In our experiments were obtained successfully from BALB/c mouse bone marrow precursors. These BM-DCs were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction (MLR). This study demonstrates the generation of an effective CTL response against gastric carcinoma cells by repeated ex vivo stimulation of T cells with tumor lysate-pulsed DCs.
DCs induce, sustain and regulate immune responses[13
]. Four stages of their development have been delineated: (1) bone marrow progenitors; (2) precursor DCs, which patrol through the blood, lymphatics and lymphoid tissues; (3) tissue-resident immature DCs, which possess high endocytic and phagocytic capacity permitting antigen (Ag)-capture; and (4) mature DCs, present within secondary lymphoid organs, expressing high levels of co-stimulatory molecules permitting Ag-presentation[14
Immature DCs are characterized by high capacity for antigen uptake but low T-cell stimulatory capacity. DCs mature because DC-mediated immune responses are more effective if DCs receive an activation signal. This signal can be microbial products such as lipopolysaccharide or unmethylated CpG motifs mimicking bacterial DNA[15,16
], inflammatory mediators such as TNFα and IL-6[17,18
], or T cell-derived signals such as CD40 ligand[19
]. Matured DCs up-regulate co-stimulatory molecules, secrete the T-cell differentiation factor IL-12[20
], and present antigens more effectively because of increased phenotypic stability and extended half-life of MHC class I- and II-molecules[21
]. Furthermore, immature DCs bear the danger of inducing non-proliferating, IL-10-producing T cells, whereas mature DCs promote the development of Th1 cells[22
In our experiment, c-kit+ hematopoietic progenitor cells from mouse bone marrow cells cultured with GM-CSF, IL-4 or TNFα for 8 d showed the character of typical mature DCs. Morphologically, these large cells with oval or irregularly shaped nuclei and many small dendrites. Phenotypically, FACS analysis showed that they had typical mature DCs phenotypic markers, and expressed high levels of Ia, DEC-205, CD11b, CD80 and CD86 antigen, and moderate levels of CD40. Functionally, these cells gained the capacity to stimulate allogeneic T cells. However, immature DCs cultured with cytokines for 5 d did not possess typical DCs phenotypic markers and could not stimulate allogeneic T cells.
The use of a DC-based tumor vaccine as a cellular adjuvant to induce tumor-specific protective immunity holds great promise for cancer patients. This is important for advanced stage tumors with poor responsiveness to chemotherapy, such as gastric cancer. Currently, no defined tumor-specific antigen is available for many tumors, including gastric cancer. Now, DCs can be pulsed with synthetic peptides or proteins derived from known tumor associated antigens (TAA) such as MUC1, Her-2/neu, tyrosinase, CEA or Melan-A/MART[9
In this study, tumor cell lysates were obtained by rapid freezing and thawing, and were regarded as tumor specific antigens. BM-derived DCs were pulsed with tumor lysates. Naïve mouse splenic T cells were primed in vitro
with SGC-7901 TP DCs or B16 TP DCs to elicit cytolytic reactivity against tumor cells. The results showed that primed T cells in vitro
with SGC-7901 TP DCs were able to induce specific CTL against SGC-7901 tumor cells, but not B16 tumor cells. Vaccination with DCs pulsed with tumor lysates has been shown to have efficient anti-tumor effects in many other tumor models and in clinical studies. Schnurr et al[23
] report that T cells specific for pancreatic carcinoma cells can be generated in vitro
by lysate-pulsed DCs and that the T-cell response can be enhanced by keyhole limpet hemocyanin (KLH). This in vitro
model can be applied to compare different strategies in the development of DC-based tumor vaccines. Primary clinical studies were performed on melanoma patients using DCs pulsed with peptides or loaded with tumor cell lysates. Kono et al[24
] report that tumor vaccination therapy with DCs pulsed with HER-2/neu-peptides may be a potential candidate for the novel treatment of gastric cancer patients. Nine gastric cancer patients with recurrent or unrescetable tumor were enrolled in the clinical trial. Vaccinations with DCs pulsed with HER-2 (p369) peptide were performed at 2-week intervals. In 3 of 9 patients, the tumor markers (CEA or CA19-9) were decreased after vaccination. Two patients had a tumor regression of more than 50%, and two presented a mixed response.
In summary, vaccination with bone marrow-derived dendritic cells pulsed with tumor cell lysates induced anti-tumor immunity specific to gastric cancer ex vivo. These results suggest that an evaluation of BM-DCs pulsed with tumor lysates against gastric cancer is an important next step in vivo. A trial evaluating this approach in mice is currently in preparation.