Immune-Reconstituted Influenza Virosome Containing CD40L Gene Enhances the Immunological and Protective Activity of a Carcinoembryonic Antig
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免疫学杂志 2005年第11期
Abstract
The correct interaction of a costimulatory molecule such as CD40L with its contrareceptor CD40 expressed on the membrane of professional APCs, provides transmembrane signaling that leads to APC activation. This process can be exploited to significantly improve the efficacy of cancer vaccines and the outcome of a possible cancer vaccine-induced, Ag-specific CTL response. Therefore, we investigated whether a novel intranasal delivery of immune-reconstituted influenza virosomes (IRIV), assembled with the CD40L gene (CD40L/IRIV), could be used to improve protective immunity and the Ag-specific CTL response against carcinoembryonic Ag (CEA) generated with a novel vaccine constituted of IRIV assembled with the CEA gene (CEA/IRIV). Our results suggest that CD40L/IRIV was able to augment CEA-specific CTL activity and CEA-specific protective immunity induced by CEA/IRIV most likely through the induction of a CTL response associated with a Th1 phenotype. In conclusion, we provide evidence that CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, acts as an immune-adjuvant that could increase the efficacy of a CEA-specific cancer vaccine, which could provide an efficacious new strategy for cancer therapy.
Introduction
Active specific immunotherapy, or vaccine therapy is a rising new strategy for the treatment of human cancer. Several approaches are being evaluated in order for inducing an Ag-specific immune response with antitumor activity in cancer patients, some of which are currently being tested in clinical trials (1, 2, 3, 4). It is already commonly accepted that it is possible to generate an efficient in vitro and in vivo immune response by delivering the target Ag directly to the APCs by using naked nucleic acids (DNA or RNA), or viral or bacterial recombinant constructs (5, 6, 7, 8). In particular, DNA immunization has been shown to be capable of inducing in vivo an effective humoral response and a cellular response simultaneously in vivo (9), restricted by class I (10, 11, 12) as well as by class II MHC+ cells (13). We have previously demonstrated the possibility of using the immune-reconstituted influenza virosomes (IRIVs) 2 as a delivery system for plasmids expressing tumor-associated Ag. We have shown that this method is able to induce a specific CTL response with antitumor activity in an in vitro human model, and also in vivo using either BALB/c mice or HLA-A2.1 transgenic mice (HHD) (14). In the present study, we investigated the possibility of enhancing the immunogenicity of a carcinoembryonic Ag (CEA) DNA vaccine by combining its administration with a plasmid expressing the coaccessory molecule CD40L in IRIV. This study was based on the fact that CTLs recognize protein Ags as small peptides produced from intracellular proteolysis operated by proteasomes bound to the class I MHC molecules on the membrane of target cells or APC. The TCRs on specific CTLs are engaged through a trimolecular interaction involving the same TCR, the peptide, and the specific MCH complex, which provides a first transmembrane signal that is necessary but not sufficient, to induce an efficient Ag-specific CTL activation with proliferation and consequent clonal expansion (15, 16). To obtain these events, it is necessary that the CTL precursors receive a second intracellular signal, which is mainly provided by a receptor/contrareceptor interaction of coaccessory molecules such as B7.1 or B7.2/CD28 or CD40L/CD40 (3, 14). CD40 is a molecule expressed on the cell membrane of partially primed APCs, which can bind to CD40L expressed on the membrane of activated CD4+ Th cells (17). This interaction is known to deliver a bidirectional second signal, which is able to simultaneously activate both the effector T cells and the APC, increasing their priming ability (17). Many researchers believe that the interaction between CD40 and CD40L is critical for helping the APC to efficiently prime CD8+-specific cytotoxic cells (17). It has already been reported that CD40L expressed as a homotrimer by a DNA plasmid can be used to enhance cellular immune response and cytolytic T cell activity in mice vaccinated with DNA encoding CEA gene (18, 19) In the present study, we investigated whether the intranasal (i.n.) administration of IRIVs containing the CD40L (CD40L/IRIV) gene plasmid could effectively improve the immunological and protective antitumor activity of a CEA-expressing IRIV vaccine, after a lethal challenge with CEA-expressing tumor cells, in an in vivo BALB/c model.
Materials and Methods
Statistics
The mean differences were statistically analyzed using StatView statistical software (Abacus Concepts). The results were expressed as the mean ± SD of three determinations made in three different experiments, and differences were determined using Bonferroni’s (all-pairwise) multiple comparison test. A value of = 0.05 was considered statistically significant.
Results
Construction and characterization of CEA and CD40L IRIV
CEA and CD40L plasmids were synthesized as described in Materials and Methods, and analyzed by restriction analysis and DNA sequencing. We cloned only a fragment of the CEA sequence (accession no. M17303) encompassing nt 1054–2205 to avoid a possible cross-reaction with the nonspecific cross-reacting Ag, a member of the CEA Ag gene family, which shares its high level of homology (42%) in the upstream sequence (22).
This CEA amino acid sequence was also chosen, because it contained a large number of epitopes potentially able to bind the most common murine and human class I and class II MHC haplotypes according to Parker’s and Remmensee’s algorithms (23). The ability of IRIV to achieve APC in vivo has already been shown in previous studies (24, 25, 26). Both CEA and murine CD40L genes were expressed in transfected cells, as shown by cytofluorometric analysis (Table I). The ability of CEA/IRIV and CD40L/IRIV to deliver the respective genes in vitro was demonstrated on several target cells including murine spleen cells, human PBMC, human and murine DCs, and P815 cell lines by means of cytofluorometric analysis performed 24, 48, 72, and 96 h after transduction (data not shown). CEA in vivo expression was indirectly confirmed by the presence of anti-CEA Ab response in mice immunized with CEA/IRIV (Fig. 1), and it was supported by the presence of the specific mRNA in the lymph nodes of mice collected 24 and 48 h after i.n. administration of CEA/IRIV (Fig. 2). However, it was not possible to detect CEA transcripts 72 h after the mice inoculation.
CEA-specific CTL response in immunized mice
We also evaluated whether the CD40L/IRIV construct was able to improve the ability of a CEA-directed cancer vaccine (CEA/IRIV) in inducing a CEA-specific CTL response. Spleens were taken from the mice vaccinated as described above, and spleen cells were isolated and pooled from the different mouse groups.
Pooled spleen cells were then in vitro stimulated with autologous irradiated splenocytes transfected with CEA gene and cultured for 10 days in a medium containing low-dose IL-2 before being tested in 51Cr release assay against the P815 target cell clone expressing CEA. These experiments showed that the spleen cells from the mice that had received treatment with CEA/IRIV showed a significant CEA-specific antitumor activity. Our results also showed that the spleen cells from the mice that had received the combined treatment with CEA/IRIV and CD40L/IRIV elicited much higher lytic activity. Significantly different values (Bonferroni’s (with control) multiple-comparison test, = 0.05) were observed by comparing the results of CTL activity of mice immunized with CEA/IRIV (group B) and CEA/IRIV plus CD40L/IRIV (group D) against CEA-expressing cells vs the remaining groups. The lytic activity of these CTLs was CEA specific and class I MHC restricted, because they were not able to kill untransfected (not expressing CEA) P815 target cells and because their lytic activity was abrogated by the addition of anti-class I MHC mAbs in the cytotoxic assay (Fig. 4). The administration of IRIVs or CD40L/IRIV did not elicit any CEA-specific CTL response, and spleen cells from mice vaccinated with these constructs only gave rise to unspecific lytic activity that was not restricted by class I MHC molecules.
The CEA-CD40L combination enhances the expression of costimulatory molecules B7-1,2
CD40L binding to CD40 (receptor) activates intracellular signaling in the APCs and DCs that improves their presenting ability and leads to the up-regulation of the other adhesion/costimulatory molecules (27) on their surface. These latter molecules can provide the second signal required to activate naive T cells, amplify the immune response, and prevent anergy or tolerance induction. Therefore, we investigated whether our CD40L/IRIV construct could enhance, through CD40/CD40L interaction, the immune response against CEA, triggered by the CEA/IRIV vaccination. FACS analysis was performed from spleen cells isolated from the immunized mice administered with different IRIV constructs, and analyzed for the expression of B7.1 and B7.2 on class II MHC+ cells. The results of these experiments revealed a greater up-regulation of the B7.1 and, particularly, B7.2 molecules in mice receiving CD40/IRIV and CEA/IRIV together (group D) ( = 0.05) (Fig. 5). Moreover, besides the higher percentage of positive cells, a higher density of B7.1 and B7.2 molecules was expressed on the cells of group D mice. IRIVs alone were able to up-regulate the expression of B7.1,2 and MHC class II molecules on APCs, as described elsewhere (28), but immunization with this combination of two plasmids resulted in a much greater increase in B7 expression. On the contrary, the administration of CEA/IRIV (without the CD40L gene) to mice did not modify the level of B7 expression. These data suggest that CD40L/IRIV enhances the immunogenic potential of pCEA/IRIV by up-regulating the costimulatory molecules on APC and DCs, determining increased Ag-processing and -presenting capability of these cells.
CEA/IRIV and CD40L/IRIV induce the production of Th1 cytokines
Finally, we evaluated whether our treatments were also able to produce proinflammatory Th1 cytokines, such as IFN-. We examined the ability of the cultured spleen cells, pooled from the different groups of mice, to produce IFN- in response to CEA by a solid-phase sandwich ELISA. Interestingly, we found high levels of this cytokine only in the mice that had been immunized with the combination of CD40L/IRIV-CEA/IRIV (group D) (0.883 ± 0.3 ng/ml) (Fig. 6). The spleen cells from the other mouse groups, including those vaccinated with CEA/IRIV alone, did not produce a detectable level of IFN- in response to CEA. No significant differences in IFN- production were observed when spleen cells of the different groups were stimulated with Con A, indicating that CEA specifically induced the expression of IFN-. These data strongly suggest that the addition of CD40L/IRIV to CEA/IRIV promotes the differentiation of lymphocytes toward a protective Th1 phenotype. The result was further supported by an increase of CEA-specific IgG2a Ab titer in the group of mice immunized with CD40L/IRIV-CEA/IRIV (40 ± 2.32 ng/ml) in comparison with the titer of mice immunized with CEA/IRIV (15 ± 3.64 ng/ml) ( = 0.05).
Discussion
The active specific immunotherapy targeted against cancer is aimed at mobilizing the immune system and destroying tumor cells (6, 29). Many tumor-associated Ags have been selected as possible targets for therapeutic cancer vaccines (3, 30), and the possibility of generating a CTL-mediated immune response directed at them, such as CEA, has been demonstrated through many different strategies (18, 31, 32, 33, 34, 35). Several hypotheses have been formulated to explain these deluding results; some are related to the lack of induction of an efficient immune response and some to mechanisms of escape, activated by tumor cells (36, 37, 38, 39). The majority of these Ags are expressed at a low level in normal cells, but their immunogenicity is too weak to give rise to an efficient immune response. Thus, cancer cells can activate alternative escape mechanisms that make them resistant to the cancer vaccine activated immune effectors. In this context, therapeutic immunization can also be hampered by inadequate activation or by a low number of professional APCs, as often observed in cancer patients and chronic diseases (40, 41).
We hypothesized that the immunogenic and therapeutic potential of Ag-directed vaccination could be significantly enhanced by the contemporary administration of agents that might increase the expression of coaccessory molecules on APCs and direct the activation and expansion of specific T cells. We believe that the CD40L molecule can modulate the immune response against human malignancies. In fact, it is known that CD40L retains a central role in initiating the immune response, although it is expressed transiently on a small proportion of cells, and it can produce long-lasting systemic immune responses, capable of blocking disease progression. CD40L, interacting with its contrareceptor (CD40), enhances the Ag-specific T cell growth by two distinct mechanisms: 1) it activates cultured DCs, which consequently express a higher amount of class I and II MHC and costimulatory molecules, and 2) it has direct stimulatory effects (coaccessory signal) on T cells. On this basis, our aim was to study the possibility of enhancing antitumor protective immunity in BALB/c mice by in vivo modulating the immune response with CD40L. In our model, we chose human CEA as the target Ag, because it has been widely tested (31, 32, 33, 34, 35), and because currently several clinical investigations are using a variety of different CEA-directed vaccine approaches. In the present study, we designed a DNA-based vaccine that is the CEA recombinant plasmid included in IRIV. This vaccine construct (CEA/IRIV) can be safely administered i.n. to mice, giving rise to an efficient CTL-mediated immune response. We also administered IRIV containing a plasmid expressing the murine CD40L, to improve the functional activity of the professional APC. Previous studies have already shown that IRIV are rapidly and efficiently taken up by many human and murine APCs, including DCs (24, 25), that they significantly up-regulate the expression of DCs maturation markers, such as MHC class I and II, ICAM-1, B7.1, B7.2, and CD40, and they are able to deliver DNA into these cells, which is then rapidly expressed. The results of this study support the hypothesis that the enhanced expression of CD40L on APCs definitely improves the protective antitumor activity of the CEA/IRIV vaccine with a mechanism that could most likely be related to a DC maturation process, due to the CD40L/CD40 interaction. CD40 activation on these cells might abolish their tolerogenic capacity or even trigger the potential for immunogenic presentation of the Ag (42, 43). In fact, we found a significant CEA-specific CD8+ T cell response in the mice coinoculated with CEA/IRIV and CD40L/IRIV, that achieved the best protective immunity against CEA+ P815 cell challenge. The splenocytes of these mice also produced high levels of IFN- in response to CEA exposure, suggesting the occurrence of a Th1 response, which could significantly improve the level and the efficiency of the immune response. Although a CEA-specific CD8+ T response was also detected in mice inoculated with CEA/IRIV alone, it was much less efficient in terms of cytotoxic activity and protective immunity. Moreover, the splenocytes of these mice did not produce IFN- in response to CEA exposure. As expected, no CEA-specific CTL activity or protective immunity was observed in mice administered with CD40L/IRIV or pVax/IRIV constructs. The histology and the molecular analysis of the tissues drawn from the animals administered with the combination of CD40L/IRIV-CEA/IRIV revealed the depletion of all cells expressing CEA, whereas the groups of mice immunized with CEA/IRIV, CD40L/IRIV, or pVax/IRIV developed much larger tumor overexpressing CEA molecules. This result appears most likely due to the immunomodulating activity of CD40L associated with CEA. Furthermore, CD40 signaling, necessary for induction of Th-dependent T cell responses induced the expression of IFN- when splenocytes of the immunized mice were pulsed with CEA, providing evidence of an Ag-specific T cell proliferation (44, 45). In fact, the CD40L-CD40 interaction controls the balance between helper and regulatory T cells in immune response, releasing immature DCs from the control of regulatory CD4+CD25+ T cells (46) and breaking the immune tolerance against Ags. The increase of B7.1,2 expression on APCs of mice coimmunized with CEA/IRIV-CD40L/IRIV indicates that these cells have primed CEA-specific CD8+ T cells providing the secondary signals necessary to activate naive T cells and enhance the immune response against the tumor by these costimulatory molecules. In conclusion, we demonstrated that the i.n. administration of CD40L/IRIV in mice concomitantly with CEA/IRIV was able to induce an efficient tumor-protective immunity against CEA. CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, is a powerful immune adjuvant that was able to increase the efficacy of a vaccine specific for a poor immunogenic Ag, that could be an efficacious strategy for cancer therapy.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Address correspondence and reprint requests to Dr. Maria Grazia Cusi, Department of Molecular Biology, Virology Section, Siena University School of Medicine, Policlinico Le Scotte, Viale Bracci, 1, 53100 Siena, Italy. E-mail address: cusi{at}unisi.it
2 Abbreviations used in this paper: IRIV, immune-reconstituted influenza virosome; CEA, carcinoembryonic Ag; i.n., intranasal(ly).
Received for publication November 19, 2004. Accepted for publication March 15, 2005.
References
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The correct interaction of a costimulatory molecule such as CD40L with its contrareceptor CD40 expressed on the membrane of professional APCs, provides transmembrane signaling that leads to APC activation. This process can be exploited to significantly improve the efficacy of cancer vaccines and the outcome of a possible cancer vaccine-induced, Ag-specific CTL response. Therefore, we investigated whether a novel intranasal delivery of immune-reconstituted influenza virosomes (IRIV), assembled with the CD40L gene (CD40L/IRIV), could be used to improve protective immunity and the Ag-specific CTL response against carcinoembryonic Ag (CEA) generated with a novel vaccine constituted of IRIV assembled with the CEA gene (CEA/IRIV). Our results suggest that CD40L/IRIV was able to augment CEA-specific CTL activity and CEA-specific protective immunity induced by CEA/IRIV most likely through the induction of a CTL response associated with a Th1 phenotype. In conclusion, we provide evidence that CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, acts as an immune-adjuvant that could increase the efficacy of a CEA-specific cancer vaccine, which could provide an efficacious new strategy for cancer therapy.
Introduction
Active specific immunotherapy, or vaccine therapy is a rising new strategy for the treatment of human cancer. Several approaches are being evaluated in order for inducing an Ag-specific immune response with antitumor activity in cancer patients, some of which are currently being tested in clinical trials (1, 2, 3, 4). It is already commonly accepted that it is possible to generate an efficient in vitro and in vivo immune response by delivering the target Ag directly to the APCs by using naked nucleic acids (DNA or RNA), or viral or bacterial recombinant constructs (5, 6, 7, 8). In particular, DNA immunization has been shown to be capable of inducing in vivo an effective humoral response and a cellular response simultaneously in vivo (9), restricted by class I (10, 11, 12) as well as by class II MHC+ cells (13). We have previously demonstrated the possibility of using the immune-reconstituted influenza virosomes (IRIVs) 2 as a delivery system for plasmids expressing tumor-associated Ag. We have shown that this method is able to induce a specific CTL response with antitumor activity in an in vitro human model, and also in vivo using either BALB/c mice or HLA-A2.1 transgenic mice (HHD) (14). In the present study, we investigated the possibility of enhancing the immunogenicity of a carcinoembryonic Ag (CEA) DNA vaccine by combining its administration with a plasmid expressing the coaccessory molecule CD40L in IRIV. This study was based on the fact that CTLs recognize protein Ags as small peptides produced from intracellular proteolysis operated by proteasomes bound to the class I MHC molecules on the membrane of target cells or APC. The TCRs on specific CTLs are engaged through a trimolecular interaction involving the same TCR, the peptide, and the specific MCH complex, which provides a first transmembrane signal that is necessary but not sufficient, to induce an efficient Ag-specific CTL activation with proliferation and consequent clonal expansion (15, 16). To obtain these events, it is necessary that the CTL precursors receive a second intracellular signal, which is mainly provided by a receptor/contrareceptor interaction of coaccessory molecules such as B7.1 or B7.2/CD28 or CD40L/CD40 (3, 14). CD40 is a molecule expressed on the cell membrane of partially primed APCs, which can bind to CD40L expressed on the membrane of activated CD4+ Th cells (17). This interaction is known to deliver a bidirectional second signal, which is able to simultaneously activate both the effector T cells and the APC, increasing their priming ability (17). Many researchers believe that the interaction between CD40 and CD40L is critical for helping the APC to efficiently prime CD8+-specific cytotoxic cells (17). It has already been reported that CD40L expressed as a homotrimer by a DNA plasmid can be used to enhance cellular immune response and cytolytic T cell activity in mice vaccinated with DNA encoding CEA gene (18, 19) In the present study, we investigated whether the intranasal (i.n.) administration of IRIVs containing the CD40L (CD40L/IRIV) gene plasmid could effectively improve the immunological and protective antitumor activity of a CEA-expressing IRIV vaccine, after a lethal challenge with CEA-expressing tumor cells, in an in vivo BALB/c model.
Materials and Methods
Statistics
The mean differences were statistically analyzed using StatView statistical software (Abacus Concepts). The results were expressed as the mean ± SD of three determinations made in three different experiments, and differences were determined using Bonferroni’s (all-pairwise) multiple comparison test. A value of = 0.05 was considered statistically significant.
Results
Construction and characterization of CEA and CD40L IRIV
CEA and CD40L plasmids were synthesized as described in Materials and Methods, and analyzed by restriction analysis and DNA sequencing. We cloned only a fragment of the CEA sequence (accession no. M17303) encompassing nt 1054–2205 to avoid a possible cross-reaction with the nonspecific cross-reacting Ag, a member of the CEA Ag gene family, which shares its high level of homology (42%) in the upstream sequence (22).
This CEA amino acid sequence was also chosen, because it contained a large number of epitopes potentially able to bind the most common murine and human class I and class II MHC haplotypes according to Parker’s and Remmensee’s algorithms (23). The ability of IRIV to achieve APC in vivo has already been shown in previous studies (24, 25, 26). Both CEA and murine CD40L genes were expressed in transfected cells, as shown by cytofluorometric analysis (Table I). The ability of CEA/IRIV and CD40L/IRIV to deliver the respective genes in vitro was demonstrated on several target cells including murine spleen cells, human PBMC, human and murine DCs, and P815 cell lines by means of cytofluorometric analysis performed 24, 48, 72, and 96 h after transduction (data not shown). CEA in vivo expression was indirectly confirmed by the presence of anti-CEA Ab response in mice immunized with CEA/IRIV (Fig. 1), and it was supported by the presence of the specific mRNA in the lymph nodes of mice collected 24 and 48 h after i.n. administration of CEA/IRIV (Fig. 2). However, it was not possible to detect CEA transcripts 72 h after the mice inoculation.
CEA-specific CTL response in immunized mice
We also evaluated whether the CD40L/IRIV construct was able to improve the ability of a CEA-directed cancer vaccine (CEA/IRIV) in inducing a CEA-specific CTL response. Spleens were taken from the mice vaccinated as described above, and spleen cells were isolated and pooled from the different mouse groups.
Pooled spleen cells were then in vitro stimulated with autologous irradiated splenocytes transfected with CEA gene and cultured for 10 days in a medium containing low-dose IL-2 before being tested in 51Cr release assay against the P815 target cell clone expressing CEA. These experiments showed that the spleen cells from the mice that had received treatment with CEA/IRIV showed a significant CEA-specific antitumor activity. Our results also showed that the spleen cells from the mice that had received the combined treatment with CEA/IRIV and CD40L/IRIV elicited much higher lytic activity. Significantly different values (Bonferroni’s (with control) multiple-comparison test, = 0.05) were observed by comparing the results of CTL activity of mice immunized with CEA/IRIV (group B) and CEA/IRIV plus CD40L/IRIV (group D) against CEA-expressing cells vs the remaining groups. The lytic activity of these CTLs was CEA specific and class I MHC restricted, because they were not able to kill untransfected (not expressing CEA) P815 target cells and because their lytic activity was abrogated by the addition of anti-class I MHC mAbs in the cytotoxic assay (Fig. 4). The administration of IRIVs or CD40L/IRIV did not elicit any CEA-specific CTL response, and spleen cells from mice vaccinated with these constructs only gave rise to unspecific lytic activity that was not restricted by class I MHC molecules.
The CEA-CD40L combination enhances the expression of costimulatory molecules B7-1,2
CD40L binding to CD40 (receptor) activates intracellular signaling in the APCs and DCs that improves their presenting ability and leads to the up-regulation of the other adhesion/costimulatory molecules (27) on their surface. These latter molecules can provide the second signal required to activate naive T cells, amplify the immune response, and prevent anergy or tolerance induction. Therefore, we investigated whether our CD40L/IRIV construct could enhance, through CD40/CD40L interaction, the immune response against CEA, triggered by the CEA/IRIV vaccination. FACS analysis was performed from spleen cells isolated from the immunized mice administered with different IRIV constructs, and analyzed for the expression of B7.1 and B7.2 on class II MHC+ cells. The results of these experiments revealed a greater up-regulation of the B7.1 and, particularly, B7.2 molecules in mice receiving CD40/IRIV and CEA/IRIV together (group D) ( = 0.05) (Fig. 5). Moreover, besides the higher percentage of positive cells, a higher density of B7.1 and B7.2 molecules was expressed on the cells of group D mice. IRIVs alone were able to up-regulate the expression of B7.1,2 and MHC class II molecules on APCs, as described elsewhere (28), but immunization with this combination of two plasmids resulted in a much greater increase in B7 expression. On the contrary, the administration of CEA/IRIV (without the CD40L gene) to mice did not modify the level of B7 expression. These data suggest that CD40L/IRIV enhances the immunogenic potential of pCEA/IRIV by up-regulating the costimulatory molecules on APC and DCs, determining increased Ag-processing and -presenting capability of these cells.
CEA/IRIV and CD40L/IRIV induce the production of Th1 cytokines
Finally, we evaluated whether our treatments were also able to produce proinflammatory Th1 cytokines, such as IFN-. We examined the ability of the cultured spleen cells, pooled from the different groups of mice, to produce IFN- in response to CEA by a solid-phase sandwich ELISA. Interestingly, we found high levels of this cytokine only in the mice that had been immunized with the combination of CD40L/IRIV-CEA/IRIV (group D) (0.883 ± 0.3 ng/ml) (Fig. 6). The spleen cells from the other mouse groups, including those vaccinated with CEA/IRIV alone, did not produce a detectable level of IFN- in response to CEA. No significant differences in IFN- production were observed when spleen cells of the different groups were stimulated with Con A, indicating that CEA specifically induced the expression of IFN-. These data strongly suggest that the addition of CD40L/IRIV to CEA/IRIV promotes the differentiation of lymphocytes toward a protective Th1 phenotype. The result was further supported by an increase of CEA-specific IgG2a Ab titer in the group of mice immunized with CD40L/IRIV-CEA/IRIV (40 ± 2.32 ng/ml) in comparison with the titer of mice immunized with CEA/IRIV (15 ± 3.64 ng/ml) ( = 0.05).
Discussion
The active specific immunotherapy targeted against cancer is aimed at mobilizing the immune system and destroying tumor cells (6, 29). Many tumor-associated Ags have been selected as possible targets for therapeutic cancer vaccines (3, 30), and the possibility of generating a CTL-mediated immune response directed at them, such as CEA, has been demonstrated through many different strategies (18, 31, 32, 33, 34, 35). Several hypotheses have been formulated to explain these deluding results; some are related to the lack of induction of an efficient immune response and some to mechanisms of escape, activated by tumor cells (36, 37, 38, 39). The majority of these Ags are expressed at a low level in normal cells, but their immunogenicity is too weak to give rise to an efficient immune response. Thus, cancer cells can activate alternative escape mechanisms that make them resistant to the cancer vaccine activated immune effectors. In this context, therapeutic immunization can also be hampered by inadequate activation or by a low number of professional APCs, as often observed in cancer patients and chronic diseases (40, 41).
We hypothesized that the immunogenic and therapeutic potential of Ag-directed vaccination could be significantly enhanced by the contemporary administration of agents that might increase the expression of coaccessory molecules on APCs and direct the activation and expansion of specific T cells. We believe that the CD40L molecule can modulate the immune response against human malignancies. In fact, it is known that CD40L retains a central role in initiating the immune response, although it is expressed transiently on a small proportion of cells, and it can produce long-lasting systemic immune responses, capable of blocking disease progression. CD40L, interacting with its contrareceptor (CD40), enhances the Ag-specific T cell growth by two distinct mechanisms: 1) it activates cultured DCs, which consequently express a higher amount of class I and II MHC and costimulatory molecules, and 2) it has direct stimulatory effects (coaccessory signal) on T cells. On this basis, our aim was to study the possibility of enhancing antitumor protective immunity in BALB/c mice by in vivo modulating the immune response with CD40L. In our model, we chose human CEA as the target Ag, because it has been widely tested (31, 32, 33, 34, 35), and because currently several clinical investigations are using a variety of different CEA-directed vaccine approaches. In the present study, we designed a DNA-based vaccine that is the CEA recombinant plasmid included in IRIV. This vaccine construct (CEA/IRIV) can be safely administered i.n. to mice, giving rise to an efficient CTL-mediated immune response. We also administered IRIV containing a plasmid expressing the murine CD40L, to improve the functional activity of the professional APC. Previous studies have already shown that IRIV are rapidly and efficiently taken up by many human and murine APCs, including DCs (24, 25), that they significantly up-regulate the expression of DCs maturation markers, such as MHC class I and II, ICAM-1, B7.1, B7.2, and CD40, and they are able to deliver DNA into these cells, which is then rapidly expressed. The results of this study support the hypothesis that the enhanced expression of CD40L on APCs definitely improves the protective antitumor activity of the CEA/IRIV vaccine with a mechanism that could most likely be related to a DC maturation process, due to the CD40L/CD40 interaction. CD40 activation on these cells might abolish their tolerogenic capacity or even trigger the potential for immunogenic presentation of the Ag (42, 43). In fact, we found a significant CEA-specific CD8+ T cell response in the mice coinoculated with CEA/IRIV and CD40L/IRIV, that achieved the best protective immunity against CEA+ P815 cell challenge. The splenocytes of these mice also produced high levels of IFN- in response to CEA exposure, suggesting the occurrence of a Th1 response, which could significantly improve the level and the efficiency of the immune response. Although a CEA-specific CD8+ T response was also detected in mice inoculated with CEA/IRIV alone, it was much less efficient in terms of cytotoxic activity and protective immunity. Moreover, the splenocytes of these mice did not produce IFN- in response to CEA exposure. As expected, no CEA-specific CTL activity or protective immunity was observed in mice administered with CD40L/IRIV or pVax/IRIV constructs. The histology and the molecular analysis of the tissues drawn from the animals administered with the combination of CD40L/IRIV-CEA/IRIV revealed the depletion of all cells expressing CEA, whereas the groups of mice immunized with CEA/IRIV, CD40L/IRIV, or pVax/IRIV developed much larger tumor overexpressing CEA molecules. This result appears most likely due to the immunomodulating activity of CD40L associated with CEA. Furthermore, CD40 signaling, necessary for induction of Th-dependent T cell responses induced the expression of IFN- when splenocytes of the immunized mice were pulsed with CEA, providing evidence of an Ag-specific T cell proliferation (44, 45). In fact, the CD40L-CD40 interaction controls the balance between helper and regulatory T cells in immune response, releasing immature DCs from the control of regulatory CD4+CD25+ T cells (46) and breaking the immune tolerance against Ags. The increase of B7.1,2 expression on APCs of mice coimmunized with CEA/IRIV-CD40L/IRIV indicates that these cells have primed CEA-specific CD8+ T cells providing the secondary signals necessary to activate naive T cells and enhance the immune response against the tumor by these costimulatory molecules. In conclusion, we demonstrated that the i.n. administration of CD40L/IRIV in mice concomitantly with CEA/IRIV was able to induce an efficient tumor-protective immunity against CEA. CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, is a powerful immune adjuvant that was able to increase the efficacy of a vaccine specific for a poor immunogenic Ag, that could be an efficacious strategy for cancer therapy.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Address correspondence and reprint requests to Dr. Maria Grazia Cusi, Department of Molecular Biology, Virology Section, Siena University School of Medicine, Policlinico Le Scotte, Viale Bracci, 1, 53100 Siena, Italy. E-mail address: cusi{at}unisi.it
2 Abbreviations used in this paper: IRIV, immune-reconstituted influenza virosome; CEA, carcinoembryonic Ag; i.n., intranasal(ly).
Received for publication November 19, 2004. Accepted for publication March 15, 2005.
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