The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocytes were T cells which were specifically cytotoxic to tumor cells, the in vivo growth of both ascitic and solid carcinoma could be suppressed by immunization with HSP70-peptides and the lifetime of tumor-bearing mice was prolonged, the in vivo immunization with HSP70-H22-peptides had no impact on the function of mouse liver and kidney, suggesting that there was no occurrence of autoimmunity in vivo after immunization.
Objective: To investigate the characteristics of specific antitumor immunity induced by antigen peptides mixture from T lymphocytic leukemia cells. Method: Antigen peptides mixtures were prepared from different leukemia cell lines and then bound with Hsp70 in vitro. Human peripheral blood mononuclear cells (PBMC) were cultured in vitro, and activated with Hsp70-antigen peptides. The activated PBMC was cultured continuously in vitro, and used as effector cells in vitro test of cytotoxicity to different target cells. Results: The antigen peptides from different leukemia cell lines were peptides mixture and could activate PBMC effectively if they were presented by Hsp70. The activated PBMC could proliferate in the presence of IL-2 and Hsp70-antigen peptides. The proliferative PBMC had specific cytotoxicity to leukemia cells corresponding to the antigen peptides. PBMC activated by antigen peptides from T lymphocytic leukemia cell lines could effectively kill T lymphocytic leukemia cells, and the cytotoxicity of these PBMC to T lymphocytic leukemia cells was significantly stronger than that of PBMC activated by antigen peptides from other leukemia cells (P < 0.05). PBMC activated by either Hut78-peptides or Molt 4-peptides could effectively kill Jurkat cells. And the cytotoxicity of PBMC activated by Hut78/Molt-4-peptides to Jurkat cells was significantly stronger than that of PBMC activated by either Hut78-peptides or Molt-4-peptides alone (P<0.05). Conclusion: Antigen peptides mixture from T lymphocytic leukemia cell lines can induce specific cytotoxic effect to T lymphocytic leukemia cells. There exists cross-reactivity among antigen peptides mixture from different T lymphocytic leukemia cell lines. The cross-reactivity could be amplified by blending of different antigen peptides from different T lymphocytic leukemia cell lines, suggesting that it is possible to prepare broad-spectrum antigen peptide vaccine against T lymphocytic leukemia by using multiple leukemia cell lines.
Objective: To investigate the feasibility of decreasing the dosage of tumor antigen peptides by dendritic cell (DC)-presenting and the characteristics of modification of DC by heat shock protein (Hsp70) and antigen peptides. Methods: Peptides were bound to Hsp70 and used to modify DC in vitro. The metabolism of the modified DC and the cytokines secreted by the modified DC were determined. The activation of lymphocytes by the modified DC and Hsp70-H22 peptides was tested. The cytotoxicity of the activated lymphocytes to H22 tumor cells was analyzed. The inhibitory effect of tumor in mice by the injection of DC and Hsp70-H22 peptides was tested. Results: 0.15μg of H22 peptides bound with Hsp70 could make 2×105 DC mature. 4×103 matured DC could activate 2×106 lymphocytes. The same amount of lymphocytes could be activated to produce similar cytotoxicity to tumor cells by either DC modified by 0.003mg of peptides bound with Hsp70 or by direct stimulation with 0.15μg of peptides bound with Hsp70. The dosage of peptides could be reduced by about 50 folds if the modified DC was used for injection instead of Hsp70-peptides. Peptides from normal hepatocytes, bound with Hsp70, could not make DC mature, nor activate lymphocytes through DC. Conclusion: The dosage of Hsp70-H22 peptides can be reduced significantly by DC-presenting to activate lymphocytes. Peptides from normal cells could not activate lymphocytes by either Hsp70-presenting or DC-presenting and they have little chance to induce autoimmunity.
