the kinetics of DC activation also impacted on in vivo priming of antigen-specific CTL responses. Bone-marrow-derived 8hDCs pulsed with TRP-2180–188 (14) , and injected into C57BL/6 mice, elicited a stronger and more efficient melanoma-specific Tc1 response than did untreated DC (0hDC) or 48hDCs. LPS-treated DCs exhibited similar in vitro survival and allostimulatory activity, as well as in vivo migration potential, but 8hDCs showed a significantly higher IL-12 production potential.
Our in vivo findings strongly support the use of recently activated DCs for the generation of potent Tc1 responses and are, therefore, relevant for the design of DC-based vaccines.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCs Preparation and in Vitro Functional Assays.
DCs were prepared as described previously (15)
. Briefly, single-cell suspensions of bone-marrow were seeded into six-well plates at 2 x 106/ml in ISCOVE supplemented with penicillin-streptomycin and 10% FCS, GM-CSF (25 ng/ml), and IL-4 (5 ng/ml; R&D Systems, Minneapolis, MN). Forty-eight, 24, 16, 8, 4, 2, 1 h and 15 min before retrieval of cells, LPS (1 µg/ml) or TNF- (50 ng/ml) was added to the culture medium. Alternatively, DCs were cocultured with mitomycin-C-treated CD40L-expressing J558 cells (kindly provided by P. Lane, Department of Immunology, University of Birmingham, Birmingham, GB) at a ratio 1:4 (10)
. On day 7 of the in vitro culture, nonadherent and loosely adherent cells were collected and were evaluated by reverse transcription-PCR for Mycoplasma contamination (positive cultures were discarded). DCs were also incubated with normal mouse serum for 15 min at 4°C, and double staining with the PE-conjugated anti-CD11c mAb and one of the following FITC-conjugated mAbs: CD3, CD4, CD8, CD19, CD40, CD80, CD86, NK/2B4, Kb, Db, or I-Ab. Samples were analyzed by a FACScan (Becton Dickinson) using propidium iodide to exclude dead cells. To measure cytokine-producing capacity, DCs were treated with Brefeldin A (10 µg/ml; 5 h at 37°C), labeled with biotin-conjugated anti-CD11c mAb followed by Cy-Chrome-streptavidin, fixed in 4% paraformaldehyde, and permeabilized with 0.1% saponin-1% FCS-PBS, incubated with PE-conjugated mAbs specific for IL-2, IL-4, or IL12p70 (clone C15.6), and FITC-conjugated IL-10 or IFN-
for 20 min at room temperature and were analyzed by FACScan. Allostimulatory activity of DCs was assessed by 2- and 3-day primary MLRs. DCs were seeded in graded doses, together with 5 x 105 splenocytes from naïve BALB/c mice, into 96-well culture plates. Culture was performed in RPMI 1640 containing 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin (tissue culture medium). T-cell proliferation was measured by adding [3H]thymidine (1 µCi/well) to the culture and by subsequent liquid scintillation counting after an overnight incubation period. To evaluate DC survival, 106 of the different DC populations, pulsed or not with the synthetic peptide OVA 257–264 (SIINFEKL; Ref. 16
; Research Genetics, Huntsville, AL), were replated with 2.5 x 106 OT-1 cells into 24-well plates. Approximately 24 h later, cells were stained with anti-CD86-FITC or anti-CD8-FITC, and biotin-conjugated anti-CD11c and Cy-chrome-streptavidin (anti-CD11c-Cy) for 15 min at 4°C. Further incubation at 37°C for 1 h was performed in the presence of CaspaTag red caspase (Intergen, Purchase, NY) according to the manufacturer’s indications. This reagent irreversibly binds to active caspases and is, therefore, useful for detecting apoptosis induction in target cells by FACScan analysis.
DC Migration Assay.
DCs were suspended in ISCOVE at 2 x 106/ml and were labeled with 2 µM fluorescent dye CMFDA (Molecular Probes, Leiden, the Netherlands) for 30 min at 37°C. After washing, DCs were incubated in ISCOVE-10% FCS for 30 min at 37°C. Cells (1 x 106) were injected into the footpad of naïve mice. Twenty-four h later, popliteal lymph nodes were collected, fragmented, and digested twice in 5 ml RPMI containing collagenase (1 mg/ml) and EDTA [500 µl of 0.1 M EDTA (pH 7.2)]. Cells were then labeled with anti-CD11c-PE and analyzed by FACScan.
Immunization Protocols.
DCs were pulsed with 2 µM TRP-2180–188 (VYDFFVWLH; Ref. 24
), OVA257–264 (Research Genetics) or gp70423–431 (SPSYVYHQF; Ref. 17
; a kind gift of A. Rosato, Padua, Dipartmento di Scienze Oncologiche e Chirurgiche, University of Padua, Italy) peptides for 1 h at 37°C, were washed, and were resuspended in PBS. Mice received one to three vaccinations (2 x 105 DCs/boost) every week. In parallel, groups of mice were given injections of unpulsed DCs 100 µl PBS or 10 µg of TRP-2 peptide in 100 µl PBS. To deplete of T cell subpopulations, mice received i.p. 300 µg of purified rat IgG (Sigma), or anti-CD4 (GK1.5 hybridoma), or anti-CD8 (53.6.72 hybridoma) mAbs 1 day before in vivo cytotoxicity assays. FACScan analysis of cells from lymphoid organs of mice receiving depleting mAbs documented a reduction of >95% of the target cell population at the time that mice were killed. For protective experiments, mice were challenged s.c. by 5 x 104 B16 cells 1 week after the last boost. Tumor size was evaluated by measuring two perpendicular diameters by a caliper. Animals were scored positive when the mean tumor diameter was >2 mm. Mice with no visible or palpable tumor 40 days after tumor challenge were scored negative. Animals were killed when the mean tumor diameter was 
10 mm. For curative experiments, naive mice were given injections s.c. with 5 x 104 B16 cells. Five days later, mice were randomly assigned to one of the following treatments: three weekly injections of unpulsed DCs (2 x 105/injection); TRP-2-pulsed DCs (2 x 105/injection), or PBS. Animals were followed thereafter as described above.
In Vivo Cytotoxicity Assay.
Naïve splenocytes were resuspended in PBS at a concentration of 107/ml and were labeled with two different concentrations (1.25 and 0.125 µM) of 5- and 6-CFSE (Molecular Probes) according to the manufacturer’s indications. Cells labeled with 1.25 µM CFSE were also pulsed with TRP-2180–188 or gp70423–431 (5 µM) for 1 h at room temperature. Both cell populations were washed, mixed (5 x 106/each), and resuspended in PBS before i.v. injection. Eighteen h later, spleens and lymph nodes were removed and processed individually. Single-cell suspensions were analyzed by FACScan, after having added propidium iodide to exclude dead cells. The specific cytolytic activity was calculated as (percentage CFSEhigh cells x 100)/percentage CFSElow cells. In each experiment, at least one naïve mouse was used to verify the survival of CFSE-labeled splenocytes 24 h after adoptive transfer. In those animals, the cytolytic activity was always below 10%.
In Vitro Cytotoxicity Assay.
Splenocytes, obtained as described above from vaccinated animals, were resuspended at 3 x 106/ml in tissue culture medium and were cultured in a T25 flask with 0.5 µM TRP-2 peptide. After 4 days, blasts were isolated on a lympholyte-M gradient (Cedarlane, Hornby, Ontario, Canada), cultured for an additional day in medium supplemented with 20 IU/ml human recombinant IL-2. At day 5, blasts were tested for cytolytic activity in a standard 4-h 51Cr release assay as described previously (15)
. Target cells were labeled with 51Cr, and left unpulsed or pulsed with 1 µM TRP-2180–188 or gp70423–431 for 30 min in ice-cold culture medium. After extensive washing, target cells (1000 target/well) were seeded with blasts at E:T ratios ranging from 6:1 to 50:1. The percentage of specific 51Cr release of triplicates was calculated as follows:
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In Vitro and in Vivo Polarization of Transgenic OT-1 Cells.
Lymph node cells from naïve OT-1 mice were cultured at 10:1 ratio with OVA257–264-pulsed DCs with or without exogenous IL-12 (3 ng/ml; R&D Systems). Seven days later, cytokine production by effector cells was assessed by FACScan after stimulation for 4 h at 37°C with PMA (10-7 M) and ionomycin (1 µg/ml). Brefeldin A (10 µg/ml) was added during the last 2.5 h of stimulation. Cells were fixed with 2% paraformaldehyde, permeabilized with PBS containing 0.5% saponin and 5% FCS, followed by staining with anti-CD8-FITC and anti-IFN--PE. In in vivo experiments, magnetic beads-purified (Mylteni Biotec, Bergish Gladbach, Germany) CD8+ cells from naïve OT-1 mice were labeled with CFSE as described above, suspended in PBS, and injected into the tail vein of C57BL/6 mice (15 x 106/animal) 24 h before vaccinating them with OVA257–264-pulsed DCs. Three days later, cells from draining lymph nodes were collected, incubated with antimouse CD8a-Cy (Ly-2), and analyzed by FACScan after having added propidium iodide to exclude dead cells. Cytokine production by effector cells was assessed by FACScan as described above using biotin-conjugated anti-IL4, anti-IL10, and anti-IFN. The incubation with primary mAbs was followed by Cy-chrome-streptavidin. IFN-, IL-4, and IL-10 producing Tc1 and Tc2 cells, induced in vitro by culturing OT-1 cells with DCs and polarizing cytokines5
were used as positive controls in each experiment.
Statistical Analysis.
Statistical analyses were performed using the Student t test and the log-rank test. Comparison of survival curves was considered statistically significant for P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Conversely, only 40% of the mice vaccinated with TRP-2180–188-pulsed 48hDCs survived tumor challenge, a result comparable with the one obtained vaccinating mice with 0hDCs (15)
. A statistically significant difference was found when the survival curves of mice vaccinated with 8hDCs and 48hDCs were compared by the log-rank test (P < 0.024). Vaccination with unpulsed DCs of all of the populations did not elicit relevant protective effects on tumor-challenged mice, when compared with animals that received injections of PBS (Fig. 1A
and not shown).
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. When vaccination started 5 days after melanoma cells injection, 75% of the animals receiving TRP-2181–188-pulsed 8hDCs were still alive when all of the control animals were dead, and showed a remarkably prolonged survival. The difference between the survival curves of mice vaccinated with 8hDCs and 48hDCs was striking (P < 0.002). Indeed, treatment with peptide-pulsed 48hDCs caused only a slight and not statistically significant increase in animal survival when compared with the relative controls. Of note, a statistically significant delay in melanoma growth was found when the survival curves of animals vaccinated with peptide-pulsed 0hDCs were compared with mice vaccinated with unpulsed 0hDCs (P < 0.005; not shown).
To assess the location and function of melanoma-specific CTLs induced by DC vaccination, we adopted an in vivo cytotoxicity assay (19)
. TRP-2180–188-pulsed and unpulsed spleen cells from naïve mice, which were differentially labeled with different concentrations of CFSE, were i.v. injected into vaccinated mice 18 h before lymphoid organ collection and FACScan analysis (i.e., day 7). The highest cytolytic activity, measured by FACScan comparing the peak of CFSEhigh TRP-2180–188-pulsed targets, relative to the internal control of unpulsed CFSElow cells, was found in all of the lymphoid organs examined (i.e., spleen, and draining and nondraining lymph nodes) of 8hDCs-vaccinated mice (Fig. 2A)
. The cytolytic activity measured in mice vaccinated with 48hDCs (Fig. 2B)
and 0hDCs (Fig. 2C)
was comparable. No specific cytotoxic activity was detected in mice that received injections of PBS (Fig. 2D)
, unpulsed DCs (lysis: 7 ± 3.2%; n = 3), or 10 µM peptide in PBS (lysis: 7 ± 3.7%; n = 3). Splenocytes from those animals were also stimulated in vitro with TRP-2180–188 and were tested 5 days later in 51Cr release assays. A good correlation was found between the results of the in vivo and in vitro cytoxicity assays (Fig. 2)
. Indeed, the highest level of TRP-2180–188-specific lysis was found in cultured splenocytes from 8hDCs-vaccinated mice (Fig 2E)
, whose lytic activity, measured by LU-30 was 2- and 3-fold higher than the one found in 48hDCs- and 0hDCs-vaccinated mice (58.1, 30.4, and 20.3 x 106, respectively). Of relevance, lysis against B16, a melanoma endogenously expressing TRP-2 (17)
, was found to be 3–7-fold higher in splenocytes from 8hDCs-injected mice (LU-10: 152 x 106; Fig. 2E
), when compared with splenocytes from mice vaccinated with 48hDCs (LU-10: 55 x 106; Fig. 2F
) and 0hDCs (LU-10: 23 x 106; and Fig. 2G
), respectively. As reported previously (15)
, splenocytes from mice that received PBS injections (Fig. 2H)
, unpulsed DCs, or peptide in PBS (not shown) did not kill any target used.
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The immune response induced in vivo against TRP-2 by different vaccines is predominantly CD4 dependent (e.g., 18 ). To better define the immune response elicited by TRP-2180–188-pulsed 8hDCs, vaccinations were performed in MHC class II-/- mice as well as in mice depleted of CD4+ cells 1 day before the injection of CFSE-labeled cells. No cytolytic activity was detected in the lymphoid organs of vaccinated MHC class II-/- mice (i.e., 5 ± 2.4%; n = 3). Because depletion of CD4+ cells during the effector phase of the immune response generated by 8hDCs did not alter the antigen-specific CTL activity (i.e., 82 ± 6.2%; n = 3), our data confirm that the immune response induced by such vaccine is also CD8 mediated and CD4 dependent.
8hDCs Skew the Antigen-specific CTL Response toward a Tc1 Response.
The above reported data strongly underline the relevance of the kinetics of DC activation in the induction of potent tumor-specific CTL responses and lend weight to the hypothesis that in vivo recently activated DCs also skew the immune response toward a Tc1 response. To evaluate the cytokine production profile of CTLs activated in vivo by 8hDCs, we took advantage of the highly cytotoxic transgenic OT-1 cells, specific for OVA257–264 (20)
. Purified CD8+ cells from OT-1 mice were labeled with CFSE and were injected i.v. into recipient mice 1 day before i.d. injection of peptide-pulsed DCs. When draining lymph node cells were analyzed by FACScan 3 days after vaccine injection, the highest percentage of proliferating OT-1 cells was found in mice vaccinated with 8hDCs (>85%; Fig. 3A–C
). Mice vaccinated with 48hDCs (Fig. 3D–F)
or 0hDCs (Fig. 3G–I)
showed a significantly lower percentage of proliferating OT-1 cells (<60 and 50%, respectively). Animals given injections with PBS showed a marginal proliferation (25%; Fig. 3L–N
). Even more strikingly, in mice vaccinated with 8hDCs, almost 50% of the proliferating cells produced IFN- (Fig. 3A)
, the paradigmatic type 1 cytokine (21)
. Conversely, <10% of the CD8+ cells produced IFN- in all of the other groups of vaccinated mice (Fig. 3, D, G, and L)
. We also measured IL-4 and IL-10 production by in vivo activated OT-1 cells. Irrespective of the experimental condition examined, we did not find significant and consistent production of IL-4 (Fig. 3B, E, H, and M)
and IL-10 (Fig. 3C, F, I, and N)
. These data, therefore, strongly suggest that on vaccination with recently activated DCs, a potent Tc1 immune response is generated.
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. Of note, the latter showed a heterogeneous expression of the indicated markers, suggesting that 0hDCs were a mixed population of immature and matured DCs. However, 8hDCs expressed similar if not lower levels of all of the indicated markers when compared with 48hDCs, therefore excluding the possibility that the higher immunogenic potential of 8hDCs versus 48hDCs depends on the amount of MHC and costimulatory molecules expressed by the two DC populations.
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and not shown). As expected, 0hDCs stimulated an alloreactive response only at the highest DC:effector ratios.
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Once migrated to the lymphoid organs, DCs may be the target of antigen-specific CTLs (22) . Therefore, we analyzed whether our DC populations were differently susceptible to CTL-mediated activation of caspases, one of the most proximal events in perforin-dependent apoptosis. Twenty-four h after incubation with naïve OT-1 cells in the presence or absence of OVA257–264, cells were stained with CD11c-Cy, CD86-FITC mAbs, and Caspatag red caspase, and were analyzed by FACScan. In all of the DC populations tested, the presence of OVA257–264 did not induce a substantial increase in caspase activation over the background (i.e., absence of the peptide): 0hDCs, 17.5 ± 2.9 and 20.7 ± 2.2%; 8hDCs, 15.2 ± 2.9 and 22.5 ± 2.8; and 48hDCs, 13.8 ± 1.9 and 18.9 ± 1.8, respectively, therefore, making highly unlikely the hypothesis of an in vivo CTL-mediated selection in favor of the 8hDCs.
The Ability of 8hDCs to Generate Tc1 Cells Depends on Their IL-12 Production.
Because IL-12 is a key cytokine for the induction of type 1 T cells (11)
, we verified whether a difference in IL-12 production could be found between 8hDCs and 48hDCs. IL-12p70 release was assessed by ELISA in the culture supernatants at the end of the period of exposure to LPS (i.e., day 7). Irrespective of the time at which LPS was added to the culture, DCs produced equal amounts of IL-12p70 (8hDC, 172 ± 33 pg/ml; 48hDCs, 187 ± 27 pg/ml; n = 3) that were much higher than those produced by 0hDCs (46 ± 3.5 pg/ml, n = 3; 8hDCs versus 0hDCs, P < 0.015; 48hDCs versus 0hDCs, P < 0.006).
Elisa tests, however, do not define the time at which IL-12 has been produced. Neither do they allow quantification of IL-12-producing DCs, nor take into account the fact that DCs can consume IL-12 produced during the culture. We, therefore, also evaluated the DC pattern of cytokine expression during LPS exposure by intracellular staining followed by FACScan analysis. In eight independent experiments we found that, different from 0hDCs, which only marginally produced IL-12 (1.5 ± 0.8%), 23.7 ± 5.1% of the 8hDCs were positive for IL-12 (Fig. 6, A, and D
; DCs not exposed to LPS versus 8hDCs, P < 0.0001). As expected, a prolonged exposure to LPS dramatically reduced the percentage of IL-12-producing DCs (12.1 ± 1.9; 8hDCs versus 48hDCs, P < 0.0004; Fig. 6
, A, D, and G). Conversely, we did not find any different production of IL-2 and IL-10, nor production of IFN- and IL-4 by the DC populations analyzed (not shown). Comparable results were obtained using CD40L as promaturation stimulus (i.e., IL-12+ cells: 0hDCs, 4 ± 2%; 8hDCs, 16.1 ± 1.5%; 48hDCs, 3.2 ± 1%; n = 3). Conversely, TNF- alone did not induce a level of IL-12 production significantly higher than the one found in 0hDCs (not shown).
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. As expected, none of the DC populations from IL-12-/- mice were not able to produce IL-12 (Fig. 6, C, F, and I)
. When BALB/c mice were vaccinated with the different populations of gp70423–431-pulsed DCs from wild-type and IL-12-/- mice, the highest level of cytotoxicity against peptide-pulsed splenocytes was found in the lymphoid organs of mice vaccinated with 8hDCs from wild-type animals (Fig. 6L)
. Vaccination with 8hDCs from IL-12-/- mice strongly reduced the induction of antigen-specific CTL (wild-type versus IL-12-/- 8hDCs, P < 0.007), therefore, suggesting that IL-12 was a key factor for the induction of antigen-specific Tc1 cells by 8hDCs. That finding was further sustained by in vitro experiments of T-cell polarization (Fig. 6M)
. Indeed, when naïve OT-1 cells were cultured with OVA257–264-pulsed DCs in the absence of exogenous IL-12, 8hDCs allowed induction of a much higher percentage of IFN--producing cells than did 48hDCs and 0hDCs (43 ± 2.8, 4.9 ± 1.2, and 2.1 ± 0.4, respectively). Conversely, all DC populations were comparably able to polarize toward Tc1 cells when exogenous IL-12 was added to the culture medium. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The kinetics of IL-12 production by 8hDCs allows DCs to reach lymphoid tissues at a time in which that cytokine is maximally produced (3) . Here, 8hDCs should rapidly induce CD4+ T cell activation (3) , possibly Th1 polarization (24) , and even faster priming of potent CTLs (25 , 26) . That narrow window of time should avoid the risk of overactivation of the immune response and/or the activation of bystander, and possibly autoreactive, T cells. Indeed, 48hDCs, although equally efficient in migrating to the draining lymph nodes, have lost most of their potential to induce powerful type 1 responses.
Th1 cells can be induced by different DC populations and/or in the presence of different promaturation stimuli (27) . Indeed, in the presence of strong viral stimuli, or low DC:T-cell ratios, even DC2 cells can promote Th1 cell induction (27) . The conditions favoring the induction of Tc1 cells are less characterized. We favor the hypothesis that, irrespective of the DC population involved, strong promaturation stimuli (e.g., LPS, bacterial CpG DNA, and double-stranded viral DNA) allow rapid and intense production of IL-12 as well as other Th1-prone factors. At later time points during the DC life span, exhaustion, probably in the presence of reduced amounts of antigen and low DC:T-cell ratios, may favor induction of Tc2 and nonpolarized T cells.
Langenkamp et al. (10) showed that "exhausted" DCs were more prone to prime Th2 and nonpolarized T cells. In in vitro experiments, we also found that 48hDCs preferentially induced nonpolarized CD8+ T cells. Furthermore, exhausted in the presence of IL-4 were the best inducers of Tc2 cells.5 In vivo, 48hDCs were not able to induce a detectable Tc1 immune response. Experiments are ongoing to verify whether Th2 cells are induced under those conditions.
It has been previously described (e.g., in Refs. 15 and 28 ) that DCs that are cultured in vitro in the presence of GM-CSF and IL-4 (i.e., 0hDCs) and pulsed with synthetic peptides are able to generate variable protective and therapeutic effects, depending on the cancer model used. Others (29) have shown that those 0hDCs exerted minimal if any therapeutic activity. Bone-marrow-derived DCs obtained by in vitro cultures in the presence of GM-CSF (and IL-4) usually exhibit different levels of maturation (e.g., in Refs. 5 and 30 ), which often depend on their manipulation. Indeed, it is well known that simple pipetting or transferring of DC cultures may cause their rapid maturation (5 , 30 , 31) . Hence, one explanation for the above reported discrepancies may be the different grade of spontaneous maturation of the DC preparations. Other possible explanations may be the different source of DCs, culture conditions, and vaccination schedule.
The effect of the kinetics of IL-12 production on DC activation was measured at time points earlier than 8 h (i.e., 15 min and 1, 2, and 4 h). We found that the percentage of IL-12+ DCs sharply increased 15 min after adding LPS to the culture, remained at similar levels for the following 4 h, and reached a peak at 8 h. A rapid decline in IL-12+ DCs was found between 24 and 48 h. The kinetic of expression of cell-surface molecules (e.g., IA-b, CD80, and CD86) was slower, with a relevant increase at 8 h or later. Therefore, we suggest that, also for mouse DCs, the more favorable window of time to obtain activated, highly IL-12-producing DCs is between 6 and 8 h after LPS stimulation.
After preclinical studies in mice, the first vaccinations in humans were conducted with DCs not exposed to promaturation stimuli (reviewed in Ref. 2 ). More recently, cancer patients have been immunized with DCs exposed to promaturation stimuli for at least 24 h (32 , 33) . It would be interesting to verify whether recently activated DCs are better cancer vaccines in humans also.
In conclusion, we show that recently activated DCs skew in vivo the antigen-specific immune response toward a Tc1 phenotype. This information is of relevance in designing DC-based vaccines. Indeed, a potent Tc1 immune response is highly desirable in the first phases of a tumor-specific immune response, when powerful CTLs are needed to eliminate all malignant cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Associazione Italiana pez la Ricerca sul Cancro, Ministero della Salute, and Ministero dell’Istruzione dell’Università e della Ricerca. 
2 Present address: Department of Environmental Science ETH-Zentrum, Wagistrasse 25/27, CH-8952 Schlieren, Switzerland. Phone: 41-16336475; E-mail: iezzi{at}biomed.umnw.ethz.ch
3 To whom request for reprints should be addressed, at Laboratory of Tumor Immunology, Cancer Immunotherapy and Gene Therapy Program, 3P-A1, Dibit, Istituto Scientifico H San Raffaele, Via Olgettina 58, 20132, Milan, Italy. Phone: 39-02-2643-4789; Fax: 39-02-2643-4786; E-mail: bellone.matteo{at}hsr.it
4 The abbreviations used are: DC, dendritic cell; LPS, lipopolysaccharide; 8hDC, DCs exposed to LPS for 8 h; 48hDC, DC exposed to LPS for 48 h; APC, antigen-presenting cell; TNF-, tumor necrosis factor(s) ; TRP-2, tyrosinase-related protein(s) 2; Tc1, CTL(s) of type 1; LU, lytic unit(s); MLR, mixed leukocyte reaction; IL, interleukin; mAb, monoclonal antibody; GM-CSF, granulocyte macrophage colony-stimulating factor; ISCOVE, Iscove’s modified Dulbecco’s medium; CMFDA, 5-chlormethylfluorescein (dye); CFSE, carboxy-fluorescein succinimidyl ester; 0hDC, DC exposed to LPS for 0 h; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PMA, phorbol 12-myristate 13-acetate.
5 G. Iezzi, A. Boni, A. Camporeale, M. Grioni, E. Degl’Innocenti, and M. Bellone. Fully activated dendritic cells and IL-4 from the instruction of regulatory CTLs, manuscript in preparation.
Received 12/16/02. Accepted 5/ 5/03.
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F. Benigni, V. S. Zimmermann, S. Hugues, S. Caserta, V. Basso, L. Rivino, E. Ingulli, L. Malherbe, N. Glaichenhaus, and A. Mondino Phenotype and Homing of CD4 Tumor-Specific T Cells Is Modulated by Tumor Bulk J. Immunol., July 15, 2005; 175(2): 739 - 748. [Abstract] [Full Text] [PDF]
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S. Herter, P. Osterloh, N. Hilf, G. Rechtsteiner, J. Hohfeld, H.-G. Rammensee, and H. Schild Dendritic Cell Aggresome-Like-Induced Structure Formation and Delayed Antigen Presentation Coincide in Influenza Virus-Infected Dendritic Cells J. Immunol., July 15, 2005; 175(2): 891 - 898. [Abstract] [Full Text] [PDF]
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S. Muraro, A. Bondanza, M. Bellone, P. D. Greenberg, and C. Bonini Molecular modification of idiotypes from B-cell lymphomas for expression in mature dendritic cells as a strategy to induce tumor-reactive CD4+ and CD8+ T-cell responses Blood, May 1, 2005; 105(9): 3596 - 3604. [Abstract] [Full Text] [PDF]
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A. Casati, V. S. Zimmermann, F. Benigni, M. T. S. Bertilaccio, M. Bellone, and A. Mondino The Immunogenicity of Dendritic Cell-Based Vaccines Is Not Hampered by Doxorubicin and Melphalan Administration J. Immunol., March 15, 2005; 174(6): 3317 - 3325. [Abstract] [Full Text] [PDF]
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S. Falcone, C. Perrotta, C. De Palma, A. Pisconti, C. Sciorati, A. Capobianco, P. Rovere-Querini, A. A. Manfredi, and E. Clementi Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3',5'-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells J. Immunol., October 1, 2004; 173(7): 4452 - 4463. [Abstract] [Full Text] [PDF]
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C. Perrotta, S. Falcone, A. Capobianco, A. Camporeale, C. Sciorati, C. D. Palma, A. Pisconti, P. Rovere-Querini, M. Bellone, A. A. Manfredi, et al. Nitric Oxide Confers Therapeutic Activity to Dendritic Cells in a Mouse Model of Melanoma Cancer Res., June 1, 2004; 64(11): 3767 - 3771. [Abstract] [Full Text] [PDF]
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R. J. A. Fijneman, M. Vos, J. Berkhof, P. Demant, and G. Kraal Genetic Analysis of Macrophage Characteristics as a Tool to Identify Tumor Susceptibility Genes: Mapping of Three Macrophage-Associated Risk Inflammatory Factors, Marif1, Marif2, and Marif3 Cancer Res., May 15, 2004; 64(10): 3458 - 3464. [Abstract] [Full Text] [PDF]
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M. Bellone, A. Camporeale, and A. Boni Dendritic Cell Activation Kinetics and Cancer Immunotherapy J. Immunol., March 1, 2004; 172(5): 2727 - 2728. [Full Text] [PDF]
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; n = 10); 8hDCs or 48hDCs 
and not shown, respectively; 5 animals/group) unpulsed (w/o) or pulsed (10 animals/group) with TRP-2180–188 (TRP-2; 
and 
, respectively). One week after the last boost, mice were challenged s.c. by 5 x 104 B16 cells. Values are expressed as percentage of surviving animals at a given day. Statistical comparison, conducted by the log-rank test, of the survival curves gave the following results: 8hDCs+TRP-2 versus 8hDCs w/o, P < 0.0006; 8hDCs+TRP-2 versus 48hDCs+TRP-2, P < 0.024; 8hDCs+TRP-2 versus PBS, P < 0.0001; 48hDCs+TRP-2 versus 8hDCs w/o, P < 0.017; 48hDCs+TRP-2 versus 48hDCs w/o, P < 0.012; and 48hDCs+TRP-2 versus PBS, P < 0.005. In all other conditions, the comparison was not statistically significant. B, in curative experiments, mice were given s.c. injections of 5 x 104 B16 cells. Five days later, mice were randomly assigned to one of the following treatments: three weekly i.d. injections of PBS (
