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Blood, 1 September 2005, Vol. 106, No. 5, pp. 1734-1741. Prepublished online as a Blood First Edition Paper on May 17, 2005; DOI 10.1182/blood-2004-10-3991.
IMMUNOBIOLOGY Efficient migration of dendritic cells toward lymph node chemokines and induction of TH1 responses require maturation stimulus and apoptotic cell interactionFrom the Centre Eugene Marquis, Département de Biologie, Rennes I University; Departement de Dermatologie, Faculte de Rennes; EFS Bretagne, Rennes; Généthon/Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 8115, Evry, France.
Dendritic cells (DCs) have the unique ability to initiate primary immune responses, and they can be conditioned for vaccinal purposes to present antigens after the engulfment of apoptotic cells. To recruit the rare antigen-specific naive T cells, DCs require a maturation step and subsequent transport toward lymph node (LN). To date, prostaglandin E2 (PGE2) is the best-characterized compound inducing this LN-directed migration in vitro, but PGE2 may skew the immune responses in a TH2 direction. We demonstrate here that on incubation with apoptotic tumor cells and tumor necrosis factor-  (TNF-) or lipopolysaccharide (LPS), human monocyte-derived DCs become fully mature and acquire high migratory capacities toward LN-directing chemokines. The migration of TNF--treated DCs occurs only after cotreatment with apoptotic cells but not with necrotic cells. DC migration requires CD36 expression and incubation with apoptotic cells in the presence of heat-labile serum components. Moreover, on treatment with apoptotic cells and LPS, the migrating DCs are able to recruit naive T cells to generate TH1 immune responses. Our results show that the cotreatment of DCs with apoptotic tumor cells and inflammatory signals is promising for the design of an antitumoral DC-based vaccine. (Blood. 2005;106:1734-1741)
Myeloid dendritic cells (DCs) have the unique ability to activate naive T cells and, as such, can be tailored to initiate an immune response against neoantigens.1 Once loaded with antigens in peripheral tissues, DCs must migrate to secondary lymphoid organs and reach a fully mature functional stage to recruit rare naive T cells.2 In the past decade, in vitro-generated DCs have been involved as cell adjuvants in multiple tumor vaccination trials to trigger antitumor immune responses and tumor regression.3 For tumor vaccination purposes, autologous DCs are generated either from CD34+ progenitors4 or from peripheral blood monocytes differentiated in the presence of granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4)5 or IL-13.6,7 DCs are then loaded with tumor antigens, matured, and reinjected into patients. Different protocols have been proposed to load DCs with tumor antigens; the most common forms of antigen are preprocessed peptides, tumor cell lysates, whole apoptotic cells, and necrotic tumor cells (for a review, see Schuler et al8). Numerous in vivo and in vitro assays have been used to compare the capacities of DCs loaded with the different forms of antigen to activate naive T cells.9-14 Antigen loading using whole apoptotic or necrotic tumoral cells and cell lysates allows the presentation of an entire set of tumor antigens by major histocompatibility complex (MHC) class I and class II molecules,15-17 which seems advantageous for recruiting large CD4 and CD8 T-cell repertoires.18 Moreover, the activation of CD4 T cells appears essential because it has been shown that CD8 activation without CD4 help may tolerize CD8 T cells19 and preclude the establishment of CD8 memory T cells.20 Indeed, the CD40/CD40L signalization pathway, probably engaged by CD4 cells, is needed to induce full DC maturation and effective activation of CD8 T cells.21 Immature DCs can internalize apoptotic bodies through multiple mechanisms, among which the recognition of altered self by CD36,22 integrins v 3 and v5,23 phosphatidylserine receptor, and the molecular complex iC3b/C1q/Calreticulin/CD9124 play crucial roles (for a review, see Savill et al25). These various receptors can mediate intracellular signaling events, thereby modifying DC responses to inflammation stimuli and influencing their migration properties24 and their antigen-presentation capacity.26
It is generally accepted that reinjected DCs loaded with tumor antigens must have the capacity to migrate to lymph nodes (LNs), encounter naive T cells, and activate them toward a TH1 phenotype8,18 to induce efficient antitumoral cytotoxic responses. This dual ability of DCs to migrate toward secondary lymphoid organs and to orient the immune response is, in our opinion, an important criterion for the design of DC-based vaccines. DC maturation agents such as tumor necrosis factor-
We report here that the preincubation of immature DCs with ApoC in the presence of TNF-
Reagents and antibodies
Culture media included RPMI (Invitrogen, Cergy Pontoise, France) supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS) for cell lines and AIMV (Invitrogen) supplemented with 500 U/mL GM-CSF and 50 ng/mL IL-13 for the DC differentiation culture, referred to as the DC medium. DC medium was supplemented with 10% autologous serum for migration experiments or 10% human AB serum for mixed-leukocyte reaction (MLR) experiments. GM-CSF (Leucomax) was obtained from Schering-Plough (Levallois-Perret, France), IL-13 was obtained from Sanofi-Synthélabo (Paris, France), TNF- Cell cultures Jurkat, a T-cell line isolated from a patient with acute myeloid leukemia (AML), was kindly donated by L. Amiot (Laboratoire d'Hématologie, Rennes, France). M44, a melanoma-derived cell line, was a kind gift from F. Jotereau (INSERM U463, Nantes, France). U251, a glioblastoma cell line, was a kind gift from V. Catros-Quemener (UPRESA CNRS 6027, Rennes, France). Buffy coats and autologous sera were collected from EFS Bretagne (Rennes, France). Immature monocyte-derived DCs were routinely generated from the CD14+-selected fraction of peripheral blood mononuclear cells (PBMCs). PBMCs isolated using the Ficoll-Methrizoate technique were purified with anti-CD14 magnetic beads, according to the manufacturer's instructions (Miltenyi Biotec, Paris, France). Alternatively, monocytes were purified from PBMCs by negative selection using anti-CD2, anti-CD3, anti-CD19, anti-CD56, anti-CD66b, and anti-glycophorin A magnetic beads (StemCell Technologies, Meylan, France). In both cases, pure monocytes (routinely more than 80% CD14/CD11c/HLA-DR triple-positive cells) were plated in DC differentiation medium at 1 x 106 cells/mL in culture bags (Stedim, Aubagne, France). Half the media were replaced on day 2 or 3, and immature DCs were harvested on day 6. Apoptosis induction of cell lines and interaction with DCs Various apoptotic stimuli have been tested on Jurkat, M44, and U251 cell lines. A 40-J/cm2 or an 80-J/cm2 ultraviolet B (UVB) stimulus was chosen for Jurkat and M44/U251 cells, respectively, rather than serum deprivation or Fas-mediated cell death. In our hands, serum deprivation induced low levels of cell death. Jurkat cells were sensitive to Fas-induced cell death, but M44 and U251 cells were not. Moreover, anti-Fas antibody (clone CH11) would have added bias to the internalization process of apoptotic bodies because of the presence of Fc-receptors on immature DCs.33 UVB irradiation had 2 main advantages: it did not add chemicals or antibodies to the culture medium, and it induced early, massive apoptosis of the cells tested (data not shown).
In some experiments, Jurkat cells were stained by incubation for 30 minutes, at 37°C, with 5 µM CMTMR (Molecular Probes). Stained or unstained Jurkat cells were plated at 1 x 106 cells/mL in AIMV in 6-well plates and were induced to apoptosis by UVB irradiation (40 J/cm2) using a UV solar simulator (Müller Elektronik, Salzkotten, Germany). Under these conditions, more than 60% of the cells were Annexin-V positive 2 hours after irradiation; 24 hours later, 100% of the cells fixed 7AAD. Irradiated Jurkat cells were then washed, resuspended in AIMV medium, and added to immature DCs to obtain the indicated DC/ApoC ratio. Immature DCs, supplemented or not supplemented with ApoC, were plated at 5 x 105 cells/mL in DC medium plus 10% complete autologous serum. When indicated, serum was heat inactivated for 30 minutes at 56°C. ApoCs and DCs were then allowed to interact for 4 hours before the addition of 250 U/mL TNF- DC phenotyping For each staining, 100 000 DCs were incubated for 30 minutes with fluorescence-labeled antibodies, in the presence of phosphate-buffered saline (PBS) and 10% AB serum, to block unspecific staining. For CCR7 detection, because of the weak expression of this antigen, we used a staining process consisting of 3 steps—unstained anti-CCR7 or isotype control antibody, anti-mouse biotinylated antibody, and PE-coupled avidin. Stained cells were analyzed using FACScan (Becton Dickinson). In some experiments, stained cells (DAPI cells, CMTMR-stained ApoCs, and CD11c-APCs) were deposed on slides by centrifugation, fixed in 2% paraformaldehyde, and mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL), for analysis on a Leica TCS SP2 confocal microscope (Leica, Mannheim, Germany), using oil immersion 40 x 1.25 NA and 100 x 1.4 NA objectives and Leica confocal software. Images were processed using Adobe Photoshop 7.0.1 (Adobe Systems, San Jose, CA). Characterization of the migratory properties of DCs DCs were suspended in AIMV media plus 10% AB serum at 1 x 106 DC/mL. To assess the migratory capacities of DCs, we used a 12 well-Transwell microplate (Corning, Amboise, France) with a 5-µm membrane pore size that forbade the passive diffusion, but allowed the active migration, of DCs. For each condition tested, lower chambers of the Transwell (Corning) were filled with 600 µL AIMV plus nothing or plus 300 ng/mL CCL19 or 250 ng/mL CCL21. DCs (1 x 105) were deposited in the upper chamber of the Transwell (Corning) and were allowed to migrate for 3 hours at 37°C. Migrating DCs were harvested from the lower chamber and were counted for 60 seconds using FACS.29 In some experiments, enumerations were performed twice to assess the reliability of the method. We never found more than 10% variation between 2 counts. Dosage of PGE2 After 2 days of incubation, DCs were spun down, and the supernatants were kept at -80°C. The PGE2 content of supernatants was measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit (R&D, Lille, France) according to the manufacturer's instructions. MLR Samples of total DCs collected before deposit in the upper chamber or migrating DCs collected in the lower chamber were plated in 96-well microplates. Because of the low number of migrating DCs, only one APC/T-cell ratio was feasible. After preliminary experiments, half-maximal T-cell stimulation was reproducibly obtained at a 1:40 APC/T-cell ratio. We thus added 100 000 allogeneic CFSE-stained CD3+ T cells from CD14- fractions to 2500 DCs. APC/T-cell mixtures were cultured in AIMV medium plus 10% human AB serum for 3 to 4 days before CD3-PC5 staining and FACS analysis. The decrease in the level of CFSE staining was used to assess T-cell division in response to DCs.
Determination of the percentage of IFN-
Total DCs or migrating DCs were counted, and 2500 DCs were plated in each well of 96-well microplates. One hundred thousand unstained allogeneic CD4+/CD45RA+ T cells, purified using the naive human stem cell CD4+ T-cell enrichment kit (greater than 95% CD4+/CD45RA+ cells), were added to the DCs and incubated in X-VIVO medium (Cambrex, Verviers, Belgium) for 6 days. After expansion in the presence of IL-2 (50 U/mL) for another 6 days, cells were stimulated with 50 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) for 4 hours. Golgi-Stop (Becton Dickinson) was added for the last 2 hours. Cells were then intracellularly stained for IFN-
DC migration after ApoC interaction and TNF- -induced maturation
In preliminary experiments, we found that maximal DC migration toward CCL21 was readily achieved after 48-hour incubation with the reference PGE2/TNF-
DCs treated with ApoC alone or with TNF-
Next we sought to determine whether the action of ApoC on DC migration was attributed to early ApoC or to late apoptotic/necrotic cells. We used 30-minute incubation at 55°C as a necrosis-inducing stimulus.34 Necrotic cells had no effect on DC migration under these conditions, in contrast to early ApoC (Figure 2A). Moreover, the induction of DC migration to LN chemoattractants by ApoC was not limited to Jurkat cells because apoptotic melanoma cell line M44 (Figure 2B) and glioblastoma cell line U251 (data not shown) induced an even higher level of DC migration than apoptotic Jurkat cells.
Finally, we sought to determine whether the effect of ApoC on DC migration was restricted to DCs that matured through TNF- by considering the role of LPS as an independent maturation stimulus acting through the TLR4 pathways. We found that LPS increased ApoC-induced migration in a manner equivalent to that for TNF- (Figure 2C), indicating that different activator pathways have a similar effect on the acquisition of migratory properties in ApoC-pulsed DCs.
Synergy between ApoC interaction, TNF-
To evaluate the possibility that ApoC-DC interaction triggers PGE2 release, either by DCs in a paracrine manner or by the cells undergoing apoptosis, we measured PGE2 content in ApoC-DC cultures and analyzed the synergy between ApoC, TNF-
To relate these functional results with DC phenotype, we measured the expression level of CCR7 on DCs under the same experimental conditions and found that TNF-
Mechanisms of DC/ApoC interactions
To further analyze the implications of molecular components involved during the tethering of ApoC on immature DCs, we assessed the roles of (1) the C1q/calreticulin/CD91 complex, which recognizes iC3b coated on the ApoC surface,24 by inactivating iC3b, which is the thermosensitive component of the complement, (2) the integrin DCs incubated with heat-inactivated autologous serum (56°C for 30 minutes) showed a 50% down-regulation in migration toward LN chemokines (Figure 3C). Anti-CD36 antibodies were used only with heat-inactivated serum to avoid complement-mediated lysis of DCs. This latter treatment again decreased migration by 40% compared with migration in heat-inactivated serum alone. The association of heat-inactivated serum and anti-CD36 resulted in more than 70% inhibition of the ApoC-mediated migration. Neither heat-inactivated serum nor anti-CD36 antibodies had an effect on PGE2-mediated migration alone. Finally, anti-CD51 antibodies had no effect (Figure 3C) on the ApoC-mediated migration of DCs. Similar results were obtained using the CCL19 chemokine as a chemoattractant (data not shown). Thus, the increase in migration capacities of DCs on ApoC challenge is partly mediated through a thermosensitive component of the serum, likely iC3b, and the CD36 receptor.
Next, to assess the effective engulfment of apoptotic cells by DCs, we incubated DCs with CMTMR-stained apoptotic cells at a 1:4 ratio and monitored by confocal microscopy the appearance of DCs containing CMTMR+ apoptotic debris in total and migratory fractions (Figure 4A). The total DC fraction contained internalized and noninternalized apoptotic cells, whereas the fraction of migrating DCs appeared devoid of free apoptotic cells. Moreover, total DCs and migrating DCs contained vesicles loaded with CMTMR+ apoptotic fragments (Figure 4A). We then assessed that we could detect these phagocytic events using FACS analysis (Figure 4B) and monitored the percentage of apoptotic cell-loaded DCs before and after DC migration toward CCL21 to determine whether ApoC-pulsed DCs would be enriched in the migrating fraction. Surprisingly, we observed no such enrichment after DC migration, in contrast to the total DC fraction before migration (Figure 4C), providing an indication that ApoC internalization is not mandatory for DC migration.
Phenotype and antigen presentation functions of migrating DCs
It has been reported that ApoC could inhibit DC maturation35 and that immature CD83- DCs expressing the Langerin marker, likely derived from epidermal Langerhans cells, were present in LNs draining chronically inflammatory skin lesions.36 These reports raised the possibility that the migrating DCs we obtained after ApoC and TNF- Polarization of naive T cells toward a TH1 response with migrating DCs
Ample evidence indicates that naive T cells must differentiate toward a TH1 phenotype to mount an efficient antitumoral response,18 leading us to test here the induction of TH1 responses with migrating DCs. DCs were matured for 48 hours in the presence of TNF-
DCs are the only cell type able to activate naive T cells, which serves as a rationale for several DC-based antitumoral therapeutic vaccines. Although the effect of the different antigen-loading protocols on the antigen-presentation capacities of DCs have been extensively documented,9-14 the effects of such treatments on this migratory property have received less attention.
In this work, we investigated the role of ApoC interaction on the migratory capacities of DCs toward LN-secreted chemokines. Because the thermosensitive iC3b component of the complement appears to be an important factor in ApoC engulfment38 and can trigger weak CCR7 expression on DCs,24 migration experiments were conducted in autologous complete serum. Under these conditions, exposure to ApoC alone, in the absence of the maturation signal, resulted in only a slight increase of CCR7 expression and promoted the migration of a small fraction of mature DCs (data not shown) in response to LN-directing chemokines. Similarly, TNF- The action of ApoC on DC migration was not correlated here with ApoC internalization, in agreement with earlier studies on ApoC interaction with macrophages.34,39 Our results also highlight the fact that CCR7 expression is not the sole determinant of DC migration. As a likely explanation of our results, ApoC interaction might promote the acquisition of highly active migratory functions in response to LN chemokines through a modification of CCR7 signaling, as reported with PGE2.30,40 It is generally accepted that ApoC has a null or an inhibitory effect on DC maturation, in contrast to necrotic cells.35 In our experimental settings, the response of DCs to maturation stimuli was not affected by the presence of ApoC, indicating that ApoC-loaded DCs were still susceptible to maturation stimuli, as previously reported.17,41-45 It can thus be presumed that DCs that encounter danger signals can overcome the suppressive effects of ApoC and can trigger an immune response against tumor antigens in the ApoC preparation.25 A positive influence of ApoC interaction on DC migration was recently reported by Ip and Lau.32 These authors showed that interaction with ApoC alone can induce DC migration toward CCL19; 5% of the DCs acquired migratory capacities, in agreement with our own data. However, we observed under these conditions an enrichment of mature DCs in the migrating fraction (data not shown), leading us to conclude that ApoC interaction allows the migration of mature DCs. Our results further demonstrate that ApoC interaction acts in synergy with inflammation stimuli and licenses monocyte-derived DCs for efficient migration toward LN-directing chemokines. Migrating DCs reach 18% of total under these conditions. In addition to the findings of Ip and Lau,32 we show here that DCs require the conjunction of ApoC interaction and maturation signaling to migrate and activate naive T cells.
Given that incompletely matured Langerhans cells have been observed in skin lesion-draining LNs in humans,36 and to ascertain that our migrating DCs are not the "semimature" tolerogenic DCs postulated by Lutz and Schuler,46 we assessed the capacities of our migrating DCs to activate naive T cells toward a TH1 phenotype. PGE2 has been shown to skew DCs to induce a TH2 type of response.31 Previous reports have addressed the role of PGE2 on DC migration and antigen presentation in bulk DC fractions.29,30 Because migrating DCs amount to 20% of total, it appeared essential to analyze phenotypic and functional maturation in these LN-migrating cells, and we assayed here the antigen-presentation properties to naive T cells using the fraction of DCs that migrated toward CCL21. We found that ApoC- and LPS-treated migrating DCs favor the induction of a TH1 response, in contrast to DCs treated with PGE2. Altogether we show here that in addition to triggering migration toward LN chemokines, ApoC interaction can induce a TH1 response in conjunction with DCs matured with strong TH1 stimuli such as LPS. In a recent antitumor vaccine trial reported by Schuler-Thurner et al,11 DCs loaded with a set of HLA class 1- and HLA class 2-restricted peptides and matured with a cytokine cocktail containing TNF- The actual knowledge of DC functions is continuously being translated into clinical applications. We believe that refinements in antigen-loading procedures may have profound consequences for the success of cancer immunotherapy trials in which DCs are used as natural vaccine adjuvants (for a review, see Schuler et al8). In antitumor vaccination protocols, various methods, such as antigenic peptides,11 tumor lysates,14 and whole necrotic or apoptotic tumor cells, have been used for antigen loading of DCs.13 All these methods have been evaluated for their capacity to allow the presentation of tumor antigens through the HLA class I pathway. We optimized the protocols by taking into account two other decisive parameters, the number of DCs that can actually reach the LNs and the capacity of these selected migratory DCs to induce a TH1 response. Our results suggest that, according to these two parameters, loading DCs with apoptotic tumor cells fulfills the most important functional criteria required to elicit efficient cytotoxic responses against tumor antigens.
We thank Stephane Robert and Ellen Etesse for technical assistance.
Submitted October 15, 2004; accepted May 9, 2005.
Prepublished online as Blood First Edition Paper, May 17, 2005; DOI 10.1182/blood-2004-10-3991.
Supported by the Association Cantonale et Intercommunale de Lutte contre le Cancer (ACIC) Baud, la Ligue Contre le Cancer, and Fondation pour la Recherche Medicale (FRM).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nicolas Bertho, Immunology Department, UMR8115 Genethon BP60, 1 bis rue de l'Internationale, 91002 Evry Cedex, France; e-mail: bertho{at}genethon.fr.
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Abstract
Introduction
(IFN-
) or apoptotic Jurkat cells (
). For clarity, specific migrations, normalized with the PGE2/TNF
