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Blood, 15 June 2007, Vol. 109, No. 12, pp. 5318-5326. Prepublished online as a Blood First Edition Paper on March 5, 2007; DOI 10.1182/blood-2006-10-053256.
IMMUNOBIOLOGY TLR3 ligand stimulates fully functional memory CD8+ T cells in the absence of CD4+ T-cell help1 Institut Pasteur, Unité de Régulation Immunitaire et Vaccinologie, Paris France; 2 Institut National de la Santé et de la Recherche Médicale (INSERM) U833, Paris, France; 3 Centre National de la Recherche Scientifique (CNRS-UMR 8147), Université Paris Descartes, Faculté le médecine René Descartes, Site Necker, Paris, France
We investigated whether Toll-like receptor ligands (TLR-Ls) can bypass the requirement for CD4+ T-cell help in the induction of fully efficient memory CD8+ T cells (cytotoxic T lymphocytes [CTLs]). "Helpless" CTLs were induced by a synthetic CD8+ T-cell epitope administered with TLR3-L and TLR9-L, but not with TLR2/6-L, TLR4-L, or TLR7-L. The up-regulation of MHC-I and costimulatory molecules by dendritic cells following TLR stimulation was not sufficient for the priming of "helpless" CTLs, which depended essentially on the induction of a strong IFN-  /ß response. The "helpless" CTLs induced by TLR-Ls differentiated into fully functional memory CTLs able to proliferate as well as their "helped" counterparts upon challenge, in the absence of CD4+ T-cell help.
The mechanisms underlying effector and memory cytotoxic T lymphocyte (CTL) activation remain unclear and a source of considerable interest. CD4+ T cells have been shown to be required for the induction of optimal primary CTL responses to minor histocompatibility and tumor antigens (Ags), soluble proteins, synthetic peptides, peptide-pulsed dendritic cells (DCs), and herpes simplex virus and influenza virus infection.1–5 However, CTLs can also be primed in the absence of CD4+ T cells ("helpless" CTLs), after infection with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus6,7 or immunization with the MHC-I–delivery vaccine vector consisting on the adenylate cyclase of Bordetella pertussis.8 The factors determining the requirement for CD4+ T-cell help in CTL priming remain a matter of debate. CD4+ T cells help CTLs by licensing Ag-bearing, CD40-expressing DCs through interaction with the CD40L expressed by CD4+ T cells.5,9,10 In "helpless" CTL responses, the requirement for DC licensing by CD4+ T cells is thought to be overcome by inflammatory mediators induced by infection.11,12 In several systems, the absence of CD4+ T-cell help during CTL induction has been shown to result in defective memory CTLs that mediate effector functions, such as cytotoxicity and cytokine secretion, upon secondary challenge but do not undergo a second round of clonal expansion in the absence of CD4+ T-cell help.4,6,7,13–15 However, other reports have shown that "helpless" memory CTLs can proliferate despite the absence of CD4+ T cells during the recall response.7 The reasons for these discrepancies are unclear, but the requirement for CD4+ T-cell help for memory CTL expansion, like that for CTL induction, may be linked to the infectious agent.
Pathogens stimulate innate immunity by signaling through TLRs, and there is strong evidence to suggest that TLR signaling in DCs predominates over other microbial stimuli. Initiation of the program leading to effector/memory CTL production following TCR activation requires a second signal, provided by costimulation, and a third signal that can be provided by IL-12,16 IFN- In the present study, we thus investigated whether various TLR-Ls could stimulate effector and memory CTLs in the absence of CD4+ T-cell help, by immunizing mice with synthetic microspheres covalently linked to the OVA257-264 CTL peptide (BOVAp) together with different TLR-Ls. We previously showed that BOVAp can transfer the CTL epitope to the MHC-I pathway of DCs in vivo, without inducing DC maturation and with no stimulatory/anergizing capacity.19 BOVAp therefore fails to induce CTL responses unless further "licensing" signals are delivered to DCs, by an agonist anti-CD40 monoclonal antibody (mAb), for example.19
In this study, we clearly demonstrate that TLR3-L and TLR9-L, but not TLR2/6-L, TLR4-L, and TLR7-L, can bypass the requirement for CD4+ T cells for the induction of fully efficient CTL responses. The ability of TLR3-L and TLR9-L to prime "helpless" CTLs depended on IFN-
Mice C57BL/6 and 129Sv mice were obtained from Charles River (L'Arbresle, France). Mice lacking MHC-II molecules were obtained from Taconic Europe A/S (Copenhagen, Denmark). 129Sv mice lacking the type I IFN receptor (IFNARko)20 and C57BL/6 mice lacking TLR3 (TLR3ko)21 were obtained from the Pasteur Institute (Paris, France). Trif–/– mice were obtained from Dr Akira and backcrossed onto a C57BL/6 background (11 backcrosses). Animals were kept under specific pathogen-free conditions. Experiments involving animals were conducted according to institutional guidelines. Reagents and immunizations Mice were immunized by intravenous or subcutaneous injection with 109 synthetic latex beads (1-µm diameter; Polysciences, Warrington, PA) covalently linked to the OVA257-264 synthetic peptide (BOVAp), as described,19 either alone or in combination with 25 µg zymosan, poly(I:C), ultrapure LPS, poly(U) (complexed to LyoVec), R837, R848, or with 5 µg CpG-B (1668), CpG-A (2216), or control oligodeoxynucleotide (ODNs) (1982, named ODN-B, and 5'-GGGGGAGCATGCTGCGGGGG-3', named ODN-A), or with 100 µg anti-CD40 antibodies (3/23) (BD Biosciences, Le Pont de Claix, France). CpGs and control ODNs were administered either free or mixed with DOTAP (30 µL) (Roche Diagnostics, Meylan, France). TLR-Ls were obtained from Invivogen (Toulouse, France), with the exception of CpGs and control ODNs, which were obtained from Genset (Paris, France). In some experiments, mice received beads coated with the following H-2b-epitope–containing peptides: E749-57 from human papilloma virus E7 protein, HY738-746 from the minor histocompatibility (H) male-specific (Y) antigen (H-Y), and LCMV33-41 from LCMV glycoprotein. All peptides were obtained from Neosystem (Strasbourg, France). Some mice were injected subcutaneously with synthetic OVA257-264 peptide (300 µg), either alone or together with poly(I:C). For recall studies, mice primed with BOVAp together with poly(I:C) or anti-CD40 mAb were boosted (day 25) with a single intravenous injection of genetically detoxified adenylate cyclase (CyaA, 50 µg) from B pertussis bearing the OVA257-264 epitope at position 224.8 In vivo killing assay Naive syngenic splenocytes were pulsed with OVA257-264 peptide (10 µg/mL; 30 minutes, 37°C), washed extensively, and labeled with high concentration (1.25 µM) of CFSE (Molecular Probes, Eugene, OR). The nonpulsed control population was labeled with low concentration (0.125 µM) of CFSE. Both CFSEhigh and CFSElow-labeled cells were mixed at a 1:1 ratio (5 x 106 cells of each population) and then injected intravenously into mice. The number of CFSE-positive cells remaining in the spleen after 20 hours was determined by fluorescence-activated cell sorting (FACS). ELISPOT
IFN- DC purification Mice were injected intravenously with PBS or 25 µg zymosan, poly(I:C), LPS, poly(U), R848, or 5 µg CpG-B. CpG was administered either free or mixed with 30 µL DOTAP. At the time indicated, spleens were harvested and treated with 400 U/mL collagenase D and 50 µg/mL DNase I (Roche Diagnostics). Spleen cell suspension was incubated with anti-CD11c antibody-coated magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and purified with anti-CD16/32 mAb (BD Biosciences). Cells were selected using an AutoMACS (Miltenyi Biotec). Flow cytometry analysis The expression of MHC-I and costimulatory molecules on the surface of DCs was analyzed by staining magnetically sorted splenic DCs with anti-CD11c-APC, anti-CD86-FITC, anti-Kb-FITC, or appropriate isotype control antibodies. For the detection of intracellular IL-12, splenic DCs were magnetically sorted 4 hours after the injection of TLR-Ls. Brefeldin A (10 µg/mL) (Sigma-Aldrich, St Louis, MO) was added during the DC enrichment procedure. Cells were first stained for extracellular markers with anti-CD11c-APC antibodies, then processed with the Fix & Perm kit (BD Biosciences), and finally stained with anti–IL-12p40-APC or an appropriate isotype control mAb. The number of OVA257-264–specific CTLs (OVATetr+) was determined by staining spleen cells first with H-2Kb-OVA257-264-tetramer-PE (Beckman Coulter France, Roissy, France) and then with anti-CD3-APC and anti-CD8-FITC. Results are expressed as the percentage of CD8+ T cells that were OVATetr+. All mAbs were obtained from BD Biosciences. The cells were acquired on a FACScalibur flow cytometer and analyzed with CellQuest Software (BD Biosciences). Cytokine ELISA assays
IFN-
Poly(I:C) and CpG promote the CTL response in the absence of CD4+ T-cell help Some TLR-Ls, such as LPS, poly(I:C), and ODN containing the CpG motif, have been shown to stimulate CTL responses against coadministered antigens.23–25 However, in all of these studies, the antigens used contained CD4+ T-cell epitopes, and the observed adjuvant effect of these TLR-Ls might have been mediated by CD4+ T-cell help. We aimed to assess the ability of various TLR-Ls to stimulate functional CTLs in the absence of any CD4+ T-cell help. C57BL/6 mice were injected intravenously with synthetic microspheres covalently linked to the OVA257-264 CTL epitope (BOVAp), either alone or together with 25 µg zymosan, poly(I:C), LPS, poly(U), R837, R848, or with 5 µg CpG-B or CpG-A. CpGs were administered either as naked ODN, or mixed with the cationic lipid formulation DOTAP (CpG(DOTAP)). Control mice were immunized with TLR-L alone or with BOVAp or control ODNs mixed with DOTAP, and CTL responses were analyzed in an in vivo killing assay or by tetramer staining. No OVA-specific CTL response was detected in control mice or in mice injected with BOVAp alone (Figure 1A and data not shown). In contrast, strong in vivo lysis was detected when BOVAp was injected with either poly(I:C), CpG-B(DOTAP), or CpG-A(DOTAP) (Figure 1A). The frequency of OVA-specific CTLs, as determined by tetramer staining (OVATetr+), was similarly high in these 3 groups (Figure 1B). Similar results were obtained when BOVAp was administered subcutaneously with poly(I:C), CpG-B(DOTAP) (Figure 1C), or CpG-A(DOTAP) (data not shown). In contrast, no CTL response was observed when BOVAp was injected with zymosan, LPS, or R837, whereas a weak response was detected in mice immunized with BOVAp and poly(U), R848, or naked CpGs (Figure 1A). The injection of larger amounts of zymosan (500 µg), LPS (100 µg), R837 (100 µg), R848 (200 µg), or naked CpGs (100 µg) did not enhance the CTL response (Figure S1, available on the Blood website; see the Supplemental Figures link at the top of the online article).
We also used adoptive transfer of OT-1 cells to assess the ability of various TLR-Ls to stimulate CTLs in the absence of any CD4+ T-cell help (Figure S2). Forty hours after immunization with BOVAp alone or together with either poly(I:C), zymosan, or LPS, we detected an early proliferation of OT-1 cells (Figure S2A). However, only poly(I:C) significantly enhanced further expansion (70 hours) of primed OT-1 cells. Expression of CD44 by transferred OT-1 cells was not affected by TLR triggering (Figure S2B). However, acquisition of other typical effector markers, such as up-regulation of CD25, down-regulation of CD62L, or production of IFN-  , was achieved only markedly upon immunization with BOVAp together with poly(I:C) but not with BOVAp alone or with zymosan or LPS (Figure S2B). A similar activation of the transferred OT-1 cells was observed in recipient mice immunized with BOVAp plus CpG-B(DOTAP) (data not shown). Neither proliferation nor activation was detected upon injection of the TLR-Ls alone (data not shown). These results show that only poly(I:C)- and CpG(DOTAP)-derived, but not TLR2/6 or TLR4 ligands–derived, signals fulfill the requirements to prime adoptively transferred OT-1 cells, confirming our previous results with normal mice. No H-2b-restricted CD4+ T-cell epitope has been identified in the OVA257-264 peptide sequence. Nevertheless, C57BL/6 WT and MHC-IIko mice were immunized (intravenously) with BOVAp and either poly(I:C) or CpG-B(DOTAP) to exclude any possible participation of CD4+ helper T cells in the induction of the CTL response against BOVAp. Both strains of mice mounted similar CTL responses upon immunization with BOVAp and poly(I:C), as shown in the in vivo killing assay (Figure 1D), and this response was similar to that induced by BOVAp in the presence of anti-CD40 mAb. The frequency of OVATetr+ CTLs was similar in WT and MHC-IIko mice after immunization with BOVAp and either poly(I:C) or CpG-B(DOTAP) (Figure 1E). Poly(I:C) also induced a strong CTL response in the absence of CD4+ T cells when injected with beads coated with 3 other peptides containing CTL epitopes (Figure 1F). Finally, we also tested the response induced by poly(I:C) in combination with soluble OVA257-264 peptide, to exclude the possibility that this ability of TLR-Ls to overcome the CD4+ T-cell requirement for CTL activation was limited to particulate antigens. In vivo lytic activity was also detected following the subcutaneous immunization of mice with the soluble OVA257-264 peptide and poly(I:C), although the CTL response was less efficient than that against particulate antigens (Figure 1G). However, no CTL response was detected if mice received the OVA257-264 peptide and poly(I:C) via the intravenous route (data not shown). Thus, TLR3 and TLR9 agonists, but not TLR2/6-L, TLR4-L or TLR7-L, can substitute for CD4+ T-cell help in the induction of an efficient CTL response. Neither the up-regulation of MHC-I and costimulatory molecules on DCs nor the production of IL-12 induced by TLR stimulation is sufficient for CD4-independent CTL priming The generation of CTL responses against exogenous cell–associated or soluble antigens requires DC licensing.2 In their resting state, DCs have only low levels of MHC and costimulatory molecules and cannot prime naive CTLs. The maturation of DCs to a functionally mature state is promoted by activated CD4+ T cells, via interactions between CD40 on DCs and CD40L on T cells, in particular.5,9,10 However, resting DCs can also be activated by inflammatory cytokines26 or stimuli from pathogens that up-regulate MHC and costimulatory molecules,27 thereby increasing the ability of these cells to stimulate T cells. We investigated whether the ability of TLR agonists to induce CTL priming in the absence of T-helper activation was dependent on their capacity to activate DCs, by injecting various TLR-Ls intravenously into C57BL/6 mice and analyzing the expression of MHC-I (H-2kb) and costimulatory molecules (CD86) on CD11chigh DCs. DCs displayed similar increases in the levels of CD86 or MHC-I molecules following in vivo stimulation with poly(I:C), LPS, poly(U), R848, and CpG-B(DOTAP) (Figure 2A). In contrast, neither zymosan (Figure 2A) nor R837 (50 µg) (Gillis Dadaglio, unpublished data, June 2005) induced the up-regulation of these molecules. The administration of CpG-B in DOTAP greatly increased the ability of this molecule to up-regulate costimulatory and MHC-I molecules in DCs (Figure 2A). Similar results were obtained when instead of CpG-B we used CpG-A (data not shown). The administration of DOTAP alone did not affect the expression of MHC-I and CD86 molecules (data not shown). These results clearly demonstrate that the expression of MHC-I and CD86 molecules by DCs following TLR-L stimulation is not correlated with the ability of TLR-Ls to promote CTL priming in the absence of CD4+ T-cell help. Similar conclusions were drawn from in vivo analyses of CD40 and CD80 expression upon TLR engagement (data not shown).
IL-12 enhances CTL responses in various experimental systems.16 We assessed the correlation between the ability of TLR-Ls to stimulate IL-12 secretion by DCs and their capacity to prime CTLs, by evaluating IL-12 production upon in vivo TLR triggering by intracellular staining in CD11chigh DCs. High levels of IL-12 production were detected in DCs from mice treated with poly(U), R848, CpG-B(DOTAP) (Figure 2B), and CpG-A(DOTAP) (data not shown), but not in DCs from mice treated with zymosan, poly(I:C), and LPS (Figure 2B). A moderate IL-12 response was detected in DCs from mice injected with naked CpGs (Figure 2B and data not shown). Our data indicate that the TLR-mediated up-regulation of MHC-I and costimulatory molecules and IL-12 production in DCs does not fulfill the requirements for CTL priming in the absence of CD4+ T-cell help.
The ability of TLR3-L and TLR9-L to induce CTL responses in the absence of CD4+ T-cell help is dependent on IFN-
Type I interferons (IFNs) are an essential link between innate and adaptive immunity.28,29 We therefore thought that the ability of some TLR-Ls to promote CTL priming in the absence of CD4+ T-cell help might be mediated by type I IFNs. We investigated the capacity of different TLR-Ls to induce IFN-
We investigated possible regulation by the type I IFN receptor (IFNAR) of the poly(I:C), or CpG-induced production of IFN- /ß in vivo, by injecting WT (129Sv) mice and mice lacking IFNAR (IFNARko) with poly(I:C) or CpG-B(DOTAP). Serum IFN-ß concentration was not significantly affected by the absence of IFNAR, and was even slightly higher in IFNARko than in WT mice upon the injection of poly(I:C) (Figure 3B). A lack of IFN-ß sequestration by IFNAR may account for the higher levels of IFN-ß in IFNARko mice. By contrast, markedly lower levels of IFN- production were induced by poly(I:C) or CpG(DOTAP) in IFNARko mice than in WT mice. Thus, the production of IFN- in response to the administration of poly(I:C) or CpG(DOTAP) is strongly regulated by IFNAR in vivo.
We then investigated whether the ability of poly(I:C) and CpG(DOTAP) to promote CTL priming in the absence of CD4+ T-cell help depends on IFN- In the absence of T-cell help, a lack of TLR3 triggering decreases the ability of poly(I:C) to promote CTL responses
We investigated whether TLR recognition was essential for poly(I:C) and CpG substitution for T-cell help in the induction of CTL priming. We investigated the involvement of TLR triggering in the in vivo production of IFN-
We then investigated whether poly(I:C) and CpGs enhanced CTL responses through their recognition by TLR3 and TLR9, respectively. TLR9ko mice were unable to mount a CTL response upon immunization with BOVAp and CpG-B(DOTAP) (Figure 4B) or CpG-A(DOTAP) (data not shown). By contrast, the immunization of TLR3ko mice with BOVAp in the presence of poly(I:C) induced a CTL response, as shown by the in vivo killing of target cells loaded with the OVA257-264 peptide. However, CTLs were much less frequent in these mice than in WT mice (Figure 4B). A similarly impaired CTL response to BOVAp and poly(I:C) immunization was observed in Trif–/– mice (Figure 5B). It should be noted that, following immunization with BOVAp in the presence of anti-CD40 mAb, TLR3ko and TLR9ko mice mounted a CTL response similar to that of control mice (Figure 4C). The impaired immune response observed in TLR3ko and TLR9ko mice injected with BOVAp and poly(I:C) or CpG(DOTAP), respectively, was due to the lack of TLR signaling rather than a general defect in these strains.
Our data show that signaling through TLR9 is strictly required for CTL priming by CpG(DOTAP), whereas an absence of TLR3 triggering significantly decreases, but does not abolish, the ability of poly(I:C) to promote CTL responses. Thus, although the adjuvant effect of poly(I:C) on CTL priming is dependent on type I IFN, IFN- In the presence of TLR3 agonist, CD4+ T-cell help is not required for the in vivo activation, persistence, and restimulation of memory CTLs
We determined whether CD4+ helper T cells, which are normally required for the activation of fully functional memory CTLs, could be replaced by signals provided through TLR stimulation. We analyzed the effector functions of memory CTLs induced by BOVAp, injected with poly(I:C). Anti-CD40 mAb, which binds CD40, leading to DC activation, was selected as a surrogate for helper T cells. After the injection of BOVAp and poly(I:C), considerable expansion of the OVA-specific CTL population was observed, reaching almost 3% of the total number of CTLs 7 days after injection. This expansion was followed by a prolonged contraction phase and then by a plateau that persisted throughout the memory phase (Figure 6A). By contrast, the expansion phase induced by BOVAp and anti-CD40 mAb was less intense (specific CTLs accounting for 2% of the total number of CTLs) and was followed by a sharp contraction phase, restoring OVATetr+ levels to levels slightly higher than those in naive mice. "Helpless" OVA-specific CTLs generated in the presence of poly(I:C) displayed full effector functions, such as in vivo lytic activity and IFN-
We then investigated whether the memory CTLs generated in the absence of CD4+ T-cell help, in response to BOVAp and poly(I:C), could undergo a second round of clonal expansion after specific restimulation. Mice were primed with BOVAp and either poly(I:C) or anti-CD40 mAb, followed by a boost 25 days later with a single intravenous injection of a MHC-I–vaccine delivery vector consisting on the adenylate cyclase (CyaA) of B pertussis carrying the OVA257-264 epitope (CyaA-OVA).8 The immunization of naive mice with CyaA-OVA induced a primary OVA-specific CTL response accounting for 2% of all CTLs 7 days after injection (Figure 6C). Upon boosting with CyaA-OVA, "helpless" and "helped" memory CTL populations underwent similar, rapid, massive expansions (Figure 6C). The peak of OVA-specific CTLs induced upon secondary immunization was about 5 times higher than the peak of the primary response (Figure 6A,C). Several studies have demonstrated that the absence of CD4+ T-cell help during CTL priming results in defective memory CTLs that do not undergo a second round of clonal expansion unless CD4+ T-cell help is provided during the recall response.4,6,7,13–15 We investigated whether memory CTLs primed by BOVAp and poly(I:C) could proliferate in response to CyaA-OVA immunization, by priming WT and MHC-IIko mice with BOVAp+poly(I:C), administering a CyaA-OVA boost on day 25 and analyzing the recall response by tetramer staining 7 days later. Poly(I:C)-derived signaling during the primary response generated fully functional memory CTLs able to undergo a second round of marked expansion despite the absence of CD4+ T cells during the recall response (Figure 6D).
Several studies have indicated that the requirement for CD4+ T-cell help for the induction of effector and memory CTLs is not absolute and depends on the nature of the stimuli present during the primary response. In particular, some viral infections elicit "helpless" CTL responses. We show here that the requirement for T-cell help to trigger CTLs can be bypassed by TLR3-L and TLR9-L, with administration in liposomes required for TLR9-L, but not by other TLR agonists, such as TLR2/6-L, TLR4-L, or TLR7-L. TLR4-L and naked TLR9-L have been shown to induce CTL responses when administered with a soluble protein.24 However, the adjuvant effect of these 2 TLR agonists used in combination with soluble antigens containing both CD4+ and CD8+ T-cell epitopes, may be due to a synergic effect between TLR triggering-derived signals and signals provided by antigen-specific CD4+ T cells. By contrast, we used an experimental system allowing the analysis of CTL responses induced by a single CTL epitope, excluding the participation of CD4+ T cells.
Unlike zymosan and R837, all the TLR-Ls tested in this study up-regulated MHC-I and costimulatory molecule levels on DCs in vivo. An increase in the capacity to deliver signal 1 (antigen) and signal 2 (costimulation) is often interpreted as the key feature in the licensing of DCs to prime an effective CTL response. However, several studies,30–32 including this one, have shown that the stimulation of naive CTLs can fail even when the DCs express large amounts of antigen and B7 ligands. It has been suggested that initiation of the program leading to effector/memory CTL activation requires a third signal that can be provided by IL-12,16 IFN-
The injection of IFN-
In cases of impaired proinflammatory response, such as after poly(I:C) injection in mice deficient for TLR3 signaling, the induction of high levels of IFN-
Microbial DNA can be sensed via TLR9-dependent and TLR9-independent pathways.32 We show here that TLR9 engagement is strictly required for CpG-promoted IFN-
LPS and TLR7-Ls do not induce CTL responses, even though they share the common adaptor proteins Trif (poly(I:C)) and MyD88 (CpG) and despite the fact that certain DC subsets have been shown to produce in vitro IFN-ß and/or IFN- No study has yet analyzed the functional properties of the "helpless" memory CTL population promoted by signals derived from TLR triggering. In this study, we show that "helpless" CTLs induced by TLR3-L differentiate into fully functional memory CTLs able to proliferate upon secondary immunization in the absence of any CD4+ T-cell help. This proliferation is equivalent to that of surrogate "helped" memory CTLs induced with anti-CD40 mAb. Our results are consistent with previous reports showing that "helpless" memory CTLs induced by Listeria monocytogenes infection can proliferate upon secondary challenge with the same agent, despite the absence of CD4+ T cells during the recall response.7
Our data clearly indicate that signals derived from TLR triggering can bypass the requirement of CD4+ T-cell help for the in vivo induction of fully efficient effector and memory CTLs. We also show that the TLR engagement-mediated up-regulation of MHC-I and costimulatory molecules and IL-12 production by DCs does not fulfill the requirements for the priming of naive CTLs in the absence of CD4+ T-cell help, as the induction of a strong IFN-
Contribution: S.H.-S. performed and coordinated most experiments and wrote the first draft of the paper; A.O. performed the experiments suggested by reviewers; F.B. initiated this work and contributed to paper writing; N.T. helped discuss data and contributed with Trifko mice; C.L. provided the original ideas, coordinated the team, obtained funding, and wrote the final version of the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Claude LECLERC, Régulation Immunitaire et Vaccinologie, Inserm U883, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France; e-mail: cleclerc{at}pasteur.fr.
This work was supported by grants from Association de la Recherche contre le Cancer and Institut National du Cancer and the European Community (Theravac Project) to C.L. S.H-S. was supported by Universidad Pública de Navarra (Spain). The authors thank Daniel Ladant for providing CyaA-OVA and Richard Lo-Man for fruitful discussions.
Submitted October 23, 2006; accepted February 26, 2007.
Prepublished online as Blood First Edition Paper, March 5, 2007
DOI: 10.1182/blood-2006-10-053256
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 USC section 1734.
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Abstract
Introduction
