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Vaccination with immature dendritic cells combined with CD40 mAb induces protective immunity against B lymphoma in hu-SCID mice
Biomedicine & Pharmacotherapy 64 (2010) 487–492
Original Article
Vaccination with immature dendritic cells combined with CD40 mAb induces protective immunity against B lymphoma in hu-SCID mice§ Yan Ge 1, Hong Xi 1, Xue-Guang Zhang * Biotechnology Research Institute, Soochow University, 708, Renmin Road, Suzhou, 215007 Jiangsu, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 October 2009 Accepted 25 January 2010 Available online 4 March 2010
Dendritic cells (DCs) pulsed by tumor antigens have been widely used as tumor vaccines to specifically trigger the cytotoxicity of CD8+ T cells. But the tumor microenviroment with enriched immunosuppressants hampered DC maturation and co-stimulation. CD40/CD40L signaling, one of the most important co-stimulatory molecules is capable of effectively skewing the immune response by promoting DCs maturation and co-stimulation. To establish a novel specific immunotherapeutic approach for the use of DC vaccine in the treatment of B lymphoma, hu-SCID mice bearing B lymphoma were vaccinated by different combination of tumor antigen pulsed DC or imDC vaccines and immuneenhancing agencies such as agonist CD40 mAb and T cells. The results of immature DCs combined with agonistic CD40 mAb were encouraging with achievement of tumor regression and induction of antigenspecific immune responses. These findings demonstrated the potential utility of imDC-based tumor vaccination combining with agonistic CD40 mAb in the treatment of malignant lymphoma. ß 2010 Elsevier Masson SAS. All rights reserved.
Keywords: CD40 mAb DC vaccine B lymphoma
1. Introduction Successful immunotherapy of tumor requires counteraction of tumor escape mechanisms and established immune tolerance. The goal is to eliminate malignant cells by a specific activation of antitumor immunity [1]. Anti-tumor immunity is mainly mediated by immune cells especially cytotoxic T lymphocytes. Dendritic cells (DCs) are most potent antigen-presenting cells that are highly effective at stimulating naı¨ve T cells to generate cytotoxic T lymphocytes [2]. Immature DC reside as sentinels in peripheral tissues where they capture antigens, mature and thereafter migrate via the afferent lymphatic vessels into the draining lymph nodes. Here, upon encounter with T cells, DCs are ‘‘licensed’’ to prime adaptive immune response through interaction between CD40 and CD40L.There is evidence that DCs play a key role in the induction of tumor-specific immune responses, especially via cross-priming which allows the transfer of antigens from tumor cells to DCs, their presentation through MHC-class I antigens and the generation of CD8+ cytotoxic T cells. Therapeutic cancer vaccination base on DCs is a new experimental immunotherapy for patients of cancer [3]. But aside from this immunogenic function,
§ This work is supported by National Key Technology Program (2009ZX09103704) and National High Technology Research and Development Program of China (2006AA02A254). * Corresponding author. E-mail address: [email protected] (X.-G. Zhang). 1 Yan Ge and Hong Xi have equally contributed to this paper.
0753-3322/$ – see front matter ß 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2010.01.004
there is increasing evidence that DCs can also induce antigenspecific unresponsiveness in lymphoid organs and in the periphery, and that, under certain circumstances, DCs may expand regulatory T-cells which are endowed with a strong capacity to suppress lymphocytes responses [4]. And except from Treg, the microenivroment of tumors is enriched with immune suppressive cytokines such as TGF-b and IL-10 that block maturation and impair differentiation of DCs [5]. Data collected from various malignancies indicated that tumor could actively recruit immature DCs to tumor site and impede their maturation and differentiation [6]. These immature DCs have reduced co-stimulatory activity and cannot induce anti-tumor immune responses. More importantly, if imDC fails to provide co-stimulatory signal, T cell tolerance or anergy will develop [7]. It has been hypothesized that whether DCs will induce immunity or tolerance to an antigen largely depends on the degree of maturation they reach [4]. Therefore, how to counteract maturation suspension and strengthen co-stimulation ability should be crucial to DC vaccination. CD40 is a transmembrane protein in the tumor necrosis factor (TNF) receptor superfamily. It is widely expressed on a variety of normal cells and a large portion of malanomas and carcinomas of the lung, breast, colon, ovary as well as all of B cell malignancies [8– 10]. Activation of CD40 signal is a promising candidate for cancer targeting therapy [11]. The agnostic mAbs targeting CD40 represent an attractive therapeutic strategy for a variety of carcinomas. Van Mierlo reported that systemic administration of agonistic anti-CD40 antibodies resulted in tumor eradication mediated via DC-induced CD8+T-cell responses. Anti-CD40 mAb in
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combination with irradiation or DCs resulted in CD8+T-celldependent immunity against B-cell lymphoma, suggesting that anti-CD40 mAb single or combining with other therapeutic agencies may provide a more potent therapeutic approach [12– 17]. ImDCs undergo phenotypic and functional changes to differentiate into their mature stage resulting in up-regulation of proteins specific for antigen presentation and T cell activation such as CD40. CD40 is among the best known signals able to induce DC maturation [18]. It was reported that CD8+ T cell could be activated without CD4+ Th1 cell through CD40 mediated signaling from DC. So, CD40 play a very important role in inducing DC maturation as well as initiating anti-tumor immunity. Therefore, we speculated that combining CD40 mAb with DC vaccination might probably get unexpected results. 5C11 is a murine mAb against human CD40 prepared by our lab which has been proved effective in tumor immunotherapy [15,19,20]. In present study, by using the hu-SCID mouse B lymphoma model, we try to modulate a novel, effective, safe and convenient DC vaccination method. Mo-DC in different maturation stages were administrated single or combined with DC-activated T cells, or CD40 agnostic mAb 5C11 to compare the efficacy of different formulation of DC vaccines. We also examined distribution of some important immune activate cells and cytokines to explore the underlying mechanism.
2.4. In vitro Cytotoxic T Lymphocyte (CTL) assays Daudi cells (2 105) were labeled with 3.7 106 Bq 51Cr according to the manufacture’s protocol. T cells activated by Daudi-pulsed DCs were added into microtiter plates containing 5000 Daudi cells/well. The ratios of T to tumor cells were 50:1. After incubation for 4 h at 5% CO2, 37 8C, supernatants were collected, and their radioactivity was measured using a gamma counter. Control groups were DCs (without tumor cell loading) induced CTL with daudi, daudi loading-DCs induced CTL with Raji and daudi loading-DCs induced CTL with XG2. 2.5. Establishment and identification of hu-SCID mice B lymphoma model Hu-SCID was established after treated with successive 4-day intraperitoneal injection of CTX (40 mg/kg/d) to inhibit the hemocytopoiesis. And then, human PBMCs (2 107) were engrafted into SCID mice. Four weeks after engraftment, B lymphoma model was established in hu-SCID by subcutaneous injection of Matri-gel (100 ml)-mixed Daudi cells (5 106/ mouse). The mice were monitored for every 2–3 days for tumor growth. 2.6. Treatment of tumor-burden mice
2. Materials and methods 2.1. Generation of immature dendritic cells Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation. CD14+ monocytes were enriched by magnetic cell separation (Miltenyi Biotec, Bergisch-Gladbach, Germany) to greater to 95% purity, as assessed by flow cytometry. The CD14+ monocytes were cultured in 24-well plates with RPMI-1640 medium containing 0.02 mmol/L L-glutamine, 100U/ml granulocyte-macrophage colonystimulating factor (GM-CSF, Berlex, Richmond, VA, USA), 500 U/ml IL-4 for 5 days at a density of 3 106 cell/ml. On day 3, fresh medium was added. The immature DCs were collected at day 5. 2.2. Preparation of dendritic cell vaccine B lymphoma cell line Daudi cells were thawed and cultured overnight at 5% CO2, 37 8C in RPMI 1640 medium (Gibco/BRL, Karlsruhe, Germany) containing 10% FCS, 2 mM L-glutamine and antibiotics. Tumor cells and immature DCs were mixed in a ratio of 3:1 and co-cultured for 16 h. After being washed by PBS, agonistic anti-CD40 mAb 5C11 (2 mg/ml) was added to the culture medium to stimulate DC maturation for the next 2 days. 2.3. Tumor cell pulsed dendritic cells promote T cell proliferation by mixed lymphocytes reaction (MLR)
Three weeks after tumor cells implantation, tumor–burdened mice were randomized to seven treatment groups (10 animals per group). A: 5C11(100 mg/mouse) i.p.; B: the mice were injected s.c. with immature DCs (1 106/mouse) + anti-CD40 agonistic mAb 5C11(20 mg/mouse); C: the mice were injected s.c. with mature DCs (1 106/mouse) + 5C11(100 mg/mouse); D: the mice were injected s.c. with mature DC (1 106/mouse) + T cells (1 107/ mouse) i.p.; E: the mice were treated by subcutaneous (s.c.) injection of mature DC (1 106/mouse); F: T cells(1 107/mouse) i.p.; G: no treatment group as blank control. Briefly:
A: 5C11; B: imDC + 5C11; C: mDC + 5C11; D: mDC + T; E: mDC; F: T; G: control.
For all the groups, the therapeutic DC vaccines were administrated once a week for 4 successive weeks. Tumor size was assessed every week by measuring the largest perpendicular diameters with a calliper and recorded as the tumor volume (V = 1/ 6pABC). Mouse survival ratios were monitored for 16 weeks. 2.7. B lymphoma rechallenge in recovered mice
T cells for mixed lymphocytes reaction (MLR) were enriched by immunomagnetic positive selection from PBMCs using a beadlabeled anti-CD3 mAb (Miltenyi Biotec, Bergisch-Gladbach, Germany) and MACS separation columns (Miltenyi Biotec, BergischGladbach, Germany). The purity of CD3+ T cells was greater than95%. In each sample, CD3+ T cells were co-cultured in 96-well round-bottom plates (Costar, Cambridg, MA) with tumor antigen loaded DCs at a ratio of 10:1. MLR cultures were incubated for 72 h at 5% CO2, 37 8C. To assess T cell proliferation, mixed cells were pulsed with (3H) thymidine (0.5 mCi/ml, The Third Atomic Energy Institute of China, China) during the last 16 h of culture. Then, the samples were transferred onto glass fiber-filter paper for analysis using a beta scintillation counter (Pharmacia, Sweden).
Mice of group B witnessed tumor elimination with the treatment of imDC + 5C11. three months after recovery, the mice were rechallenged with the same tumor burden to observe the tumor resistance. 2.8. Evaluation of IFN-g secretion One week after the last treatment, mouse serum was collected from the peripheral blood to evaluate the concentrations of human serum IFN-g during immunotherapy. Human IFN-g levels were determined by ELISA kit (BD Bioscience, San Jose, CA, USA).
Y. Ge et al. / Biomedicine & Pharmacotherapy 64 (2010) 487–492
2.9. Statistical analysis Numerical data were expressed as means standard deviation (S.D.). ANOVA and Chi2 tests were performed to determine the differences in the means among the various treatment groups. p < 0.05 was considered statistically significant. SPSS 17.0 software package was used for analysis. The Kaplan–Meier survival curve was analyzed by the log-rank test with the Graphpad Prism 4.02 software.
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Table 1 T cell phenotypic analysis before and after Daudi-pulsed dendritic cells (DCs) stimulation for 24 h CD3+ T cells enriched by immunomagnetic positive selection from PBMCs were co-cultured with Daudi loaded DCs at a ratio of 10:1 for 24 h. Apoptostic Daudi-pulsed DCs could effectively stimulate T cell activation evidenced by up-regulating CD25 and CD28 expression. (%)
CD3
CD4
CD8
CD25
CD28
Before After
64.2 6.9 97.3 1.5*
43.8 2.2 48.0 5.6
23.4 5.1 49.3 4.3*
7.6 1.8 55.3 2.9#
47.4 4.0 64.8 1.7*
* p < 0.05; # p < 0.01.
3. Results 3.1. Tumor specific Mo-dendritic cells inducing and T lymphocyte priming Human monocytes were cultured with GM-CSF and IL-4 for 5 days followed by another 2 days in the presence of apoptostic Daudi cells together with anti-CD40 agnostic mAb 5C11. When analyzing by microscopy, cell cultures showed typical features of mature DCs: non-adherent, clustered and protruding veils. Flow cytometry analysis on day 7 revealed a strong expression of CD80, CD86 and CD83, which are the phenotypes of mature DCs. These results suggested that apoptostic Daudi cells in combination with 5C11 have the ability to induce the maturation of DCs. Tumor antigen loaded mature DCs were co-cultured with T cells for MLR. (3H) thymidine incorporation was performed to assess T cell proliferation (Fig. 1). Apoptostic Daudi-pulsed DCs could effectively stimulate CD4+ T cell and CD8+ T cell proliferation and upregulate the expression of CD25 and CD28 (Table 1). 3.2. Cytotoxicity of tumor specific CTLs primed by apoptotic Daudi pulsed dendritic cells CTLs that primed by Daudi-pulsed DCs were co-cultured with Daudi cells. Daudi-primed CTLs co-cultured respectively with Raji and XG2 were set up as control groups. Another control is DCs (without tumor cell loading) induced CTL co-cultured with daudi. Twenty-four hours later, Daudi cells of experiment group tend to conglomerate and 24.3 3.9% Daudi cells died. Forty-eight to 72 hours later, almost all the tumor cells died while no obvious difference could be detected by microscope for the three control groups. The 51Cr-release assay revealed that the most potent CTL response was induced by DCs pulsed by apoptostic Daudi cells (with 57.6 5.3% killing activity). The CTL responses in the two control groups were relatively limited. (Raji: 29.5 3.4%, XG-2:
Fig. 1. Daudi specific Mo-dendritic cells (Mo-Dcs) inducing and T lymphocyte priming. CD3+ T cells isolated from PBMCs were co-cultured with apoptostic Daudi loaded DCs or DC at a ratio of 10:1 or 20:1. T cell proliferation were evaluated by mixed lymphocytes reaction (MLR). Apoptostic Daudi-pulsed DCs could most effectively stimulate T cell proliferation at the ratio of 1:10.
Fig. 2. Cytotoxicity of tumor specific CTLs primed by apoptotic tumor cell pulsed dendritic cells (DCs). CTLs that primed by Daudi-pulsed DCs were co-cultured with Daudi cells to testifiy the cytotoxicity (lane1). Daudi loading-DCs induced CTL cocultured with Raji (lane3) and XG-2 (lane4), DCs (without tumor cell loading) induced CTL co-cultured with Daudi (lane1) were set as controls. The results indicated that CTLs primed by apoptotic Daudi-pulsed DCs were most strongly activated showing high specificity and potent efficiency to B lymphoma cell line Daudi. The CTL responses in the three control groups were relatively limited.
20.1 4.0%). The killing activity of CTL stimulated by DCs without tumor loading was only 18.5 1.2%. (Fig. 2) The results indicated that CTLs primed by apoptotic Daudi-pulsed DCs were most strongly activated showing high specificity and potent efficiency to tumor. 3.3. Dendritic cell vaccination decreases tumor growth and increases survival ratio Three weeks after tumor cell implantation, tumor–burdened mice were administrated with therapeutic DC vaccines once a week and for four successive weeks. As shown in Fig. 3, three weeks after treatment with imDC+5C11, tumor shrinked with more lymphocyte infiltration. Six weeks later, tumor disappears almost. Compared with control group, without any treatment, tumor grew constantly with obvious bleeding and necrosis. Survival ratio showed that the complete regression ratio of group B (imDCs + 5C11) was 80%. Group C, D, E, F (C: mDC + 5C11 D: mDC + T, E: mDC, F: T) also witnessed 20–40% complete regression. While for group G (blank control), no complete regression was found. Four weeks after tumor transplantation, mouse of control group began to die and no survival by the end of week 8. For the therapeutic groups, mouse began to die at week 6 and survival periods were obviously prolonged. Among them, mouse treated by imDC + 5C11 could live more than 6 months. (Fig. 4). The tumor growth of each therapeutic groups (except group a: 5C11 only) was obviously lowered. Tumor volume shrinked. Group B (imDCs + 5C11) got the best effects (Fig. 5).
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Fig. 3. Tumor bearing hu-SCID mice treated by imDC + 5C11. A: before treatment; B: 3 weeks after first treatment; C: 6 weeks after first treatment; D: no treatment.
3.4. B lymphoma resistance in recovered mice Three month after recovery, five recovered mice of group B (daudi pulsed imDC + 5C11) were tumor rechallenged by subcutaneous injection of Matri-gel (100 ml)-mixed Daudi cells (5 106/mouse) to observe the tumor resistance. Tumor could be touched 8–10 weeks later, much prolonged than primary transplantation which was 2–3 weeks, indicating that Daudi pulsed imDC + 5C11 vaccinated mice have established specific anti-tumor immune response.
ELISA. During immunotherapy, the serum concentrations IFN-g increase significantly in most animals. One week after last treatment, IFN-g of mDC + T group was significantly higher than other groups, reaching the highest in the 2nd week and decreased thereafter. While IFN-g of imDC + 5C11 group witnessed a continuous elevation and reached almost the peak in the 3rd week. (Fig. 6). 4. Discussion
Serum IFN-g obtained from the animals’ peripheral blood 1week, 2 weeks and 3 weeks after treatment were determined by
As the most potent antigen presenting cells (APC), DCs are highly effective at stimulating T cell immunity. Therefore, DCs are increasingly being utilized for anti-tumor therapy due to their unsurpassed potency to initiate immune responses [21]. Recent strategies for developing therapeutic vaccines have focused on the ability to deliver antigen to DCs in a targeted and prolonged
Fig. 4. Survival ratios of tumor-burdened hu-SCID mice 1, 2, 4, 8 and 16 weeks after vaccination. Three weeks after Daudi implantation, tumor-burdened mice were randomized to seven treatment groups (10 animals per group). A: 5C11(100 mg/ mouse) i.p.; B: the mice were injected s.c. with immature dendritic cells (DCs) (1 106/mouse) + anti-CD40 agonistic mAb 5C11(20 mg/mouse); C: the mice were injected s.c. with mature DCs (1 106/mouse) + 5C11(100 mg/mouse); D: the mice were injected s.c. with mature DC (1 106/mouse) + T cells (1 107/mouse) i.p.; E: the mice were treated by subcutaneous (s.c.) injection of mature DC (1 106/ mouse); F: T cells(1 107/mouse) i.p.; G: no treatment group as blank control. Tumor–burdened mice were administrated with therapeutic DC vaccines once a week and for 4 successive weeks. Survival ratio showed that the complete regression ratio of group B (imDCs + 5C11) was 80%, obviously higher than other vaccination and control groups.
Fig. 5. Tumor volumes of tumor-burdened hu-SCID mice 1, 2, 3 and 4 weeks after vaccination. Daudi cells (5 106/mouse) were injected s.c. into the left inguinal region of hu-SCID. Three weeks after tumor implantation, tumor growth was palpable to approximately 1 mm3. The mice were randomized into seven groups for vaccination. A: 5C11; B: imDC + 5C11; C: mDC + 5C11; D: mDC + T; E: mDC; F: T; G: control. The tumor growth of each therapeutic groups (except group A: 5C11 only) was obviously lowered. Tumor volume shrinked. Group B (imDCs + 5C11) got the best effects.
3.5. Successful dendritic cell therapy is accompanied by enhanced IFN-g production
Y. Ge et al. / Biomedicine & Pharmacotherapy 64 (2010) 487–492
Fig. 6. Concentrations of IFN-g in tumor-burdened hu-SCID sera 1, 2 and 3 weeks after vaccination Serum IFN-g obtained from peripheral blood 1 week, 2 weeks and 3 weeks after vaccination were determined by ELISA. During immunotherapy, the serum concentrations IFN-g increase significantly in most animals. One week after last treatment, IFN-g of mDC + T group was significantly higher than other groups, reaching the highest in the 2nd week and decreased thereafter. While IFN-g of imDC + 5C11 group witnessed a continuous elevation and reached almost the peak in the 3rd week.
manner more specifically and induce the subsequent activation of T-cell immunity. Targeting DCs with an antigen-delivery system provides tremendous potential in developing new vaccines. However, antigen delivery to, and activation of, DCs is a complex problem, involving DC migration, maturation, antigen presentation. The complex ‘‘maturation’’ process involves not only the upregulation of co-stimulatory surface proteins and the optimization of antigen presentation capacities, but also the production of cytokines and chemokines that profoundly influence the outcome of the T cell response. Some inhibitory cytokines such as TGF-b, IL10, VEGF in tumor microenvironment inhibit DC maturation and antigen presentation [22]. Although immature DCs are able to migrate into tumor beds and to capture tumor-derived antigens for presentation to specific T cells, adaptive immune responses are often hampered in cancer patients by inhibit DC maturation and migration to lymph nodes where anti-tumor immunity was carried on. Therefore, reagents that can stimulate DC maturation are promising in order to obtain a more balanced immune response and to increase the efficacy of vaccines [23]. Current approaches prefer culture conditions that include maturation stimuli. But excessive stimulation during the ex vivo cell manipulations may exhaust DCs, making them incapable of secreting IL-12 later on upon encounter with T lymphocytes in the organism [24]. And finally, maturation stimuli reduce DC capacity to capture antigens, which will have a negative impact. So how to induce DC maturation and migration to lymph nodes similar as possible as it is in the environment in vivo remains a matter of investigation. CD40, member of tumor necrosis superfamily is among the best-known signals able to induce DC maturation. CD40 signaling could exclusively stimulate DC maturation in vitro. Maturing DCs rapidly migrate to afferent lymph nodes where they improve their antigen presentation and immunostimulatory capacity. Upon encounter with helper T-cells, DCs are ‘‘licensed’’ to prime the immune response through interaction between CD40 and CD40L [25]. CD40 engagement strongly induces CD25 expression on DCs and polarizes T cell responses toward Th1 and mediate anti-tumor cellular immunity [26]. So, CD40 agonistic mAb might be an efficient anti-tumor reagent in the way it induces DC maturation and enhances CTL responses. In our study, to establish a novel specific immunotherapeutic approach in the treatment of B lymphoma, tumor–burdened huSCID mice were vaccinated by different formulae of therapeutic DC vaccines such as immune-enhancing agencies agonist CD40 mAb
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(a) and ex vivo activated tumor specific T cells (f), tumor antigen pulsed mature DC (e), and different combination of immature DC + CD40 mAb (b), mature DC + CD40 mAb (c) as well as mature DC + ex vivo activated T cells (d). The results showed that compared with control group, some effectiveness have been got among the above vaccination groups. Immature DC + anti-CD40 agonistic mAb 5C11 witnessed the highest 80% complete regression ratio and the longest survival period. Other groups except 5C11 single (a) had 20–30% complete regression ratio. DCs from tumor-bearing mice are immature, tolerogenic, defective in antigen presentation and are poor stimulators of cellular immunity. We treat the mice with imDCs with antiCD40 mAb which could on the one hand stimulate DC maturation during their migration to lymph nodes, prevent the possible reverse differentiation and dysfunction due to lack of cytokines and on the other hand, CD40 signaling could also counteract the inhibitory effect by tumor-induced IL-10. Immunohistochemistry detected DC-activated T cell infiltration in tumor tissues, liver and spleen, indication CD40 mAb activated DC in vivo. Mounting data showed that T cell-mediated tumor rejection depend not only upon perforin, granzyme and Fas/FasL but on some soluble cytokines such as IFN-g [27]. During our immunotherapy, the serum concentrations IFN-g increase significantly in most animals, reaching the highest level after 2 weeks of treatment. IFN-g production of group b (imDC + 5C11) is the highest than that of other groups, suggesting CD40 signaling promote DC-primed T cell polarization to Th1. ImDC + 5C11 vaccinated mouse has more tumor specific DCs and DC-induced tumor specific cytotoxic T cells as well as high level IFN-g. Giving the same volume of tumor burden, these immune components could rapidly take part in anti-tumor responses and prolong the latent period of tumor. So, imDCs are able to trigger more powerful anti-tumor immunity than mDCs and exert the best outcome. To testify the anti-tumor effects in complete regressed therapeutic model, we randomly select five complete regressed mice to give them B lymphoma cell transplantation again. For the secondly injection, tumor formulation began 8–10 weeks later, much longer than the 1st time (2–3 weeks), indicating that the DC vaccinated mouse have specific anti-tumor imminity which could effective prohibit tumor growth in vivo (data not shown). For a newly formulated vaccine, data regarding safety should be required, next to data of pharmaceutical quality and efficacy. In this way, we sampled some organs from mouse of complete regressed therapeutic group (imDC + 5C11) for pathological examination. No tissue damage was found on heart, liver, lung, spleen, kidney and other important organs (data not shown). All these indicated that our vaccine was safety. This study illustrates that tumor-mediated immunosuppression can be circumvented by ex vivo manipulation of DCs and describes a means of improving the therapeutic efficacy of CD40based tumor vaccines. ImDC combined with anti-CD40 mAb could directly targeting malignant B lymphoma cells and effectively inhibit growth of B lymphoma which might eventually provide a novel idea for tumor therapy, not just for B lymphoma but also for many other malignant tumors. References [1] Berntsen A, Geertsen PF, Svane IM. Therapeutic dendritic cell vaccination of patients with renal cell carcinoma. Eur Urol 2006;50:11–3. [2] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–52. [3] Allgeier T, Garhmmer S, Nossner E, et al. Dendritic cell-based immunogens for B-cell chronic lymphocytic leukemia. Cancer Lett 2007;245:275–83. [4] Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 2007;7:610–21. [5] Pries R, Nitsch S, Wollenberg B. Role of cytokines in head and neck squamous cell carcinoma. Expert Rev Anticancer Ther 2006;6:1195–203.
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Original Article
Vaccination with immature dendritic cells combined with CD40 mAb induces protective immunity against B lymphoma in hu-SCID mice§ Yan Ge 1, Hong Xi 1, Xue-Guang Zhang * Biotechnology Research Institute, Soochow University, 708, Renmin Road, Suzhou, 215007 Jiangsu, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 October 2009 Accepted 25 January 2010 Available online 4 March 2010
Dendritic cells (DCs) pulsed by tumor antigens have been widely used as tumor vaccines to specifically trigger the cytotoxicity of CD8+ T cells. But the tumor microenviroment with enriched immunosuppressants hampered DC maturation and co-stimulation. CD40/CD40L signaling, one of the most important co-stimulatory molecules is capable of effectively skewing the immune response by promoting DCs maturation and co-stimulation. To establish a novel specific immunotherapeutic approach for the use of DC vaccine in the treatment of B lymphoma, hu-SCID mice bearing B lymphoma were vaccinated by different combination of tumor antigen pulsed DC or imDC vaccines and immuneenhancing agencies such as agonist CD40 mAb and T cells. The results of immature DCs combined with agonistic CD40 mAb were encouraging with achievement of tumor regression and induction of antigenspecific immune responses. These findings demonstrated the potential utility of imDC-based tumor vaccination combining with agonistic CD40 mAb in the treatment of malignant lymphoma. ß 2010 Elsevier Masson SAS. All rights reserved.
Keywords: CD40 mAb DC vaccine B lymphoma
1. Introduction Successful immunotherapy of tumor requires counteraction of tumor escape mechanisms and established immune tolerance. The goal is to eliminate malignant cells by a specific activation of antitumor immunity [1]. Anti-tumor immunity is mainly mediated by immune cells especially cytotoxic T lymphocytes. Dendritic cells (DCs) are most potent antigen-presenting cells that are highly effective at stimulating naı¨ve T cells to generate cytotoxic T lymphocytes [2]. Immature DC reside as sentinels in peripheral tissues where they capture antigens, mature and thereafter migrate via the afferent lymphatic vessels into the draining lymph nodes. Here, upon encounter with T cells, DCs are ‘‘licensed’’ to prime adaptive immune response through interaction between CD40 and CD40L.There is evidence that DCs play a key role in the induction of tumor-specific immune responses, especially via cross-priming which allows the transfer of antigens from tumor cells to DCs, their presentation through MHC-class I antigens and the generation of CD8+ cytotoxic T cells. Therapeutic cancer vaccination base on DCs is a new experimental immunotherapy for patients of cancer [3]. But aside from this immunogenic function,
§ This work is supported by National Key Technology Program (2009ZX09103704) and National High Technology Research and Development Program of China (2006AA02A254). * Corresponding author. E-mail address: [email protected] (X.-G. Zhang). 1 Yan Ge and Hong Xi have equally contributed to this paper.
0753-3322/$ – see front matter ß 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2010.01.004
there is increasing evidence that DCs can also induce antigenspecific unresponsiveness in lymphoid organs and in the periphery, and that, under certain circumstances, DCs may expand regulatory T-cells which are endowed with a strong capacity to suppress lymphocytes responses [4]. And except from Treg, the microenivroment of tumors is enriched with immune suppressive cytokines such as TGF-b and IL-10 that block maturation and impair differentiation of DCs [5]. Data collected from various malignancies indicated that tumor could actively recruit immature DCs to tumor site and impede their maturation and differentiation [6]. These immature DCs have reduced co-stimulatory activity and cannot induce anti-tumor immune responses. More importantly, if imDC fails to provide co-stimulatory signal, T cell tolerance or anergy will develop [7]. It has been hypothesized that whether DCs will induce immunity or tolerance to an antigen largely depends on the degree of maturation they reach [4]. Therefore, how to counteract maturation suspension and strengthen co-stimulation ability should be crucial to DC vaccination. CD40 is a transmembrane protein in the tumor necrosis factor (TNF) receptor superfamily. It is widely expressed on a variety of normal cells and a large portion of malanomas and carcinomas of the lung, breast, colon, ovary as well as all of B cell malignancies [8– 10]. Activation of CD40 signal is a promising candidate for cancer targeting therapy [11]. The agnostic mAbs targeting CD40 represent an attractive therapeutic strategy for a variety of carcinomas. Van Mierlo reported that systemic administration of agonistic anti-CD40 antibodies resulted in tumor eradication mediated via DC-induced CD8+T-cell responses. Anti-CD40 mAb in
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combination with irradiation or DCs resulted in CD8+T-celldependent immunity against B-cell lymphoma, suggesting that anti-CD40 mAb single or combining with other therapeutic agencies may provide a more potent therapeutic approach [12– 17]. ImDCs undergo phenotypic and functional changes to differentiate into their mature stage resulting in up-regulation of proteins specific for antigen presentation and T cell activation such as CD40. CD40 is among the best known signals able to induce DC maturation [18]. It was reported that CD8+ T cell could be activated without CD4+ Th1 cell through CD40 mediated signaling from DC. So, CD40 play a very important role in inducing DC maturation as well as initiating anti-tumor immunity. Therefore, we speculated that combining CD40 mAb with DC vaccination might probably get unexpected results. 5C11 is a murine mAb against human CD40 prepared by our lab which has been proved effective in tumor immunotherapy [15,19,20]. In present study, by using the hu-SCID mouse B lymphoma model, we try to modulate a novel, effective, safe and convenient DC vaccination method. Mo-DC in different maturation stages were administrated single or combined with DC-activated T cells, or CD40 agnostic mAb 5C11 to compare the efficacy of different formulation of DC vaccines. We also examined distribution of some important immune activate cells and cytokines to explore the underlying mechanism.
2.4. In vitro Cytotoxic T Lymphocyte (CTL) assays Daudi cells (2 105) were labeled with 3.7 106 Bq 51Cr according to the manufacture’s protocol. T cells activated by Daudi-pulsed DCs were added into microtiter plates containing 5000 Daudi cells/well. The ratios of T to tumor cells were 50:1. After incubation for 4 h at 5% CO2, 37 8C, supernatants were collected, and their radioactivity was measured using a gamma counter. Control groups were DCs (without tumor cell loading) induced CTL with daudi, daudi loading-DCs induced CTL with Raji and daudi loading-DCs induced CTL with XG2. 2.5. Establishment and identification of hu-SCID mice B lymphoma model Hu-SCID was established after treated with successive 4-day intraperitoneal injection of CTX (40 mg/kg/d) to inhibit the hemocytopoiesis. And then, human PBMCs (2 107) were engrafted into SCID mice. Four weeks after engraftment, B lymphoma model was established in hu-SCID by subcutaneous injection of Matri-gel (100 ml)-mixed Daudi cells (5 106/ mouse). The mice were monitored for every 2–3 days for tumor growth. 2.6. Treatment of tumor-burden mice
2. Materials and methods 2.1. Generation of immature dendritic cells Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation. CD14+ monocytes were enriched by magnetic cell separation (Miltenyi Biotec, Bergisch-Gladbach, Germany) to greater to 95% purity, as assessed by flow cytometry. The CD14+ monocytes were cultured in 24-well plates with RPMI-1640 medium containing 0.02 mmol/L L-glutamine, 100U/ml granulocyte-macrophage colonystimulating factor (GM-CSF, Berlex, Richmond, VA, USA), 500 U/ml IL-4 for 5 days at a density of 3 106 cell/ml. On day 3, fresh medium was added. The immature DCs were collected at day 5. 2.2. Preparation of dendritic cell vaccine B lymphoma cell line Daudi cells were thawed and cultured overnight at 5% CO2, 37 8C in RPMI 1640 medium (Gibco/BRL, Karlsruhe, Germany) containing 10% FCS, 2 mM L-glutamine and antibiotics. Tumor cells and immature DCs were mixed in a ratio of 3:1 and co-cultured for 16 h. After being washed by PBS, agonistic anti-CD40 mAb 5C11 (2 mg/ml) was added to the culture medium to stimulate DC maturation for the next 2 days. 2.3. Tumor cell pulsed dendritic cells promote T cell proliferation by mixed lymphocytes reaction (MLR)
Three weeks after tumor cells implantation, tumor–burdened mice were randomized to seven treatment groups (10 animals per group). A: 5C11(100 mg/mouse) i.p.; B: the mice were injected s.c. with immature DCs (1 106/mouse) + anti-CD40 agonistic mAb 5C11(20 mg/mouse); C: the mice were injected s.c. with mature DCs (1 106/mouse) + 5C11(100 mg/mouse); D: the mice were injected s.c. with mature DC (1 106/mouse) + T cells (1 107/ mouse) i.p.; E: the mice were treated by subcutaneous (s.c.) injection of mature DC (1 106/mouse); F: T cells(1 107/mouse) i.p.; G: no treatment group as blank control. Briefly:
A: 5C11; B: imDC + 5C11; C: mDC + 5C11; D: mDC + T; E: mDC; F: T; G: control.
For all the groups, the therapeutic DC vaccines were administrated once a week for 4 successive weeks. Tumor size was assessed every week by measuring the largest perpendicular diameters with a calliper and recorded as the tumor volume (V = 1/ 6pABC). Mouse survival ratios were monitored for 16 weeks. 2.7. B lymphoma rechallenge in recovered mice
T cells for mixed lymphocytes reaction (MLR) were enriched by immunomagnetic positive selection from PBMCs using a beadlabeled anti-CD3 mAb (Miltenyi Biotec, Bergisch-Gladbach, Germany) and MACS separation columns (Miltenyi Biotec, BergischGladbach, Germany). The purity of CD3+ T cells was greater than95%. In each sample, CD3+ T cells were co-cultured in 96-well round-bottom plates (Costar, Cambridg, MA) with tumor antigen loaded DCs at a ratio of 10:1. MLR cultures were incubated for 72 h at 5% CO2, 37 8C. To assess T cell proliferation, mixed cells were pulsed with (3H) thymidine (0.5 mCi/ml, The Third Atomic Energy Institute of China, China) during the last 16 h of culture. Then, the samples were transferred onto glass fiber-filter paper for analysis using a beta scintillation counter (Pharmacia, Sweden).
Mice of group B witnessed tumor elimination with the treatment of imDC + 5C11. three months after recovery, the mice were rechallenged with the same tumor burden to observe the tumor resistance. 2.8. Evaluation of IFN-g secretion One week after the last treatment, mouse serum was collected from the peripheral blood to evaluate the concentrations of human serum IFN-g during immunotherapy. Human IFN-g levels were determined by ELISA kit (BD Bioscience, San Jose, CA, USA).
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2.9. Statistical analysis Numerical data were expressed as means standard deviation (S.D.). ANOVA and Chi2 tests were performed to determine the differences in the means among the various treatment groups. p < 0.05 was considered statistically significant. SPSS 17.0 software package was used for analysis. The Kaplan–Meier survival curve was analyzed by the log-rank test with the Graphpad Prism 4.02 software.
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Table 1 T cell phenotypic analysis before and after Daudi-pulsed dendritic cells (DCs) stimulation for 24 h CD3+ T cells enriched by immunomagnetic positive selection from PBMCs were co-cultured with Daudi loaded DCs at a ratio of 10:1 for 24 h. Apoptostic Daudi-pulsed DCs could effectively stimulate T cell activation evidenced by up-regulating CD25 and CD28 expression. (%)
CD3
CD4
CD8
CD25
CD28
Before After
64.2 6.9 97.3 1.5*
43.8 2.2 48.0 5.6
23.4 5.1 49.3 4.3*
7.6 1.8 55.3 2.9#
47.4 4.0 64.8 1.7*
* p < 0.05; # p < 0.01.
3. Results 3.1. Tumor specific Mo-dendritic cells inducing and T lymphocyte priming Human monocytes were cultured with GM-CSF and IL-4 for 5 days followed by another 2 days in the presence of apoptostic Daudi cells together with anti-CD40 agnostic mAb 5C11. When analyzing by microscopy, cell cultures showed typical features of mature DCs: non-adherent, clustered and protruding veils. Flow cytometry analysis on day 7 revealed a strong expression of CD80, CD86 and CD83, which are the phenotypes of mature DCs. These results suggested that apoptostic Daudi cells in combination with 5C11 have the ability to induce the maturation of DCs. Tumor antigen loaded mature DCs were co-cultured with T cells for MLR. (3H) thymidine incorporation was performed to assess T cell proliferation (Fig. 1). Apoptostic Daudi-pulsed DCs could effectively stimulate CD4+ T cell and CD8+ T cell proliferation and upregulate the expression of CD25 and CD28 (Table 1). 3.2. Cytotoxicity of tumor specific CTLs primed by apoptotic Daudi pulsed dendritic cells CTLs that primed by Daudi-pulsed DCs were co-cultured with Daudi cells. Daudi-primed CTLs co-cultured respectively with Raji and XG2 were set up as control groups. Another control is DCs (without tumor cell loading) induced CTL co-cultured with daudi. Twenty-four hours later, Daudi cells of experiment group tend to conglomerate and 24.3 3.9% Daudi cells died. Forty-eight to 72 hours later, almost all the tumor cells died while no obvious difference could be detected by microscope for the three control groups. The 51Cr-release assay revealed that the most potent CTL response was induced by DCs pulsed by apoptostic Daudi cells (with 57.6 5.3% killing activity). The CTL responses in the two control groups were relatively limited. (Raji: 29.5 3.4%, XG-2:
Fig. 1. Daudi specific Mo-dendritic cells (Mo-Dcs) inducing and T lymphocyte priming. CD3+ T cells isolated from PBMCs were co-cultured with apoptostic Daudi loaded DCs or DC at a ratio of 10:1 or 20:1. T cell proliferation were evaluated by mixed lymphocytes reaction (MLR). Apoptostic Daudi-pulsed DCs could most effectively stimulate T cell proliferation at the ratio of 1:10.
Fig. 2. Cytotoxicity of tumor specific CTLs primed by apoptotic tumor cell pulsed dendritic cells (DCs). CTLs that primed by Daudi-pulsed DCs were co-cultured with Daudi cells to testifiy the cytotoxicity (lane1). Daudi loading-DCs induced CTL cocultured with Raji (lane3) and XG-2 (lane4), DCs (without tumor cell loading) induced CTL co-cultured with Daudi (lane1) were set as controls. The results indicated that CTLs primed by apoptotic Daudi-pulsed DCs were most strongly activated showing high specificity and potent efficiency to B lymphoma cell line Daudi. The CTL responses in the three control groups were relatively limited.
20.1 4.0%). The killing activity of CTL stimulated by DCs without tumor loading was only 18.5 1.2%. (Fig. 2) The results indicated that CTLs primed by apoptotic Daudi-pulsed DCs were most strongly activated showing high specificity and potent efficiency to tumor. 3.3. Dendritic cell vaccination decreases tumor growth and increases survival ratio Three weeks after tumor cell implantation, tumor–burdened mice were administrated with therapeutic DC vaccines once a week and for four successive weeks. As shown in Fig. 3, three weeks after treatment with imDC+5C11, tumor shrinked with more lymphocyte infiltration. Six weeks later, tumor disappears almost. Compared with control group, without any treatment, tumor grew constantly with obvious bleeding and necrosis. Survival ratio showed that the complete regression ratio of group B (imDCs + 5C11) was 80%. Group C, D, E, F (C: mDC + 5C11 D: mDC + T, E: mDC, F: T) also witnessed 20–40% complete regression. While for group G (blank control), no complete regression was found. Four weeks after tumor transplantation, mouse of control group began to die and no survival by the end of week 8. For the therapeutic groups, mouse began to die at week 6 and survival periods were obviously prolonged. Among them, mouse treated by imDC + 5C11 could live more than 6 months. (Fig. 4). The tumor growth of each therapeutic groups (except group a: 5C11 only) was obviously lowered. Tumor volume shrinked. Group B (imDCs + 5C11) got the best effects (Fig. 5).
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Fig. 3. Tumor bearing hu-SCID mice treated by imDC + 5C11. A: before treatment; B: 3 weeks after first treatment; C: 6 weeks after first treatment; D: no treatment.
3.4. B lymphoma resistance in recovered mice Three month after recovery, five recovered mice of group B (daudi pulsed imDC + 5C11) were tumor rechallenged by subcutaneous injection of Matri-gel (100 ml)-mixed Daudi cells (5 106/mouse) to observe the tumor resistance. Tumor could be touched 8–10 weeks later, much prolonged than primary transplantation which was 2–3 weeks, indicating that Daudi pulsed imDC + 5C11 vaccinated mice have established specific anti-tumor immune response.
ELISA. During immunotherapy, the serum concentrations IFN-g increase significantly in most animals. One week after last treatment, IFN-g of mDC + T group was significantly higher than other groups, reaching the highest in the 2nd week and decreased thereafter. While IFN-g of imDC + 5C11 group witnessed a continuous elevation and reached almost the peak in the 3rd week. (Fig. 6). 4. Discussion
Serum IFN-g obtained from the animals’ peripheral blood 1week, 2 weeks and 3 weeks after treatment were determined by
As the most potent antigen presenting cells (APC), DCs are highly effective at stimulating T cell immunity. Therefore, DCs are increasingly being utilized for anti-tumor therapy due to their unsurpassed potency to initiate immune responses [21]. Recent strategies for developing therapeutic vaccines have focused on the ability to deliver antigen to DCs in a targeted and prolonged
Fig. 4. Survival ratios of tumor-burdened hu-SCID mice 1, 2, 4, 8 and 16 weeks after vaccination. Three weeks after Daudi implantation, tumor-burdened mice were randomized to seven treatment groups (10 animals per group). A: 5C11(100 mg/ mouse) i.p.; B: the mice were injected s.c. with immature dendritic cells (DCs) (1 106/mouse) + anti-CD40 agonistic mAb 5C11(20 mg/mouse); C: the mice were injected s.c. with mature DCs (1 106/mouse) + 5C11(100 mg/mouse); D: the mice were injected s.c. with mature DC (1 106/mouse) + T cells (1 107/mouse) i.p.; E: the mice were treated by subcutaneous (s.c.) injection of mature DC (1 106/ mouse); F: T cells(1 107/mouse) i.p.; G: no treatment group as blank control. Tumor–burdened mice were administrated with therapeutic DC vaccines once a week and for 4 successive weeks. Survival ratio showed that the complete regression ratio of group B (imDCs + 5C11) was 80%, obviously higher than other vaccination and control groups.
Fig. 5. Tumor volumes of tumor-burdened hu-SCID mice 1, 2, 3 and 4 weeks after vaccination. Daudi cells (5 106/mouse) were injected s.c. into the left inguinal region of hu-SCID. Three weeks after tumor implantation, tumor growth was palpable to approximately 1 mm3. The mice were randomized into seven groups for vaccination. A: 5C11; B: imDC + 5C11; C: mDC + 5C11; D: mDC + T; E: mDC; F: T; G: control. The tumor growth of each therapeutic groups (except group A: 5C11 only) was obviously lowered. Tumor volume shrinked. Group B (imDCs + 5C11) got the best effects.
3.5. Successful dendritic cell therapy is accompanied by enhanced IFN-g production
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Fig. 6. Concentrations of IFN-g in tumor-burdened hu-SCID sera 1, 2 and 3 weeks after vaccination Serum IFN-g obtained from peripheral blood 1 week, 2 weeks and 3 weeks after vaccination were determined by ELISA. During immunotherapy, the serum concentrations IFN-g increase significantly in most animals. One week after last treatment, IFN-g of mDC + T group was significantly higher than other groups, reaching the highest in the 2nd week and decreased thereafter. While IFN-g of imDC + 5C11 group witnessed a continuous elevation and reached almost the peak in the 3rd week.
manner more specifically and induce the subsequent activation of T-cell immunity. Targeting DCs with an antigen-delivery system provides tremendous potential in developing new vaccines. However, antigen delivery to, and activation of, DCs is a complex problem, involving DC migration, maturation, antigen presentation. The complex ‘‘maturation’’ process involves not only the upregulation of co-stimulatory surface proteins and the optimization of antigen presentation capacities, but also the production of cytokines and chemokines that profoundly influence the outcome of the T cell response. Some inhibitory cytokines such as TGF-b, IL10, VEGF in tumor microenvironment inhibit DC maturation and antigen presentation [22]. Although immature DCs are able to migrate into tumor beds and to capture tumor-derived antigens for presentation to specific T cells, adaptive immune responses are often hampered in cancer patients by inhibit DC maturation and migration to lymph nodes where anti-tumor immunity was carried on. Therefore, reagents that can stimulate DC maturation are promising in order to obtain a more balanced immune response and to increase the efficacy of vaccines [23]. Current approaches prefer culture conditions that include maturation stimuli. But excessive stimulation during the ex vivo cell manipulations may exhaust DCs, making them incapable of secreting IL-12 later on upon encounter with T lymphocytes in the organism [24]. And finally, maturation stimuli reduce DC capacity to capture antigens, which will have a negative impact. So how to induce DC maturation and migration to lymph nodes similar as possible as it is in the environment in vivo remains a matter of investigation. CD40, member of tumor necrosis superfamily is among the best-known signals able to induce DC maturation. CD40 signaling could exclusively stimulate DC maturation in vitro. Maturing DCs rapidly migrate to afferent lymph nodes where they improve their antigen presentation and immunostimulatory capacity. Upon encounter with helper T-cells, DCs are ‘‘licensed’’ to prime the immune response through interaction between CD40 and CD40L [25]. CD40 engagement strongly induces CD25 expression on DCs and polarizes T cell responses toward Th1 and mediate anti-tumor cellular immunity [26]. So, CD40 agonistic mAb might be an efficient anti-tumor reagent in the way it induces DC maturation and enhances CTL responses. In our study, to establish a novel specific immunotherapeutic approach in the treatment of B lymphoma, tumor–burdened huSCID mice were vaccinated by different formulae of therapeutic DC vaccines such as immune-enhancing agencies agonist CD40 mAb
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(a) and ex vivo activated tumor specific T cells (f), tumor antigen pulsed mature DC (e), and different combination of immature DC + CD40 mAb (b), mature DC + CD40 mAb (c) as well as mature DC + ex vivo activated T cells (d). The results showed that compared with control group, some effectiveness have been got among the above vaccination groups. Immature DC + anti-CD40 agonistic mAb 5C11 witnessed the highest 80% complete regression ratio and the longest survival period. Other groups except 5C11 single (a) had 20–30% complete regression ratio. DCs from tumor-bearing mice are immature, tolerogenic, defective in antigen presentation and are poor stimulators of cellular immunity. We treat the mice with imDCs with antiCD40 mAb which could on the one hand stimulate DC maturation during their migration to lymph nodes, prevent the possible reverse differentiation and dysfunction due to lack of cytokines and on the other hand, CD40 signaling could also counteract the inhibitory effect by tumor-induced IL-10. Immunohistochemistry detected DC-activated T cell infiltration in tumor tissues, liver and spleen, indication CD40 mAb activated DC in vivo. Mounting data showed that T cell-mediated tumor rejection depend not only upon perforin, granzyme and Fas/FasL but on some soluble cytokines such as IFN-g [27]. During our immunotherapy, the serum concentrations IFN-g increase significantly in most animals, reaching the highest level after 2 weeks of treatment. IFN-g production of group b (imDC + 5C11) is the highest than that of other groups, suggesting CD40 signaling promote DC-primed T cell polarization to Th1. ImDC + 5C11 vaccinated mouse has more tumor specific DCs and DC-induced tumor specific cytotoxic T cells as well as high level IFN-g. Giving the same volume of tumor burden, these immune components could rapidly take part in anti-tumor responses and prolong the latent period of tumor. So, imDCs are able to trigger more powerful anti-tumor immunity than mDCs and exert the best outcome. To testify the anti-tumor effects in complete regressed therapeutic model, we randomly select five complete regressed mice to give them B lymphoma cell transplantation again. For the secondly injection, tumor formulation began 8–10 weeks later, much longer than the 1st time (2–3 weeks), indicating that the DC vaccinated mouse have specific anti-tumor imminity which could effective prohibit tumor growth in vivo (data not shown). For a newly formulated vaccine, data regarding safety should be required, next to data of pharmaceutical quality and efficacy. In this way, we sampled some organs from mouse of complete regressed therapeutic group (imDC + 5C11) for pathological examination. No tissue damage was found on heart, liver, lung, spleen, kidney and other important organs (data not shown). All these indicated that our vaccine was safety. This study illustrates that tumor-mediated immunosuppression can be circumvented by ex vivo manipulation of DCs and describes a means of improving the therapeutic efficacy of CD40based tumor vaccines. ImDC combined with anti-CD40 mAb could directly targeting malignant B lymphoma cells and effectively inhibit growth of B lymphoma which might eventually provide a novel idea for tumor therapy, not just for B lymphoma but also for many other malignant tumors. References [1] Berntsen A, Geertsen PF, Svane IM. Therapeutic dendritic cell vaccination of patients with renal cell carcinoma. Eur Urol 2006;50:11–3. [2] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–52. [3] Allgeier T, Garhmmer S, Nossner E, et al. Dendritic cell-based immunogens for B-cell chronic lymphocytic leukemia. Cancer Lett 2007;245:275–83. [4] Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 2007;7:610–21. [5] Pries R, Nitsch S, Wollenberg B. Role of cytokines in head and neck squamous cell carcinoma. Expert Rev Anticancer Ther 2006;6:1195–203.
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