Inducing long-term survival with lasting anti-tumor immunity in treating B cell lymphoma by a combined dendritic cell-based and hydrodynamic plasmid-encoding IL-12 gene therapy

Hsin-Wei Chen1, Yi-Ping Lee1, Yu-Fen Chung1, Yan-Chung Shih1, Jy-Ping Tsai1, Mi-Hua Tao2 and Chou-Chik Ting1

1 Immunology Group, National Health Research Institutes, Taipei, Taiwan 2 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

The first two authors contributed equally to this work
Correspondence to: C.-C. Ting, Cooperative Laboratory, Veterans General Hospital, National Health Research Institutes, 201 Shih-Pai Road, Section 2, Taipei, Taiwan. E-mail: gting{at}nhri.org.tw
Transmitting editor: A. Singer


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In a previous study we showed that immunization with dendritic cells (DC) pulsed with idiotype (Id) fused with CD40 ligand (CD40L) could break the tolerance to Id which is expressed on B lymphoma cells and restored the responsiveness of Th cells, and, subsequently, induced IgG antibody response. However, this treatment had no therapeutic effect. In the present study, we found that using a hydrodynamic transfection-based technique, a high level of IL-12 production was noticed as early as 7 h after administering plasmid encoding IL-12 (pIL-12) and persisted at a detectable level for at least 9 days. In evaluating the efficacy of DC-based and/or IL-12 gene-based therapy in the treatment of 38C13 B cell lymphoma, it was found that either treatment alone was ineffective. However, a combined treatment induced 100% long-term survival. Furthermore, a long-lasting anti-tumor immunity was induced in these mice which resisted further tumor challenge at 58 days after initial inoculation. The surviving mice showed a strong IFN-{gamma}-producing Th cell response and humoral antibody response, but there were no detectable cytotoxic T lymphocytes. The antibody from the immune sera mediated a complement-dependent lysis of tumor cells that was tumor specific. Furthermore, immunization of mice with DC-based vaccine and pIL-12 treatment elicited higher levels of anti-Id IgG titer and an enhanced IgG2a response which increased the efficacy in mediating 38C13 tumor lysis. On examining the mechanism for this isotype change, we found that IFN-{gamma} production by CD4+ T cells is not the only determining factor for achieving a successful therapy. DC-based treatment alone could induce the increase of IFN-{gamma} production, but lacked any therapeutic effect. The deciding factor appears to be the abrogation of IL-4 production that was achieved by combing with IL-12 gene therapy. Our study provides a basis for exploring the combined use of cytokines or cytokine genes in DC-based treatment for achieving effective cancer immunotherapy.

Keywords: CD40 ligand, hydrodynamics-based transfection, idiotype, immunotherapy, tumor vaccine


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The primary goal of tumor immunotherapy is aimed at activating the body’s own immune defense system to fight and then to eliminate the existing tumor. However, immunotherapy has encountered a number of inherent difficulties in achieving a successful cancer treatment. They include the antigenic resemblance between the tumor and normal cells, the rapid growth of tumor cells with low immunogenicity, and the evasive nature of tumor cells to escape immune surveillance. These obstacles pose a serious problem for developing an effective tumor vaccine. It is often more difficult to perform immunotherapy once the tumor growth is established. Even if a therapeutic effect can be obtained in some approaches, the survivors may lack lasting immunity and the residual tumor may recur. We have been searching for an effective way or ways to deal with these issues. In the present study, the 38C13 B lymphoma was used as the tumor model. The idiotype (Id) of the Ig expressed on the surface of 38C13 B cell lymphoma can serve as a unique tumor-specific antigen. Dendritic cells (DC) play a crucial role in the immune system (1,2). It has been shown that DC are potent inducers for eliciting primary immune responses because they are highly efficient in antigen presentation (3,4) and in breaking T cell tolerance (5,6). Thus, there is a great expectation in utilizing DC as facilitators for inducing anti-tumor immunity. In previous work (7), we have demonstrated that DC pulsed with 38C13 Id–CD40 ligand (CD40L) fusion protein, consisting of 38C13 Id protein and CD40L extracellular domains, were able to up-regulate co-stimulatory molecule expression and IL-12 production. Mice immunized with DC pulsed with 38C13 Id–CD40L fusion protein induced Id-specific immune responses and the growth of tumor was significantly retarded (7). Due to the aggressive nature of the 38C13 B cell lymphoma, it is often difficult to completely eradicate this tumor in the in vivo hosts. Recently, cytokine therapy has been shown to give promising results in preclinical studies, such as with IL-12 (8,9) or Flt3 ligand (10,11). The exact mechanism of action has yet to be determined. Their effect appears to be mediated through augmenting the immune responses against tumors. Thus, exploiting the anti-tumor properties of these cytokines offers hope for a more effective cancer immunotherapy. To improve the therapeutic effect, we explored a new approach to combine DC-based therapy with plasmid encoding (pIL-12) in the treatment of 38C13 B cell lymphoma. The mechanism that is accounted for raising the combined treatment to a therapeutic threshold has been carefully examined.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and cell line
Female 6- to 8-week-old C3H/HeN mice were purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan. All mice were housed at the Laboratory Animal Facility, Veteran General Hospital—Taipei. All of the animal studies were approved by the Animal Committee of the National Health Research Institutes and were performed according to their guidelines. The carcinogen-induced 38C13 B cell lymphoma has be described previously (12).

Preparation of pIL-12
The procedure for the construction of pIL-12 has been previously described (13). The control plasmid was pTCAE that contained two cytomegalovirus promoters and two bovine growth hormone polyadenylation sequences to drive p35 and p40 gene expression. The backbone of the control plasmid is the same as the IL-12 cDNA-containing plasmid. Plasmid DNA was purified from transformed Escherichia coli DH5{alpha} by EndoFree Plasmid Mega Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Determination of IL-12 gene expression in vivo
Mice were injected with 20 µg of pIL-12 or control plasmid in 1.6 ml of saline through the tail vein within 10 s. Serum samples were collected from the tail veins at 7 h, and 1, 2, 4, 6 and 9 days after plasmid DNA delivery. Serial dilutions of serum samples were tested for IL-12 levels by ELISA assays (R & D Systems, Minneapolis, MN) according to the manufacturer’s protocol.

Preparation of DC
DC were generated as previously described (7). Briefly, bone marrow cells were depleted of T, B and MHC class II+ cells by a cocktail of mAb and rabbit complement for 30 min at 37°C. The mAb used were GK1.5 (anti-CD4), 53-6.7 (anti-CD8), RA3-3A1 (anti-B220) and M5/114 (anti-MHC class II) (TIB-207, TIB-105, TIB-146 and TIB-120 respectively; ATCC, Manassas, VA). Cells (1 x 106) were dispensed in 24-well plates in 1 ml medium supplemented with 500 U/ml recombinant murine granulocyte macrophage colony stimulating factor (mGM-CSF) and 1000 U/ml recombinant mIL-4. Medium was replenished after 2 days by gently swirling the plates, aspirating the medium and then adding back fresh medium with cytokines. After 4 days of culture, non-adherent and loosely adherent cells were harvested by gently pipetting and replated at 1 x 106 cells/ml in fresh medium supplemented with 500 U/ml recombinant mGM-CSF and 3000 U/ml recombinant mIL-4. Medium with cytokines was replenished on day 6; cells were cultured overnight in the presence of medium alone or the Id–CD40L fusion proteins at 100 nM. The Id–CD40L fusion protein was prepared as described previously (7). On the following day, non-adherent and loosely adherent DC were harvested, washed and then resuspended in PBS for immunization.

In vivo immunotherapy of B cell lymphoma 38C13
C3H/HeN mice were challenged by s.c. inoculation with 500 B lymphoma cells 38C13. They were then divided into three groups—treated with DC pulsed with Id–CD40L then boosted with Id–CD40L fusion protein, pIL-12 or both (Fig. 2A). Tumor growth was observed 3 times a week during the first month and then the surviving mice were observed for up to 200 days.



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Fig. 2. Combined DC-based and IL-12 gene therapy. (A) Experimental protocols. Groups of C3H mice (n = 4–5) were challenged with 500 38C13 B lymphoma cells on day 0. Following challenge, mice received DC-based, plasmid pIL-12 or both treatments as indicated. Tumor size was assessed 3 times a week and the animals were sacrificed when the tumor reached 3000 mm3 in size. (B) Tumor incidence and (C) tumor volume of combined therapy. The tumor size of individual mice is plotted as a function of time after tumor cell inoculation. A representative of two experiments is shown. Giving DC-based or pIL-12 treatment alone had no therapeutic effect. Mice that received both DC-based and pIL-12 treatments induced long-term survival. The surviving animals were rechallenged on day 58 and remained tumor free for >100 days.

 
Measurement of Th function for IFN-{gamma} and IL-4 production
Survivors were boosted with 500 38C13 tumor cells at 190 days after primary challenge. One week later, mice were sacrificed and spleens were removed. Splenocytes were cultured in 24-well tissue culture plates (4 x 106/2 ml/well) with irradiated 38C13 tumor cells (20,000 rad) at a responder: stimulator ratio (R:S) of 20:1 or 100:1. Three days later, supernatants were harvested from three wells for each treatment to determine IFN-{gamma} production. To determine the profiles of the IFN-{gamma}- or IL-4-producing cells from mice receiving DC-based or combined DC-based plus pIL-12 treatment, splenocytes were obtained at 2 weeks after boost and were incubated with GK1.5 (anti-CD4) or 53-6.7 (anti-CD8). Subsequently, these cells were incubated with goat anti-rat IgG microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and separated by LS separation columns (Miltenyi Biotec) according to the manufacturer’s instruction. The CD4+, CD8+ or unfractionated splenocytes were then stimulated with 20,000 rad X-irradiated 38C13 cells in a 24-well plate. For each well, 1 ml of medium contained 5 x 105 of CD4+, CD8+ or unfractionated cells and 1.5 x 104 of irradiated 38C13 cells (stimulator). In addition, 1 x 106 naive splenocytes were added as feeder cells. Three days later, supernatants were harvested and the production of INF-{gamma} or IL-4 was determined by ELISA kits (R & D Systems) according to the manufacturer’s instruction.

Cell-mediated cytotoxicity assay
As described above, after 5 days of incubation, the remaining cells were harvested and incubated with 51Cr-labeled 38C13 target cells in 96-well U-bottom tissue culture plates at the specified E:T, at 37°C/5% CO2 in a humidified incubator. After 5 h, the plate was spun and 0.1 ml supernatant was removed from each well to measure 51Cr release. The percentage of cytotoxicity was determined as (sample c.p.m. – spontaneous c.p.m.)/(total c.p.m. – spontaneous c.p.m.) x 100%.

Measurement of humoral antibody response
The levels of anti-Id IgG in the serum samples were determined by titrating the samples in ELISA plates coated with purified 38C13 Id. Bound IgG was detected with horseradish peroxidase-conjugated goat anti-mouse IgG Fc (ICN, Aurora, OH). Color was generated by adding 2,2'-azino-bis(ethylbenzthiazoline sulfonic acid) (Sigma) and the absorbance at 405 nm was measured by an ELISA reader. The purified monoclonal anti-38C13 Id antibody was used as the reference. For measurement of IgG1 and IgG2a anti-Id isotypes, biotin-conjugated rat anti-mouse IgG1 (A85-1; PharMingen, San Diego, CA) and IgG2a (R19-15; PharMingen) were used as detectors. Streptavidin–horseradish peroxidase conjugate (PharMingen) was then added. Color was developed as described above. End-point titers were defined as the highest serum dilution that resulted in an absorbance value >0.1.

Complement-dependent antibody-mediated lysis
Aliquots of 50 µl of cell suspension containing 1 x 104 of 51Cr-labeled 38C13 or P815 target cells were added to a 96-well U-bottom microplate containing 10 µl of medium alone or serum samples at final dilutions of 1:20, 1:100 or 1:500. The plate was then incubated on ice for 30 min to allow antibody to bind to target cells. Then, the plate was spun for 5 min at 200 g. The supernatant was carefully removed and 0.2 ml of 1:10 dilution of rabbit complement was added to each well. The plate was incubated for 1 h at 37°C/5% CO2 in a humidified incubator. Following incubation, the plate was spun and 0.1 ml supernatant was removed from each well to measure 51Cr release. The percentage of cytotoxicity was determined as (sample c.p.m. – spontaneous c.p.m.)/(total c.p.m. – spontaneous c.p.m.) x 100%.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-12 gene expression in vivo
Transfection with pIL-12 has been shown to induce the production of biologically active murine IL-12 (13). In a previous study, we also found that intramuscular injection of pIL-12 into mice was able to elevate serum levels of IL-12 (14). Liu et al. and others (15,16) have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. In the present study, this procedure was adopted and pIL-12 was introduced by tail vein injection. To evaluate the expression of the murine IL-12 gene in vivo, we determined the time course of IL-12 serum levels after delivering the hydrodynamics-based plasmid encoding murine IL-12. As shown in Fig. 1, following plasmid DNA injection, a high level of IL-12 at 370 ± 185 ng/ml was reached at 7 h after DNA injection and then the levels declined quickly, but still maintained at a significantly higher level than control or naive mice (41 versus < 15 pg/ml) at 9 days after DNA injection. These results indicate that administering pIL-12 was able to induce IL-12 production in vivo. Although IL-12 gene therapy was found to be very effective in the treatment of a variety of tumors (8,9), we found that pIL-12 gene therapy alone was insufficient to induce significant therapeutic effect in the treatment of B cell lymphoma 38C13, a very aggressive tumor growth (Fig. 2B and C).



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Fig. 1. Time course of IL-12 expression after hydrodynamic plasmid pIL-12 delivery. Mice were injected with 1.6 ml of saline containing 20 µg of pIL-12 or control plasmid DNA. Sera were subsequently collected at the indicated time points. The IL-12 level was determined by ELISA assay. Each time point represents the mean and SD of four mice.

 
Combined treatment in B cell lymphoma immunotherapy
In a previous study (7) we found that mice which were immunized with DC pulsed with Id–CD40L could break the tolerance to Id and restored the responsiveness of Th cells to produce IFN-{gamma} upon stimulation with Id, and, subsequently, induced anti-Id IgG antibody response. However, this treatment has little therapeutic effect. Experiments were conducted to examine the anti-tumor efficacy of plasmid-encoding IL-12 combined with DC-based therapy for treating B cell lymphoma. The treatment protocol is shown in Fig. 2(A). Mice were challenged s.c. with 500 38C13 B lymphoma cells on day 0. Then they were divided into three groups: (i) 2 days later, one group of mice was immunized with DC pulsed with Id–CD40L (1 x 105 DC in 0.2 ml of PBS/mouse, by i.v. injection) and boosted with Id–CD40L fusion protein (10 µg/mouse) at 9 days after tumor inoculation, (ii) mice received pIL-12 treatment at 5 days after tumor inoculation or (iii) a combination of the two treatments. Tumor growth was observed 3 times a week. A representative experiment is shown in Fig. 2(B and C). The tumor volume of individual mice is plotted as a function of time after tumor cell inoculation. All mice treated with DC-based treatment alone or pIL-12 alone developed progressive tumor growth. In contrast, mice that received the combined treatment remained tumor free. The difference is statistically significant (P < 0.01, Wilcoxon rank-sum test). The 38C13 B cell lymphoma is a poorly immunogenic, highly metastatic tumor. To test whether the anti-tumor immunity was sustained in the surviving mice, they were re-challenged with 38C13 B lymphoma at 58 days after the first challenge. All mice resisted the second tumor challenge and no tumor growth was observed for >100 days after this challenge, whereas all the naive mice, which were challenged with 38C13 at the same time, developed progressive tumor growth (Fig. 2B and data not shown). These results indicate that long-lasting immunity was induced in a combined pIL-12 gene therapy and DC-based treatment.

Cell-mediated immunity
In vitro assays were performed to determine whether tumor-specific cellular immunity was elicited in surviving mice. As shown in Fig. 3(A), splenocytes obtained from surviving mice after re-challenge produced high levels of IFN-{gamma}, 786 ± 38 and 959 ± 22 pg/ml for R:S = 20:1 and 100:1 respectively. Induction of IFN-{gamma} production by Th cells in these mice was tumor specific (7). Stimulation with 38C13 tumor cells of splenocytes from naive mice produced negligible IFN-{gamma}. However, we were unable to demonstrate the existence of cytotoxic T lymphocytes (CTL) in these mice (Fig. 3B). Even the long-term survivors who were repeatedly boosted with viable 38C13 tumor cells in vivo and further stimulated with X-irradiated 38C13 tumor cells in vitro failed to show any CTL response. These results indicated that CTL might not play an important role in eradicating 38C13 tumor cells. Other factors are involved in 38C13 B lymphoma destruction in vivo.



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Fig. 3. Cell-mediated immunity in long-term survivors. Immune splenocytes were stimulated in vitro with irradiated 38C13 tumor cells at R:S = 20:1 or 100:1. (A) Stimulation for 3 days. IFN-{gamma} level in the supernatants was determined by ELISA assay. The results are plotted as mean ± SD of triplicate wells. (B) Five days later, cell-mediated cytotoxicity was performed at indicated E:T cell ratios. Cytotoxicity was determined by the percentage of 51Cr release. Each point represents the average of triplicate wells.

 
IL-12-modulated anti-Id humoral antibody response
Immunization with DC pulsed with Id–CD40L induced an anti-Id antibody response (7). Humoral responses were further determined in the long-term survivors. As shown in Fig. 4(A), serum samples from these mice contained high titers of anti-Id IgG antibody. To determine the effect of pIL-12 gene therapy on the anti-Id immune response to the vaccination with DC-based treatment, we examined the quantity (antibody titer) and quality (isotype profile) of anti-Id IgG antibodies after immunization. Mice were immunized with DC pulsed with Id–CD40L, then boosted with Id–CD40L fusion protein 1 week later or combined DC-based treatment with pIL-12. Mice without any treatment served as the control group. Serum samples were collected at 6 days after the last immunization. Anti-Id IgG antibodies were analyzed by ELISA assay. As shown in Fig. 4(B), vaccination of mice with combined treatment induced higher levels of anti-Id IgG antibodies than just receiving DC-based treatment alone (4610 ± 960 versus 2840 ± 940 ng/ml, P < 0.05, Wilcoxon rank-sum test). The profile of the anti-Id IgG antibody isotype is shown in Fig. 4(C). Both DC-based and DC-based plus pIL-12 treatment induced high and comparable levels of anti-Id IgG1. Nevertheless, the combined treatment induced significantly higher titers of anti-Id IgG2a than mice that received DC-based treatment alone (P < 0.01, Wilcoxon rank-sum test). It is shown that the ratios of anti-Id IgG1 to anti-Id IgG2a titer obtained from the sera of DC-based and DC-based plus pIL-12-treated mice are 24.3 and 0.8 respectively. These results indicate that pIL-12 injection enhanced IgG2a response.



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Fig. 4. (A) Antibody response in long-term survivor. Serum samples were collected from surviving animals of Fig. 2(B) at 190 days after primary challenge. Anti-Id IgG levels in the serum samples were determined by titrating the sera on ELISA plates coated with purified 38C13 Id. There was no detectable anti-Id IgG antibody in the serum samples from naive animals. Antibody response to DC-based and combined DC-based plus pIL-12 treatment. Mice were immunized with DC pulsed with Id–CD40L with or without pIL-12 and then boosted with Id–CD40L fusion protein. Sera were obtained at 6 days after immunization, and (B) anti-Id IgG levels and (C) anti-Id IgG1:IgG2a isotype profile were determined by ELISA assay. (D) Profiles of IFN-{gamma} and IL-4 production in splenocytes from mice receiving DC-based or combined DC-based plus pIL-12 treatment. Two weeks after the boost immunization, mice were sacrificed. Splenocytes were selected by immunomagnetic beads and stimulated with irradiated 38C13 tumor cells at R:S = 100:1. After 3 days, IFN-{gamma} and IL-4 levels in the supernatants were determined by ELISA assay.

 
To determine which lymphocyte population was responsive to 38C13 for inducing cytokine production, splenocytes were separated by immunomagnetic selection into CD4+ and CD8+ subpopulations. In all immunized groups, CD8+ T cells produced negligible or undetectable levels of IFN-{gamma}. In contrast, CD4+ T cells produced significant levels of IFN-{gamma} (Fig. 4D). These results indicate that CD4+ cells accounted for the bulk of IFN-{gamma} production. In mice that received combined pIL-12 and DC-based treatment, the IL-4 production was nearly completely shut-off.

Anti-tumor activity of the anti-Id antibody
Mice that received a combined DC-based treatment with pIL-12 elicited higher anti-Id IgG responses than mice treated with DC-based treatment alone. Immune sera obtained from DC-based treatment alone or combined with pIL-12 were adjusted to contain the same titer of anti-Id IgG. Experiments were performed to determine whether the antibodies from the immune sera could mediate lysis against the tumor cells. Serial dilutions of serum samples were incubated with 51Cr-labeled 38C13 or P815 cells. After adding complement, lysis of tumor cells was determined by measuring 51Cr release. As shown in Fig. 5, specific killing of 38C13 tumor cells was seen in the complement-dependent antibody-mediated lysis with the sera obtained from DC-based vaccination with or without pIL-12 treatment, but not sera from naive animals. In contrast, there was no significant lysis of P815 cells by incubation with sera from immunized or naive mice. Thus, the lysis was tumor specific. In addition, sera obtained from mice that received DC-based plus pIL-12 treatment were more effective in mediating 38C13 lysis than sera obtained from mice that received DC-based treatment alone. These results correlate with the therapeutic efficacy (Fig. 2B).



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Fig. 5. Complement-dependent cytotoxicity. Serum samples were obtained from DC-based, DC-based plus pIL-12 treatment and naive mice. Sera from the mice that received combined treatment were adjusted to contain the same titer of anti-Id IgG, by adding naive sera, as the sera from the mice that received DC-based treatment alone. Serial dilutions of sera were incubated in vitro with 51Cr-labeled 38C13 or P815 cells and rabbit complement. One hour later, complement-mediated cytotoxicity was determined by 51Cr release into supernatant. Each point represents the average of triplicate wells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we demonstrate that hydrodynamic administration of the IL-12 gene was able to modulate the immune response elicited by DC-based treatment. Combination of pIL-12 with DC-based treatment produces a synergistic effect that changes both the quantity (Fig. 4B) and quality (Fig. 4C) of the immune response that may lead to more effective tumor destruction (Fig. 5) and the induction of a long-lasting immunity (Fig. 2B).

The Id expressed on the B lymphoma cells is largely non-immunogenic and is tolerated by the host. Linking Id with CD40L enhances the immunogenicity (Huang, H. I., Wu, P. Y., Teo, C. Y., Chen, M. N., Chen, Y. C. and Tao, M. H. 2002. Enhanced antitumor immunity induced by fusion of CD40L to a self tumor antigen (submitted)). DC were found to be the most effective vehicle for delivering and processing antigen. Mice immunized with DC pulsed with Id–CD40L could elicit anti-Id T cell and humoral responses, but failed to produce any therapeutic effect against the tumor growth (7). These results indicate that increasing the immunogenicity of tumor vaccine combined with an efficient antigen delivery system may still be insufficient to generate an adequate anti-tumor immunity. Thus an adjuvant may be needed to overcome this deficiency. Trimmerman et al. have shown that linked Id with keyhole limpet hemocyanin could provide a needed adjuvant effect in the mouse 38C13 lymphoma model (17) and human clinical trials (18).

In many instances, cytokines play an important role in modulating the immune response and serve as a physiological adjuvant. They not only modulate the quantity of immune responses, but also the quality. A variety of cytokines were shown to exert the anti-tumor effect in the treatment of animal tumors (10,11,1923). Very often, these cytokines work at strengthening the effector arm of the immune response. Therefore, we explored the use of cytokines as an adjuvant for use in a DC-based immunotherapy of B cell lymphoma.

In our study, IL-12 gene therapy alone failed to curtail the growth of B lymphoma 38C13 (Fig. 2B). Nevertheless, a combination of DC-based and pIL-12 therapy led to a successful treatment of this aggressive tumor. IL-12 modulates a variety of immunoregulatory functions in the immune system to promote cell-mediated immunity (24). IL-12 can promote specific CTL activity and thus it has been used as a vaccine adjuvant to enhance the cell-mediated immune response (25). However, the CTL response could not be detected in our study, even after exhaustive searching by repeated in vivo challenge and in vitro stimulation (Fig. 3B and data not shown). Others have reported that DC-based vaccines (7,18), adenovirus-based vaccines (26) and DNA vaccines (27) were able to induce protection against 38C13 tumor challenge, but failed to detect cell-mediated cytotoxic activity. The in vivo adoptive transfer and T cell-depletion experiments also failed to demonstrate the existence of Id-directed cytotoxic cellular immunity (26,27). These results indicated that cytotoxic effector T cells did not seem to play a dominant role in 38C13 tumor cells destruction in vivo. It should be noted that 38C13 tumor cells are susceptible to the killing by activated T cells (28). In fact, under specialized conditions, CTL against 38C13 tumor cells could be induced by a different immunization protocol (29,30). Thus, the failure to show in most cases that a non-cell-mediated mechanism for the anti-tumor activity against 38C13 lymphoma is apparently not due to the resistance of 38C13 to CTL-mediated killing.

One consistent finding in our study is the maintenance of a high level of anti-Id antibody titers in the long-term survivors (Fig. 4A). The immune sera obtained from these mice were capable of mediating specific killing of 38C13 tumor cells through complement-dependent antibody-mediated lysis (Fig. 5). Campbell and others demonstrated that adoptive transfer of immune sera to naive recipients was protective against tumor challenge (27,31). Although we failed to detect enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC) by these sera (data not shown), its role cannot be excluded because ADCC may take place in the in vivo environment to participate in the eradication of tumor cells. These results indicate that the humoral antibody response appears to play a major role in mediating anti-tumor immunity in the 38C13 B lymphoma tumor model.

It has been shown that IL-12 can positively or negatively modulate the humoral immunity, depending on the Ig isotype. Administration of recombinant IL-12 with protein antigen enhanced Th1-associated IgG2a, IgG2b and IgG3 antibody responses (32,33), whereas the Th2-associated IgG1 response was suppressed (34). Co-administration of DNA vaccines with the IL-12 gene was found to selectively increase IgG2a antibody, but had little effect or even decreased the total antibody titer (3538). In our study using hydrodynamic administration of pIL-12, it is shown that combining the DC-based immunization with pIL-12 treatment elicited a 30-fold higher anti-Id IgG2a titer than immunization with DC-based treatment alone. In contrast, there is no significant effect on anti-Id IgG1 antibody elicited by DC-based immunization with or without pIL-12 treatment (Fig. 4C). In adoptive immunotherapy of 38C13 B lymphoma with anti-Id mAb by using isotype-switched variants, it has been shown that the IgG2a anti-Id mAb induced much stronger in vitro ADCC activity against the 38C13 tumor than anti-Id mAb of other IgG subclasses. Furthermore, anti-Id antibodies of the IgG2a subclass were 100-fold more potent in conferring in vivo tumor protection than their class-switched IgG1 counterparts (39). In the present study, we show that the switch of the Id-specific IgG isotype can be achieved under a more physiological condition by introducing IL-12 gene therapy combined with DC-based treatment (Fig. 4C). The reversal of the anti-Id IgG1:IgG2a ratio may be a key factor contributing to the increase of the anti-tumor efficacy of the combined treatment. This possibility is further supported by the fact that the immune sera obtained from mice that received combined treatment were more efficient in mediating tumor cell lysis than sera from mice that received DC-based treatment alone (Fig. 5). Furthermore, serum samples from long-term survivors contained high titers of anti-Id IgG antibody (Fig. 4A). These sera also contained a higher proportion of anti-Id IgG2a isotype and the Id-specific IgG1:IgG2a ratio was usually <1 (data not shown)

On examining the mechanism for the antibody isotype change that leads to tumor eradication, we found that IFN-{gamma} production by CD4+ T cells is not the only determining factor for achieving a successful therapy. DC-based treatment could induce IFN-{gamma} production (Fig. 4D), but any lacked therapeutic effect (Fig. 2B and C). The deciding factor appears to be the abrogation of IL-4 production that was achieved by combining with IL-12 gene therapy (Fig. 4D). It is likely that in the absence of IL-4, IFN-{gamma} further drives the Th pathway toward Th1 with the production of more IgG2a antibody. Thus, it appears that DC-based treatment increases the magnitude of immune response (quantity change) and IL-12 gene therapy promotes the isotype switch of antibody production (quality change). In the end, the combined anti-tumor efficacy is raised to a therapeutic threshold for the eradication of tumor growth.

Taken together, these results demonstrate that treating B cell lymphoma with a combined DC-based and pIL-12 therapy produced not only long-term survival, but also a long-lasting anti-tumor immunity. The mechanism for this anti-tumor effect appears to be mediated by a T cell-dependent humoral immunity. One consistent finding is that the long-term survivors of the combined therapy group maintained a high level of anti-Id antibody titers and the presence of antigen-specific IFN-{gamma}-producing CD4+ T cells. These results provide a basis for exploring the use of other cytokines or cytokine genes in a combined DC-based treatment for achieving a successful cancer immunotherapy.


    Acknowledgements
 
This study is supported by a supplement grant CA-091-SG-03 from the National Health Research Institutes.


    Abbreviations
 
ADCC—antibody-dependent cell-mediated cytotoxicity

CD40L—CD40 ligand

CTL—cytotoxic T lymphocyte

DC—dendritic cells

Id—idiotype

mGM-CSF—murine granulocyte macrophage colony stimulating factor

pIL-12—plasmid-encoding IL-12

R:S—responder:stimulator cell ratio.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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