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- Material and methods
Generation of antigen-specific CD8+ T cell responses is considered optimal for an effective immunotherapy against cancer. In this study, we provide a proof of principle that in vitro observed diminished CD8+ T cell response provided a strong in vivo tumor protection. Immunization with an adenovirus vaccine containing ovalbumin (OVA) gene (Ad5-OVA) strongly induces antigen-specific CD8+ T cell responses measured in vitro using various immunological assays. However, in an attempt to augment the antigenic CD8+ T cell response, coinjection of a TLR9 agonist CpG ODN with the viral vaccine unexpectedly reduced the CD8+ T cell responses measured in vitro but provided a remarkably enhanced tumor protection compared to the CD8+ T cell response generated by Ad5-OVA vaccine alone. Interestingly, despite reduced ex vivo/in vitro CD8+ T cell responses following Ad5-OVA+CpG immunization, immunodepletion studies revealed that the augmented anti-tumor immunity was primarily dependent on CD8+ T cells. The magnitude and effector function of anti-OVA CD8+ T cells remain low following primary and secondary antigenic challenge, presenting a dichotomy between in vitro CD8 T cell responses and in vivo anti-tumor immunity. To examine the impact of CpG ODN, we observed that presence of CpG suppresses the CD8+ T cell proliferation both in vitro and in vivo. These data demonstrate that coadministration of adenovirus vaccine with a TLR9 agonist can generate potentially effective tumor-reactive CD8+ T cells in vivo. In addition, the results indicate that widely used standard immune parameters may not predict the vaccine efficacy containing a TLR9 agonist as adjuvant. © 2007 Wiley-Liss, Inc.
Therapeutic vaccinations are designed to stimulate an excess accumulation of antigen-specific T cells with effector functions that result in the destruction of antigen-bearing tumors. Since healthy individuals harbor a repertoire of auto-reactive T cells, use of cancer vaccines with tumor cell lines or tumor-specific antigens has been successful in breaking immune tolerance to these self-antigens.1, 2 There are multiple choices for the delivery of these antigens, such as RNA, DNA, whole protein, peptides, viral vectors or dendritic cells (DC). Immunizations using DCs loaded with the antigen strongly influence the effector T cell function with induced anti-tumor immunity.3, 4 Use of recombinant adenovirus as a genetic delivery system containing a gene of interest has been an attractive approach to elicit the immune response because of their ability to infect a broad range of cell types and a high propensity to transport the tumor antigens.5, 6 It has been demonstrated that use of adenovirus as a vehicle for antigen delivery as well as vaccine carrier strongly induces cytotoxic T cell response.7, 8, 9 They are further capable of modulating DC maturation by an increase in the expression of MHC antigens and costimulatory molecules.10
Current strategies to further improve the effectiveness of a vaccine formulation against the targeted tumor cells include combining adenovirus vaccines with an adjuvant capable of enhancing antigen presentation to DCs. A variety of adjuvants have been evaluated in immunization protocols but none have been successful in mediating the regression of the disease in advanced stages. Discovery of Toll-like receptor 9 (TLR9) ligands (commonly known as CpG ODN), containing nonmethylated CpG motifs have provided a family of novel and relatively safe Th1-promoting adjuvants.11, 12 Use of CpG as a monotherapy or an adjuvant has successfully demonstrated an induction of innate or adaptive T cell immunity where CpG induced IL-12 secretion by antigen presenting cells (APCs) which in turn activate NK cells to secrete IFN-γ, hence generation of an effective immune response depends on the presentation of antigen via APCs. Therefore, activation of APCs via CpG ODN appeared to be a prerequisite to stimulate functional antigen-reactive T cells.13, 14, 15
Various models have successfully demonstrated that addition of CpG ODN with the antigen during primary immunization cause an induced T cell function including tumor clearance and CD8 T cell proliferation.13, 16, 17 Several investigators observed that, in addition to its immune stimulatory effect, CpG ODN can also reduce the magnitude of CD8+ T cells and IFN-γ secretion.18, 19 However, these studies did not examine the impact on in vivo immune responses against the tumor challenge. Recently, we examined the sensitivity of such in vivo responses and demonstrated that despite diminished CD8+ T cell responses measured in vitro, there was an augmented anti-tumor response in vivo following immunization with Ad5-PSA and CpG ODN.20 Here in this study, we transform such observations in details to understand the basic parameters of clinical importance in a mouse tumor model using ovalbumin (OVA) as a model antigen. The ability of a combined approach of immunization (Ad5-OVA+CpG) was examined at multiple time points to analyze the magnitude and effector function of CD8+ T cells following primary (prophylactic) as well as secondary antigenic challenge (post-tumor analysis). The results conclude that clinical trials should be carefully designed when using the vaccine strategy in combination with TLR9 agonist as adjuvant.
- Top of page
- Material and methods
Various therapeutic approaches are being developed and tested to improve the treatment for cancer patients. Along with the vaccines, use of adjuvants is well recommended to further enhance the immune response both qualitatively as well as quantitatively. In this context, CpG ODNs are considered potent stimulators of innate and adaptive immune responses and are widely used in pre-clinical and clinical studies.13, 15, 22, 23 Here, we demonstrated that immunization with Ad5-OVA alone strongly induced the generation of antigen-specific CD8+ T cells, while in an attempt to further augment the T cell immune response, addition of CpG ODN to the Ad5-OVA vaccine suppresses the anti-OVA CD8+ T cell responses. When comparing the strength of generated immune responses following immunization with Ad5-OVA±CpG, it appeared that despite reduced anti-OVA CD8+ T cell response because of coinjection of CpG, mice developed an augmented anti-tumor immunity. These observations extend our previous study in a number of ways that (i) the observation is not unique to the PSA vaccine, but occurs with an Ad5-OVA vaccine in an identical manner; (ii) there is a reduction in total number of antigen-specific CD8+ T cells, not only T cells involved in CTL function and IFN-γ production; (iii) using cell depletion experiments, identifies the cells responsible for in vivo tumor destruction as CD8+ T cells; (iv) demonstrate that the reduced in vitro activity was observed from early to very late after immunization; (v) demonstrate by tetramer staining that mice immunized with Ad5-OVA + CpG had little to no expansion and contraction of antigen-specific CD8+ T cells as did mice immunized with Ad5-OVA alone; (vi) the reduction in anti-OVA CD8+ T cells is independent of the presence of OVA-expressing tumors; and (vii) that the reduction in anti-OVA CD8+ T cells is a function of reduced cell proliferation.
Various studies have shown that generation of powerful CTL response, trafficking of antigen-reactive T cells in circulation or into the lymph nodes, migration of antigen-reactive T cells to the site of infection, are certain possible mechanisms documented to eliminate the tumor development.24 While examining the trafficking of effector cell population to other lymphoid organs because of CpG ODN, it was evident that the decrease in antigen-specific CD8+ T cell response from the spleen is not due to the migration of anti-OVA CD8+ T cells to lymph nodes following immunization in presence of CpG. Similarly, a lower number of antigenic CD8+ T cells were observed in the blood from the Ad5-OVA+CpG immunized mice (unpublished data).
CpG ODN has the potential to activate a variety of different immune cells including NK or B cells that can contribute to anti-tumor activity.25 In experiments performed to determine the necessity of antibodies against OVA for the enhanced anti-tumor activity, we were not able to detect the level of IgA, IgM, IgG2a, b and c antibodies in serum samples from the group of mice immunized with Ad5-OVA+CpG (unpublished data). Such observations exclude the involvement of antibody mediated T cell cytotoxicity to the effector T cell function in the present scenario. Analyzing the participation of T cells in tumor clearance, immunodepletion studies showed that enhanced antigen-specific tumor clearance was predominantly CD8+ T cell-dependent whereas CD4+ T cell depleted mice were fully protected against the tumor challenge. However, a minor contribution of NK cells in tumor clearance was observed.
Although a correlation between in vitro immune response and in vivo tumor protection was not evident, it was possible that the magnitude of the CD8+ T cell response could be influenced by the vaccination in presence of CpG because of very dynamic nature of T cells.26, 27, 28 An initial burst of primary response determines the larger size of adaptive T cell response at the later stage and may provide improved anti-tumor activity.29 Alternatively, CpG ODN can also drive the CD4+ T cell response during the primary antigenic challenge to provide a base to augment the CD8+ T cell response during the secondary antigenic challenge (post tumor inoculation). In both cases, neither a higher antigenic CD4+ T cell response following primary immunization nor an enhanced anti-OVA CD8+ T cell response following tumor (secondary) challenge was observed.
In clinical settings, one of the approaches to immunize patients is to transfer the autologous tumor reactive T cells that produce higher IFN-γ and possess higher cytotoxic activity against the antigen-bearing tumor cells in vitro.30 However, due to the low success rate of immunotherapy, there has been a growing interest in exploring the number and nature of effector CD8+ T cells in vitro and in vivo. Some studies revealed that following immunization the patients that showed regression of tumors actually have low number of tumor reactive T cells compared to the patients having higher number of reactive T cells without any tumor regression.31, 32 It is also reported that fully functional effecter CD8+ T cells generated in vitro were less effective in vivo compared to the naïve early effector CD8+ T cells.33, 34 Retrospective analyses in melanoma patients revealed that generated CD8+ T cells for enhanced IFN-γ and cytolysis induced the clinical response only in a small minority of the patients.35, 36 In the present study, we clearly demonstrated a phenomenon that theoretically so called fully functional CD8+ T cells with higher CTL and IFN-γ production (in vitro) generated because of Ad5-OVA immunization paradoxically possess less in vivo anti-tumor immunity compared to the CD8+ T cells with reduced in vitro CTL activity and IFN-γ production following Ad5-OVA+CpG immunization. These data demonstrate that higher frequencies of tumor-specific IFN-γ-secreting CD8+ T cells do not necessarily ensure an augmented tumor protection, and should not be considered as a surrogate marker for vaccine efficacy.
DCs are the key regulators of immune effector function and can promote or suppress anti-tumor T cell responses depending upon the cytokine milieu and the signaling cascades that are activated. DC-based immunotherapeutic studies using tumor antigen to induce protective immune responses against the antigen-expressing tumor cells have been well documented.4, 37, 38 Both of the reagents used in the present study (adenovirus vaccine and CpG ODN) are separately proficient at enhancing DC maturation and stimulatory effects.10, 23, 39 To the best of our knowledge, the presence of CpG ODN in subcutaneous immunization protocols such as ours uniformly has been reported to promote the T cell expansion.16, 40, 41 Similarly, the presence of CpG ODN in intravenous or intraperitoneal immunization settings has been shown to induce CD8 T cell response.17, 42 In clinical studies, use of CpG ODN generates a strong peptide-specific CD8+ T cell response.12, 22, 43 To gain insight into the impact of CpG on CD8+ T cells, we observed that CpG ODN suppresses the rate of proliferation of antigen-specific CD8+ T cells both in vitro and in vivo. These observations at least partly explained the lower number of IFN-γ secreting CD8+ T cells, since production of IFN-γ depends on the rate of T cell proliferation.28, 44 A decrease in antigen-specific CD8+ T cells has also been reported using a mixture of adenovirus containing OVA or carcinoembryonic antigen (CEA) and CpG ODN.18, 19 In recent studies a critical role was reported for the route of the CpG injection in determining whether CpG would have immune suppressive effects (mediated via Treg induction) or immune stimulatory effects.18, 45
In contrast to adenoviral vaccine, co-injection of CpG ODN with plasmid vectors has been associated with increase CTL response.46, 47, 48 A few reports in prime-boost protocols described the use of recombinant vaccinia virus following antigenic peptide and CpG ODN to promote the CTL responses.49, 50 It will be interesting to understand if the dichotomy (suppressed CD8+ T cell responses of enhanced anti-tumor immunity) is unique to adenovirus in combination with CpG ODN since adenovirus is a DNA virus and may activate TLR9 on its own or other TLR ligands that interact with TLR9 pathway. DCs and macrophages are known to be able to cross-present virus like particles. It may be possible that virus like particles (as adenovirus in this case) facilitate cross-presentation by APCs, which are being simultaneously activated by CpG. This may lead to the presentation of antigen by activated APCs to induce an optimal CD8+ T cell response.51, 52, 53 Therefore, powerful induction of protective anti-tumor response in combinations of adenovirus with CpG may be due to altered processing capabilities of DCs and macrophages. Nonetheless, use of such a combined approach of immunization may permit the extension of this protocol in association with solid tumors of defined antigen to potentially provide a unique strategy for the generation of more effective in vivo adaptive T cell immunity, despite their lower in number.
Conclusively, this study highlights several unique observations with clinical importance that: (i) the combination of vaccine and CpG improves both survival and tumor growth kinetics; (ii) standard techniques for monitoring the immunological effects of vaccination failed to predict a benefit for the combination approach; (iii) coinjection of CpG diminishes the adaptive CD8+ T cell response by suppressing the anti-OVA CD8+ T cell expansion; (iv) challenges the assumption that a bigger T cell response is better in vivo. It is likely that the immunization mixture (Ad5-OVA+CpG) produced a microenvironment to select the CD8+ T cells that are advantageous in pathological conditions. Unnecessarily higher number of CD8+ T cells can cause a damage to host tissue system or can contribute towards the autoimmune disease, and a higher amount of IFN-γ may exert a pro-apoptotic effect on CD8+ T cell expansion.54, 55 It is apparent that developing an immunization protocol to selectively induce antigen-specific CD8+ T cell responses and enhanced anti-tumor immunity could prove a novel strategy in the field of immunotherapy where labor intensive, cost effective and potentially risky protocols are used to generate in vitro effector CD8+ T cells. We continue to explore the nature of the CD8+ T cells optimally required to generate effector function in vivo following this combined approach of immunization. It is possible that other soluble factors secreted by various immune cells play a critical role to provide microenvironment in favor of CD8 T cells. It remains a mystery though how a small proportion of antigen-specific CD8+ T cells can have higher in vivo effector function than a larger proportion.