| Objective: In order to estalish an efficient anti-tumor immunity , we designed different experimental strategies with or without DCs and evaluated the therapeutic effect in murine acute lymphocytic leukemic model (L615) /plasmacytoma model (SP2/0).Methods: (1) Murine BM mononuclear cells were adjusted at 1 x 106/ml and cultured with GM-CSF(10 ng/ml) and IL-4( 10 ng/ml) for 7 to 8 days. Then the floating and loosely adherent cells were pooled and readjusted at a density of 1 x 106/ml. LPS( 1 ug/ml) or whole tumor lysates were added for additional 24 hours to further induce the maturity. These mature DCs were analyzed by morphological characteristics and FACS for phenotypes. Further experiments were perfonned to test whether they have potent antigen presenting capacity and stimulatory capacity towards allogeneic naive T cells or not by N/TTT assay. (2) Murine spleen derived mononuclear cells uere adjusted at 5-7x 106/ml and cultured with IFN- y 1000U/ml (1 00ng/ml) tor one day, then IL-1 (3 (5ng/ml), anti-CD3 mab (lug/ml) were added for additional one day. After that, the cells were expanded with the addition of low dose human EL-2 . Then the cells were pooled and termed as CATs when they had been cultured for 5 to 8 days. CAT expansion was analyzed by eveiy other day cell count. Next, the lytic activity against various syngeneic and allogeneic target cells by CATs was analyzed by 3lCr release assay and then Winn assay was performed. The therapeutic effects of CAT adoptive transfer incombination with CY were evaluated in L615 leukemia mice and SP2/0 plasmacytoma model. (3) CATs of 5 days were co-cultured with BM derived DC pulsed with whole tumor lysates for additional 7 days and then they were termed as DC-CATs. Their cytotoxiciry was analyzed by 3lCr release assay and winn assay was perfomied. (4) 2 x 105BM derived DC pulsed with WTL were injected into each mouse.. Seven days later , these mice were sacrificed and their spleens were removed. CATs were generated from these spleen cells and tenned as SET cells (specific effector T cells). Their lytic activity in vitro . therapeutic effect in vivo and winn assay were performed as above.Results: (1) Fully mature DCs can be generated from murine BM mononuclear cells with typical morphological characteristics and high expression of CD lie, CD40, CD80, CD86 and I-A. Moreover , these DCs have potent stimulatory abilities towards allogeneic naive T cells and high Ag presentation abilities. (2) CATs can be generated from spleen cells and phenotypic analysis revealed that they were Thy 1.2 positive. Morphologically, the expanded cells were large, granular, and vacuolated when they were cultured for 5 to 8 days. These CATs didn't have any significant lytic activity against various allogeneic and syngeneic target cells including Yac-1, Sp2/0 and L615 regardless of culture days by DlCr release assay. Winn assay showed CATs hadn't any protective effect. When they were injected into leukemic mice, no therapeutic effect can be observed. However, when the\ \\ere injected in combination with CY, 83.3% leukemic mice can be cured. The observed therapeutic effects were specifically affected by CY timing, culture days of CATs, CATs number for therapy and the number of L615 leukemic cells for initiating leukemia. Some of the cured mice were rechallenged again with L615 leukemic cells, and all the rechallenged mice died without survival prolongation. (3) DC-CATs showed enhanced lytic activity against specific target cells. Winn assay revealed these DC-CATs have potent anti-tumoreffect in L615 leukemic mice. (4) SETs still lacked quick (4 hours) lysis activity. Winn assay revealed that they can prolong the survival days of L615 leukemic mice and avoid the formation of SP2/0 tumor. When they were injected into L615 leukemic mice in combination with CY, low number of CATs as 5 x 106 cells can be effective. Moreover, one single injection of CATs can suppress the development of SP2/0 myeloma.Conclusion: (1) Spleen cells derived CATs don't have lytic activity in v... |
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