Murine β-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-γ production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL. (Cancer Res 2006; 66(2): 1169-76)
- innate immunity
- acute lymphoid leukemia
- NK cells
The key to developing an effective antitumor response is to understand why, initially, the immune system is unable to detect transformed cells and is subsequently tolerant of tumor growth and metastasis. Ineffective antigen presentation limits the adaptive immune response; however, we are now learning that the innate immune system of the host may first fail to recognize the tumor as posing a danger ( 1). For many years, innate immunity has been considered as a separate entity from the adaptive immune response and has been regarded as to be of secondary importance in the hierarchy of immune functions. However, it has become increasingly clear that the innate immune system has a much more important and fundamental role in host defense. By contrast with adaptive immunity, innate immune recognition is mediated by pattern recognition receptors (PRR), which recognizes components commonly found on the pathogen and not normally found in the host ( 2, 3). Cellular PRRs are expressed on effector cells of the innate immune system, including dendritic cells that function as professional antigen-presenting cells (APC) in adaptive immunity. This expression profile allows PRR to mediate events that are instructive to the subsequent adaptive immune response, such as inducing the expression of proinflammatory cytokines and chemokines and up-regulating costimulatory molecules necessary for T-cell activation ( 2, 4– 6). Thus, the innate immune system is essential for the activation of the adaptive immune response and its direction into a particular effector type and allows the adaptive immune response to discriminate between self and nonself ( 7, 8). However, up to date, immunotherapy strategies of malignancies using innate immunity activation mechanisms are still few.
β-Defensins are small antimicrobial peptides of the innate immune system produced in response to microbial infection of mucosal tissue and skin ( 9). Recently, Biragyn et al. showed that murine β-defensin 2 (MBD2) not only are chemotactic for immature dendritic cells through chemokine receptor CCR6 but also acted directly on immature dendritic cells as an endogenous ligand for Toll-like receptor 4 (TLR4), a PRR ( 10), inducing up-regulation of costimulatory molecules and dendritic cell maturation ( 11). In vivo, injection of mice with plasmid-containing cDNA for MBD2 linked to HIV ENV antigen induced mucosal as well as systemic immunity ( 12). DNA immunization with a fusion construct of MBD2 and a tumor antigen elicit antitumor immunity ( 13). Thus, MBD2 may point the way toward novel therapeutic approaches against tumor. However, up to date, the antitumor mechanisms of MBD2, its efficacy in various tumor models and by various administration strategies, remain largely unknown.
Despite intensive chemotherapy regimens, adult patients with acute leukemia achieve long-term survival in only one third of cases. Immunotherapy with acute leukemia cells modified to express immunomodulators is a promising approach for the treatment of leukemia ( 14– 22). Many leukemic cells are easy to harvest for manipulation. The state of minimal residual disease achieved after conventional chemotherapy is an ideal setting for trials of leukemia cell vaccines. To attempt a novel approach for overcoming the formidable mechanisms of tumor immune tolerance, here, we engineered a murine lymphoid leukemia cell line L1210 to secrete MBD2, the endogenous ligand for PRR, and evaluated the in vivo antileukemia activity of the MBD2-based tumor cell vaccine. We showed that MBD2-modified leukemia cells showed significantly reduced leukemogenicity and induced protective and therapeutic immunity. The antileukemia activity of MBD2 required CD8+ T cells, and of interest, natural killer (NK) cells were also essential for lymphoid leukemia rejection.
Materials and Methods
Mice. Female DBA/2 mice (H-2d), which were 6 to 8 weeks old, were purchased from Center for experimental animals, Chinese Academy of Medical Sciences. The animals were kept at the animal facility of Institute of Hematology according to the institute's guidelines.
Cell lines and cell culture. L1210, a murine lymphoid leukemia cell line of DBA/2 origin, was a generous gift of Prof. Sheng-Guo You (Laboratory of Immunology, Institute of Hematology, Peking Union Medical College, Tianjin, China). P815, a murine mastocytoma cell line, was purchased from Cell Bank of Chinese Academy of Medical Sciences. Both cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2.
Plasmid construction. The pIRES expression plasmid was purchased from BD Biosciences Clontech (Palo Alto, CA). pIRES vector carrying secretable MBD2 was created by the following procedure. Gene for mature MBD2 was cloned from lipopolysaccharide (10 ng/mL)–treated DBA/2 mice skin by reverse transcription-PCR (RT-PCR) using the following primers: def1 5′-GAACTTGACCACTGCCACACC-3′ and def2 5′-CCGACGCGTTCATTTCATGTACTTGCAAC-3′. The cDNA encoding the mature MBD2 fused with a mouse Igκ signal sequence was obtained by PCR amplification using a 78-bp 5′ primer corresponding to mouse Igκ signal sequence (63 bp) and 15 bp of annealing to the mature mouse MBD2 coding region. Recognition sequence of XhoI and MluI was added to the sense and antisense primers, respectively. The sequence of sense primer containing Igκ signal sequence was 5′-ATGGAGTCAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGAACTTGACCACTGC-3′. The PCR product was digested with XhoI and MluI and cloned into the same sites of pIRES vector, which contains a selective marker (the neomycin phosphotransferase gene). The construct was verified by the DNA sequencing and purified using a plasmid purification kit (Qbioscience, Irvine, CA).
Transfection of L1210 cells. The MBD2 expression plasmid or empty plasmid vector was transduced into L1210 cells using LipofectAMINE 2000 reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instruction. The cells were selected in medium containing 0.45 mg/mL G418 (Life Technologies) for 3 weeks. Independent G418-resistant clones were isolated and screened for MBD2 expression.
Expression of MBD2. After identifying MBD2 mRNA expression by RT-PCR, each transfectant (1 × 106 cells/mL) was cultured for 48 hours, and secreted MBD2 protein in the culture supernatant was analyzed by SDS-PAGE using a 16.5% Tris-Tricine running gel. Then, the gels were visualized by staining with Coomassie brilliant blue R250. For Western blot analysis, proteins were transferred electrically onto polyvinylidene difluoride membrane (Millipore, Bedford, MA) after SDS-PAGE. The membrane was first blocked with 5% nonfat milk in TBST [150 mmol/L NaCl, 25 mmol/L Tris, and 0.1% Tween 20 (pH 7.5)] for 1 hour at room temperature. Then, the membrane was incubated with diluted goat monoclonal antibody (mAb) against MBD2 (Santa Cruz Biotechnology, Santa Cruz, CA) in 0.5% nonfat milk-TBST at 4°C overnight followed by incubation with horseradish peroxidase–conjugated rabbit anti-goat mAb in 0.5% nonfat milk-TBST for another 2 hours at room temperature. Finally, the membrane was visualized by incubation with 3,3′-diaminobenzidine. Extensive washes were done with TBST between each two steps.
Biofunctional assay of MBD2. The bioactivity of MBD2 produced by L1210 transfectants was determined by the ability of cell supernatants to chemoattract immature dendritic cells. Isolation of murine immature dendritic cells was described previously ( 23). Briefly, bone marrow was collected from tibias and femurs of DBA/2 mice. Erythrocytes were lysed with ammonium chloride lysis buffer for 3 minutes at 37°C. The precursors were plated in RPMI 1640-5% FCS with 15 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ) and 10 ng/mL interleukin (IL)-4 (Peprotech). Three days later, the floating cells were removed and the adherent cells were replenished with fresh GM-CSF and IL-4-containing medium. Nonadherent cells were harvested on day 5. To confirm the phenotype of the immature dendritic cells, expression of cell surface markers, such as B7.2, CD40, CD11c, and I-Ad, were analyzed by flow cytometry. The FITC-CD86, FITC-CD40, FITC-CD11c, and FITC-I-Ad mAb and their isotype controls were purchased from Biolegend (San Diego, CA).
The migration of immature dendritic cells was assessed using 5-μm pore Transwells (Costar, Cambridge, MA). Immature dendritic cells (50 μL; 106/mL) were added to the upper compartment, and transfectant supernatants (600 μL) were added to the lower compartment. Cells were incubated for 2 hours at 37°C in 5% CO2. The Transwells were then removed, and immature dendritic cells that had migrated to the lower compartment were collected and counted with a reversed microscope. The rate of migration was expressed in percentages of migrated cells as a fraction of the total number of cells placed in the upper compartment.
Cell surface molecule expression analysis by flow cytometry. To examine the effect of MBD2 transfection on MHC class I and II and costimulatory molecule expression of L1210 cells, MBD2-transduced L1210 cells (L1210-MBD2), L1210 cells transfected with empty plasmid (L1210-p), and parental L1210 cells were stained by the following mAbs for flow cytometry analysis: FITC-H-2Kd, FITC-H-2Dd, FITC-I-Ad, FITC-CD80, and FITC-CD86 (Biolegend).
Growth curve analysis. To determine if the transfection of MBD2 had effects on growth of L1210 cells in vitro, a growth curve was generated by seeding cell lines at 105 cells/mL and counting cells using a hemocytometer under light microscopy by trypan blue exclusion method at 12-hour intervals for 84 hours.
In vivo immunization studies. For evaluation of leukemogenicity of genetically modified L1210 cells, 1 × 105 live L1210-MBD2 cells were injected i.p. into DBA/2 mice. L1210-p or wild-type L1210 cells were injected as controls. Cells were diluted in 200 μL PBS.
For evaluation of the induction of protective immunity by L1210-MBD2 cells, mice that had achieved long-term survival after the initial inoculation of L1210-MBD2 cells were rechallenged i.p. with 1 × 105 parental L1210 cells. Age-matched naive mice received the same amount of parental L1210 cells as controls.
To evaluate the therapeutic effect of L1210-MBD2 cells, 1 × 104 parental L1210 cells were inoculated i.p., and mice were treated with s.c. injections of 1 × 106 irradiated (10 Gy) L1210-MBD2 cells twice weekly, saline, or the same amount of irradiated L1210-p or parental L1210 cells were used as controls.
In vivo depletions. For in vivo antibody depletion experiments, DBA/2 mice were injected i.p. with 1 × 105 L1210-MBD2, L1210-p, or wild-type L1210 cells on day 0. Depletions of CD4+ T cells (hybridoma GK1.5; American Type Culture Collection, Manassas, VA) or CD8+ T cells (hybridoma 53-6.72; American Type Culture Collection) were done by i.p. injections of 150 μg mAb on days −3, −2, and −1 and twice weekly for 2 weeks ( 14). Efficiency of the depletion procedure was checked by flow cytometry on splenocytes from one mouse per group. Control mice received the same amount of rat IgG. NK depletions were done by i.p. injections of 20 μL anti–asialo GM1 (Wako Pure Chemical Industries, Osaka, Japan) on days −2, 0, 3, 7, and 14. To verify depletion of NK, splenocytes from one mouse per group were used in a NK assay against YAC-1 targets. Mice injected i.p. with normal rabbit serum at the same dose and schedule were used as controls.
CTL and NK cell assays. Spleens were collected, single-cell suspensions were prepared, and the erythrocytes were depleted with ammonium chloride lysis buffer. The cells were used as NK effector cells. For preparation of CTL effector cells, splenocytes (5 × 106) were cocultured with irradiated (100 Gy) L1210 cells (1 × 106) in 5 mL RPMI 1640 in the presence of recombinant IL-2 (20 units/mL). Five days later, splenocytes were harvested. The CTL and NK cytotoxicity was determined by a CytoTox96 Assay kit (Promega, Madison, WI). L1210 or irrelevant syngeneic control P815 tumor cells were used as targets for CTL, and YAC-1 cells were used as targets for NK cytotoxicity. Effector and target cells were seeded into the 96-well microtiter plate at various E:T ratios.
IL-12 ELISA. The splenocytes (5 × 106 cells/mL) from mice with different inoculations were cultured for 1 day. Supernatants were collected and assayed for IL-12 production by ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).
ELISPOT assay. IFN-γ-producing cells were determined by IFN-γ ELISPOT kit (R&D Systems) following the manufacturer's instructions. Freshly isolated splenocytes were plated at 106 cells per well in the presence or absence of 104 irradiated (100 Gy) L1210 cells. The plate was incubated for 24 hours at 37°C in a CO2 incubator and then incubated overnight at 4°C with detection antibody followed by a 2-hour incubation with streptavidin-alkaline phosphatase conjugate. The spots were visualized by adding alkaline phosphatase substrate and were counted by two independent investigators with background spots subtracted.
Statistical analysis. The statistical significance of differences between experimental groups of animals was determined using Student's t test. Survival curves were plotted using Kaplan-Meier estimates. The statistical survival analysis was done using the standard Mantel-Cox log-rank test. Differences were considered significant when P < 0.05.
Characterization of MBD2 transfectants. L1210 cells were stably transfected with the expression vector of MBD2 or the empty vector (designated as L1210-MBD2 and L1210-p, respectively). The supernatant of each transfectant cultured for 2 days was analyzed by Western blot with MBD2-specific mAb ( Fig. 1A ). Secreted MBD2 was observed in culture supernatant of L1210-MBD2 but not in that of L1210-p or parental L1210 cells.
To investigate the functional activities of secreted MBD2, the culture supernatant from L1210-MBD2, L1210-p, or L1210 was tested for its ability to chemoattract murine bone marrow–derived dendritic cells, which are known to express CCR6 ( 24, 25). The purity of murine bone marrow–derived dendritic cells and their immature phenotype was confirmed by expression of CD11c+ (57.21 ± 3.56%) and low levels of CD40 (11.52 ± 2.48%), B7.2 (14.83 ± 2.67%), and I-Ad (22.78 ± 3.11%). The results showed that the L1210-MBD2 supernatant chemoattracted the immature dendritic cells in a dose-dependent manner, whereas supernatants from L1210-p or L1210 did not induce chemotaxis of the immature dendritic cells ( Fig. 1B).
To determine if the transfection of MBD2 influence the expression of MHC and costimulatory molecules, parental and gene-modified L1210 cells were analyzed for MHC class I, class II, B7.1, and B7.2 expression. The data showed that parental L1210 cells expressed MHC class I H-2Kd molecules (50.85 ± 9.42%), whereas the expression level of H-2Dd, I-Ad, B7.1, and B7.2 was very low (0.54 ± 0.58%, 4.43 ± 0.71%, 2.48 ± 0.13%, and 0.20 ± 0.18%, respectively). Transfection of L1210 with MBD2 or empty vector did not show significant effect on the surface expression of these molecules (data not shown).
As shown in Fig. 1C, the in vitro growth characteristics of L1210-MBD2 were similar to those of L1210-p and parental L1210 cells. Furthermore, in preliminary study, we found that 10 Gy irradiation did not abrogate MBD2 expression for at least 4 days (data not shown).
Potent antileukemia activity of MBD2 with enhanced NK cell and CTL cytotoxicity and augmented IL-12 and IFN-γ production. The L1210 murine lymphoid leukemia model is highly aggressive. Cells (1 × 105) injected i.p. can cause a death of all mice within 16 days. As few as 1 × 102 L1210 cells result in uniform mortality within 32 days. Mice that survive for >50 days after inoculation of L1210 cells may be regarded as long-term survivors ( 26).
To evaluate the antileukemia response induced by MBD2 gene transfer, groups of syngeneic DBA/2 mice (n = 10) were injected i.p. with 105 live L1210-MBD2 cells or control L1210-p and wild-type L1210 cells and followed for survival ( Fig. 2 ). We observed that 80% of mice injected with L1210-MBD2 rejected the leukemia cells and survived until day 50 without developing any clinical signs of toxicity. In contrast, all mice inoculated with control L1210-p or parental L1210 cells died within 16 days. Mice injected with L1210-MBD2 cells showed significantly reduced leukemogenicity (P < 0.001, log-rank test).
To examine the mechanism underlying the antileukemia activity of MBD2, naive DBA/2 mice were inoculated i.p. with 1 × 105 live L1210-MBD2 or L1210-p or parental L1210 cells. Seven days after inoculation, splenocytes were isolated. For NK activity, the splenocytes were used directly in cytolytic assays against YAC-1 cells ( Fig. 3A ). Greatly augmented NK cell cytotoxicity was observed compared with those inoculated with L1210-p and wild-type L1210 cells (P < 0.05, Student's t test).
Specific CTL activity was observed in the isolated splenocytes following a 5-day coculturing with irradiated L1210 cells (100 Gy). Inoculation with L1210-MBD2 was sufficient to generate anti-L1210 CTL activity, significantly higher than that of L1210-p or parental L1210 group (P < 0.05). Lysis against irrelevant syngeneic control P815 cells was <10% at all E:T ratios, showing specificity ( Fig. 3B).
We then analyzed IL-12 and IFN-γ production of the isolated spleen cells. The secretion of IL-12 was markedly elevated in mice inoculated with L1210-MBD2 but undetectable in the rest of the groups ( Fig. 3C), and the number of IFN-γ-producing cells in L1210-MBD2 inoculated mice was 5-fold higher than those inoculated with L1210-p or parental L1210 cells ( Fig. 3D).
These results suggest that MBD2 has a strong in vivo antileukemia activity with augmented NK and CTL activity and enhanced IL-12 and IFN-γ production.
Protective immunity to parental tumor cells. Systemic protective immunity against parental L1210 rechallenge was analyzed in animals receiving L1210-MBD2 cells. Mice that had achieved long-term survival after initial inoculation of L1210-MBD2 cells were rechallenged with i.p. injection of 1 × 105 parental L1210 cells and followed for survival. All the mice remained alive for >6 months, whereas all the age-matched naive mice receiving the same amount of L1210 cells died within 15 days ( Fig. 4A ).
Splenocytes from the mice that survived the rechallenge of parental L1210 cells for 3 months were assayed for CTL activity. Splenocytes from naive mice were used as a control. Significantly augmented CTL activity against parental L1210 cells but not against irrelevant syngeneic tumor P815 was seen in spleen cells obtained from mice recovered from inoculation with L1210-MBD2 ( Fig. 4B). These results suggest that MBD2 induces a potent protective immunity, and memory CTLs were generated by inoculation with the L1210-MBD2 tumor vaccine.
Therapeutic effect of L1210-MBD2. To evaluate the therapeutic effect of irradiated L1210-MBD2 cells, mice were first injected i.p. with 104 live parental L1210 cells and treated on the same day with eight subsequent s.c. injections of irradiated (10 Gy) L1210-MBD2 cells delivered twice weekly. Saline and the same amount of irradiated L1210-p and parental L1210 cells were used as controls. The results showed that injections of irradiated L1210-MBD2 led to a significant therapeutic effect; 50% mice achieved long-term survival (P < 0.001, log-rank test). We also found that, compared with saline, irradiated L1210-p and parental L1210 cells could slightly prolong the survival time in some mice; the difference was not found to be statistically significant ( Fig. 5 ).
Involvement of NK cells and CD8+ but not CD4+ T cells in the induction of antileukemia activity by MBD2. To further investigate the role of CD4+ and CD8+ T cells and NK cells in mediating rejection of L1210-MBD2 cells, in vivo depletion was carried out by i.p. injection of anti-CD4 or anti-CD8 mAb or anti–asialo GM1 antiserum before and after L1210-MBD2 inoculation. Mice treated with an irrelevant rat monoclonal IgG or normal rabbit serum at the same dose and schedule were included as controls. Flow cytometry showed that the efficiency of CD4+ and CD8+ cell depletion was >98%, and NK depletion could abrogate 95% of the lytic activity against YAC-1 targets (data not shown). As shown in Fig. 6 , the administration of anti-CD8 and anti–asialo GM1 antibodies completely abrogated the antileukemia effect induced by L1210-MBD2 cells; all CD8+ and NK-depleted vaccinated mice developed leukemia at the comparable time as control animals, whereas with depletion of CD4+ T cells only 20% of the mice injected with L1210-MBD2 developed leukemia. These results suggest that NK cells and CD8+ T cells play critical roles in the induction of antileukemia activity by MBD2, but CD4+ T cells are not essential for it.
The host immune response to foreign challenge requires the coordinated action of both innate and acquired arms of the immune system. The innate immune response not only provides the first line of defense against microorganisms but also the biological context (the “danger signal”) that instructs the adaptive immune system to mount a response ( 27). Defensins comprise a family of cysteine-rich cationic polypeptides that contribute significantly to host defense against the invasion of microorganisms in animals, humans, insects, and plants. β-Defensins are constitutively expressed in the epithelial compartment and can be induced to higher levels of expression on infection and inflammation ( 28). In the present report, we examined the ability of MBD2-modified leukemia cells to induce antileukemic immune responses in a murine model of acute lymphoid leukemia (ALL). We found that ectopic expression of MBD2 in murine leukemia cell line L1210 did not affect the growth of leukemia cells and the expression of MHC and costimulatory molecules. Transduced L1210 cells secreted biologically functional MBD2. Most (80%) mice inoculated with 105 live L1210-MBD2 cells rejected the leukemic cells, suggesting that local secretion of MBD2 by L1210 cells can elicit effective antileukemic activity.
Although it is conceivable that antigens presented by the genetically modified leukemia cell vaccines are also expressed by normal hematopoietic cells, none of the immunized mice have noted the development of undesirable side effects, such as bone marrow aplasia or peripheral cytopenia (data not shown). Thus, no toxicity related to autoimmunity is anticipated as a result of MBD2 leukemia cell vaccine. Furthermore, spleen cells removed from immunized mice did not show any major changes in cell population (data not shown), suggesting that secretion of MBD2 modulates the immune system without causing overt systemic side effects.
A whole-cell vaccine approach by ex vivo gene transfer in acute leukemia would have many advantages (e.g., to date, only a few leukemia-associated antigens have been identified). The clinical usefulness of these antigens remains to be determined. In contrast, genetically modified leukemia cells would likely result in the presentation of multiple leukemia-associated antigens without requiring knowledge of the identity of these antigens. The efficacy of the MBD2-based leukemic cell vaccines provides evidence that leukemia-specific antigens exist on L1210 ALL cells and the antigens recognized by the immune system do not cross-react with epitopes on normal hematopoietic progenitors.
We also found that increased number of inoculated tumor vaccine cells may lead to decreased survival rate of mice: when injected with 106 L1210-MBD2, 50% of mice could achieve long-term survival (data not shown), suggesting that the kinetics between tumor cell growth and the induction of tumor-specific immune responses is a critical factor in the rejection of tumor cells in murine model, consistent with other's observations ( 29).
All the mice that had rejected initial L1210-MBD2 inoculation were resistant to the rechallenge of wild-type L1210 cells. Three months later, survived mice were assayed for CTL activity. Significantly enhanced CTL activity against L1210 but not the irrelevant syngeneic P815 cells was observed. These results suggest the surviving animals developed long-lasting protective immunity, which was specific for the L1210 leukemia line and did not cross-react with other syngeneic tumor lines. It was based on tumor-specific immune T-cell memory.
In therapeutic studies, we found that treatment with irradiated L1210-MBD2 could significantly prolong survival time and 50% mice achieved long-term survival. When the irradiated L1210-MBD2 cells were injected 1 week after the parental L1210 cells, 30% mice achieved long-term survival most likely due to a larger tumor burden (data not shown). The less efficient of therapeutic experiments than inhibition of leukemogenicity may be explained by the high malignancy of the L1210 cell line. As few as 1 × 102 L1210 cells result in 100% mortality within 32 days and may be related to the specific need of strong systemic immunity in therapeutic experiments. We also found that, compared with saline, irradiated L1210-p and parental L1210 cells could slightly prolong the survival time in some mice. Probably, irradiation of L1210 leukemia cells leading to increased apoptosis may facilitate tumor antigen take-up and processing; however, without the immunomodulatory role of MBD2, its therapeutic function is much less potent.
All the CD8+-depleted mice injected with L1210-MBD2 cells developed leukemia, and inoculation of naive mice with L1210-MBD2 resulted in enhanced CTL activity, suggesting the requirement for CD8+ T cells in leukemia rejection. In vitro studies have shown that MBD2 acts directly on immature dendritic cells as an endogenous ligand for TLR4, inducing up-regulation of costimulatory molecules and dendritic cell maturation, and MBD2-activated dendritic cells exhibited Th1-polarized responses, such as the production of IL-12 ( 11). The effective antileukemia activity and induction of potent T-cell immunity in the present study validates these in vitro observations. Enhanced IL-12 and IFN-γ production was found in mice inoculated with MBD2-modified leukemia cell vaccine. Because IL-12 stimulates cytotoxicity and proliferation of CTL and NK cells, IL-12 and the IFN-γ that it induces play central roles in the development of Th1-type immune responses and generation of CTL; the elevated production of IL-12 and IFN-γ may be involved in the MBD2 antileukemia activity.
Here, we found that NK cells were also critical in rejection of leukemia and markedly augmented NK cell cytotoxicity was observed in mice injected with L1210-MBD2. Based on recent clinical and experimental data, NK cells seem to play a crucial role in eradication of acute myeloid leukemia; in contrast, ALL often display a high degree of resistance, the mechanisms of which have not been fully elucidated ( 30). Thus, the requirement for NK cells in the antileukemia response of MBD2 in our murine model of ALL may be of interest. NK cell activation is regulated by the balance of signals that originate from a variety of activating and inhibitory NK-cell receptors ( 31– 34). Recently, it is reported that NK-cell resistance of ALL cells involves deficient engagement of activating natural cytotoxic receptors rather than activation of inhibitory receptors on NK cells ( 35). MHC class I chain-related antigen (MICA/B), the ligands for NK-cell activating receptor NKG2D, has been found to play important roles in NK-mediated killing in ALL ( 35). In another observation, the involvement of PVR and Nectin-2, the ligands for NK-cell activating receptor DNAM-1, has also been reported in the NK-mediated eradication of ALL ( 36). Whether MBD2 can up-regulate expression of ligands for NK-cell activating receptors directly or indirectly deserves further investigation. In addition, our observation that all CD8+ and NK-depleted vaccinated mice developed leukemia at the comparable time with control suggests that both CD8+ and NK cells are essential in the rejection of leukemia. It is possible there exist an indirect NK/T cell interaction mediated by dendritic cells in the antileukemia response induced by MBD2. It has been reported that activated NK induced by tumor cells can induce dendritic cell maturation and indirectly promote antitumor T-cell responses ( 37). Reciprocally, mature dendritic cells stimulate T cells that could directly activate NK cells through IL-2 secretion ( 38). Additional studies are necessary to elucidate the molecular mechanism underlying the antileukemia activity induced by MBD2.
Although it is generally accepted that priming of antitumor CD8+ T cells needs help, which can be provided by CD4+ T cells, CD4+ T cells were found not to be required for the antileukemia activity by MBD2. The unnecessary of participation of CD4+ T cells in tumor rejection has also been reported by other studies ( 39, 40), and by skewing the cytokine milieu to Th1 phenotype, in vivo elimination of CD4+ T cells may actually enhance the antitumor effect in cytokine gene therapies ( 41). Recently, Adam et al. showed that interplay between dendritic cells and NK cells can completely replace CD4+ T-cell help in the induction of CD8+ CTLs. They found that NK cells, which are activated by dendritic cells to secrete IFN-γ, might induce IL-12 expression in dendritic cells, which then prime a potent CTL response ( 42). Based on the data that inoculation of mice with L1210-MBD2 resulted in elevated production of IL-12 and IFN-γ, we speculate that dendritic cells may be activated by MBD2 to secrete IL-12, which activates NK cells to produce IFN-γ, and these cytokines may replace CD4+ T-cell help and are critical for CTL generation.
In conclusion, the potent antileukemia activity of MBD2 described in this study suggests that MBD2 provides a critical link between the innate immune system and the adaptive immune response. In addition, its activation of both innate and adaptive immunity may provide a novel therapeutic approach for the treatment of human lymphoid leukemia.
Grant support: National Natural Science Foundation of China grant 30471964 and Natural Science Foundation of Tianjin grant 05YFJMJC 02200.
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.
- Received August 15, 2005.
- Revision received October 30, 2005.
- Accepted November 10, 2005.
- ©2006 American Association for Cancer Research.