同基因BMT后CpG+GM-CSF抗小鼠白血病免疫治疗的实验研究
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摘要
目的
荷红白血病(EL9611)小鼠同基因骨髓移植(syn-BMT)后，经采用非甲基化胞嘧啶鸟嘌呤二核苷酸(CpG-ODNs)联合GM-CSF和／或肿瘤疫苗进行免疫治疗，观察其诱导syn-BMT后抗白血病特异性免疫能力的效应，并初步探讨其免疫机理。为CpG+GM-CSF用于自体造血干细胞移植(auto-HSCT)后白血病免疫治疗提供实验依据。
方法
1、8-10周龄、健康、SPF级、雌性Balb／c小鼠接种EL9611细胞后行syn-BMT，移植后分别以PBS液(A组)；CpG+GM-CSF(B组)；CpG+GM-CSF+肿瘤疫苗(C组)进行免疫治疗。
2、免疫治疗后检测小鼠的血清IFN-γ和IL-4浓度；脾细胞对EL9611靶细胞和ConA激活的淋巴母细胞(C朋)靶细胞的细胞毒活力。
3、免疫治疗结束后，所有存活小鼠用EL9611细胞进行二次攻击，同时以相同剂量EL9611攻击正常小鼠(设为D组)，观察其生存时间和存活率。
4、分别在免疫治疗结束后和EL9611细胞再攻击后14天取外周血检测T细胞亚群。
5、通过外周血涂片和病理形态观察明确小鼠死因。接种EL9611细胞后超过60天，外周血涂片未见有幼红细胞者，为长期无病生存。
结果
1、所有死亡小鼠经外周血涂片和病理检查均死于红白血病。
2、荷红白血病小鼠syn-BMT后60天，对照组11／20只死亡，B组4／20只死亡，C组3／20只死亡(p=0.011)，其平均生存时间：A组32.9±3.7天，B组40.8±4.9天，C组42.6±3.2天(p=0.000)。将长期生存小鼠用EL9611细胞再攻击后，A组5／5只死亡，B组6／12只死亡，C组4／13只死亡(p=0.009)，其平均生存时间：A组21.8±1.9天，B组30.8±2.1天，C组34.5±3.1天(p=0.000)。说明荷红白血病小鼠
s叩一BMT后，用印G＋GM｛SF免疫治疗可以显著延长小鼠的生存时间和提
高小鼠的无病存活率。
3、荷红白血病小鼠syn－BMT及免疫治疗后，其血清IFN－Y水平明
显升高，m组224．二土15，6 Pg／ml，C组274．6士19．IPg／ml，均显著高
于A组134．4土28．3Pg加1，P值分别为0．011和0．001）。而血清IL－4
水平与对照组比较则显著下降m组49．8士6．7 pg／ml，C组 47．9士
5．3Pg／ml，均显著低于A组70．3土4．5 Pg加1中值分另为0．013和0．008）。
说明荷红白血病小鼠syn于见后，CpG仆CSF免疫治疗诱导了W 型免
疫反应。
4、荷红白血病小鼠syn－BMT及免疫治疗后，长期生存的小鼠的脾细
胞对 EL961靶细胞的细胞毒活力显著高于其对 CAB靶细胞的细胞毒活力
（P＝0．000）。用CPG＋GM－CSF免疫治疗的小鼠脾细胞对EL9611细胞的特
异性杀伤活力明显增强，C组：83．5 ig．l％，B组：72．8f5．0％均显著
高于 A组：52．6土 8．4％…值分别为 0．000和 0．005）。对 CAB靶细胞的
杀伤活力，三组间比较无显著差异。
5、荷红白血病小鼠syn－BMT及免疫治疗后，外周血T细胞亚群三组
之间比较无显著差异。但经 EL961细胞二次攻击后 14天，A组小鼠 T
细胞亚群已有明显改变，CD3＋T细胞％，CD4＋T细胞％明显下降h值分
别为：o．0站和0．024），ms八细胞明显上升（p0．022）：而此时8和C
组T细胞亚群尚无明显变化；同时，三组CD4＋／CD45RA十和＊＋／CD45RA＋T
细胞在免疫治疗后和EL96ll细胞二次攻击后均未见明显的变化。
小结
1、荷红白血病小鼠syn－BMT后，使用CpG＋GM－CSF免疫治疗不仅可以明
显改善移植后生存率及生存时间，而且可以保护大于 50％小鼠在致
死剂量 EL961细胞H次攻击时获得长期生存。
2、荷红白血病小鼠syn－BMT后，使用CpG＋GM－CSF兔疫治疗可以明显增
强小鼠脾细胞的细胞毒活力，明显增加血清IFN－Y的水平及降低
IL－4的水平，说明其通过诱导Thl型免疫反应而增强了特异性抗白
血病免疫能力。
．3－
3、荷红白血病小鼠syn－BMT及使用CpG＋GM－CSF免疫治疗后，虽然外
周血T细胞亚群的比例与对照组长期生存的小鼠比较无显著差异，但
致死剂量EL9611细胞攻击后，对照组的T细胞亚群则出现变化，而
免疫治疗组的T细胞亚群却无明显变化。syn－BMT后早期免疫治疗的
机理可能未涉及到初始T细胞的变化。
由此说明，syn－BMT不仅可以清除绝大部分的肿瘤细胞，而且可以
激发一定程度的抗白血病免疫效应；syn亿MT后的 CpG川M｛SF免疫治疗，
可以诱导仆 免疫反应，从而显著增强抗白血病免疫能力及具有一定的
持续性。本研究为CpG＋GM－CSF用于auto－SCT后白血病的免疫治疗提供
了实验依据。
Objective:
To study the effects of immunotherapy with synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODNs) and GM-CSF (CpG+GM-CSF) in the erythroleukemia-bearing mice after syngeneic bone marrow transplantation (syn-BMT).
Methods:
1. The erythroleukemia-bearing mice (Balb/c) were immunized with PBS (A group),CpG+GM-CSF (B group),CpG+GM-CSF+tumor vaccines (C group) after syn-BMT.
2.Peripheral blood serums were harvested for the measurement of IFN-Y and IL-4 and spleen cells were harvested to measure the cytotoxicity from the mice after immunotherapy.
3. Subsets of CDS,CD4,CDS and CD45RA in peripheral blood cells were assessed after the immunotherapy and at the 14d after the mice were rechalleged with EL9611 cells at a lethal dose.
4. After syn-BMT and immunotherapy,all of the survival transplant mice with syn-BM cells and the normal mice were rechallenged with EL9611 cells at a lethal dose. The survival time and long-term survival rate were observed.
5.Peripheral blood cell smears and pathology determined whether the cause of death was due to erythroleukemia. The mice survived without erythroleukemia sixty days after EL9611 rechallenging were considered as long-term survivors.
Results:
1. All the mice died of erythroleukemia.
2.1mmunotherapy could prolong the survival time of erythroleukemia-bearing mice receiving syn-BMT. Within 60 days,in the
control group 11/20 mice died,B group 4/20 died,C group 3/20 died (p=0.011),the average survival time was as follows:the control 32.9+3.7 days,B group 40.8+4.9 days,C group 42.6+3.2 days (p=0.000). Then the long-term survivors were rechallenged with EL9611 cells at a lethal dose. The control group 5/5 died,B group 6/12 died,C group 4/13 died (p=0.009) .The average survival time was as follows:the control 21.8+1.9 days,B group 30.8+2.1 days,C group 34.5+3.1 days (p=0.000).
3.The average serum level of IFN- Y of the B and C group was significantly higher than the level of the control group (B group:224.1 + 15.6 pg/ml,C group:274.6 + 19. Ipg/ml vs A group:134.4+28.3 pg/ml,p= 0.011 and 0.001,respectively). The average serum level of IL-4 from B group and C group was significantly less than the level of the A group (B group:49.8+ 6.7 pg/ml and C group:47.9+5.3pg/ml,p= 0.013 and 0.008,respectively).
4. The cytotoxic activity of spleen cells to EL9611 cells targets from the erythroleukemia-bearing mice were significantly higher than the Concanavalin A (ConA) lymphoblast (CAB) targets after the transplantation. After immunotherapy the spleen cells from B and C group showed higher cytotoxic activity to the EL9611 target cells than that from the control group. The average cytotoxic activity of spleen cells was as follows:B group:72.8 +5.0 %,C group:83.5 + 9.1% vs E group:52.6+8.4%,(p= 0.005 and 0.000,respectively. The cytotoxicity to CAB target cells of spleen cells from all groups were similar.
5.There was no significantly change in the CD3+%,CD3+CD4+%,CD3+CD8+%,CD4+/CD45RA+,CD8+/CD45RA+ among the three groups after immunotherapy. But the CD3+%,CD3+CD4+% decreased significantly (p=0.048 and 0.024,respectively)and the CD3+CD8+% increased significantly(p=0.022) hi A group at the 14d after the EL9611 cells rechallege,while no significant change was seen in the B and C group. There was no significant difference of the CD4+/CD45RA+ and CD8+/CD45RA+ among the three groups.
Conclusion
1. After syn-BMT,imunotherapy with CpG+GM-CSF not only prolong the survival time and improve the long-term survival rate of the erythroleukemia-bearing mice,but also protect more than 50% of the mice from the rechallege with EL9611 cells at a lethal dose.
2. Immunotherapy with CpG+GM-CSF could significantly increase the cytotoxicity activity of spleen cells and significantly increase the level of IFN- y and reduce IL-4 after the erythroleukemia-bearing mice were transplantated with syngeneic bone marrow.
3. There was no significantly change in the T cell subsets among the three groups after immunotherapy. But the T cell subs
荷红白血病(EL9611)小鼠同基因骨髓移植(syn-BMT)后，经采用非甲基化胞嘧啶鸟嘌呤二核苷酸(CpG-ODNs)联合GM-CSF和／或肿瘤疫苗进行免疫治疗，观察其诱导syn-BMT后抗白血病特异性免疫能力的效应，并初步探讨其免疫机理。为CpG+GM-CSF用于自体造血干细胞移植(auto-HSCT)后白血病免疫治疗提供实验依据。
方法
1、8-10周龄、健康、SPF级、雌性Balb／c小鼠接种EL9611细胞后行syn-BMT，移植后分别以PBS液(A组)；CpG+GM-CSF(B组)；CpG+GM-CSF+肿瘤疫苗(C组)进行免疫治疗。
2、免疫治疗后检测小鼠的血清IFN-γ和IL-4浓度；脾细胞对EL9611靶细胞和ConA激活的淋巴母细胞(C朋)靶细胞的细胞毒活力。
3、免疫治疗结束后，所有存活小鼠用EL9611细胞进行二次攻击，同时以相同剂量EL9611攻击正常小鼠(设为D组)，观察其生存时间和存活率。
4、分别在免疫治疗结束后和EL9611细胞再攻击后14天取外周血检测T细胞亚群。
5、通过外周血涂片和病理形态观察明确小鼠死因。接种EL9611细胞后超过60天，外周血涂片未见有幼红细胞者，为长期无病生存。
结果
1、所有死亡小鼠经外周血涂片和病理检查均死于红白血病。
2、荷红白血病小鼠syn-BMT后60天，对照组11／20只死亡，B组4／20只死亡，C组3／20只死亡(p=0.011)，其平均生存时间：A组32.9±3.7天，B组40.8±4.9天，C组42.6±3.2天(p=0.000)。将长期生存小鼠用EL9611细胞再攻击后，A组5／5只死亡，B组6／12只死亡，C组4／13只死亡(p=0.009)，其平均生存时间：A组21.8±1.9天，B组30.8±2.1天，C组34.5±3.1天(p=0.000)。说明荷红白血病小鼠
s叩一BMT后，用印G＋GM｛SF免疫治疗可以显著延长小鼠的生存时间和提
高小鼠的无病存活率。
3、荷红白血病小鼠syn－BMT及免疫治疗后，其血清IFN－Y水平明
显升高，m组224．二土15，6 Pg／ml，C组274．6士19．IPg／ml，均显著高
于A组134．4土28．3Pg加1，P值分别为0．011和0．001）。而血清IL－4
水平与对照组比较则显著下降m组49．8士6．7 pg／ml，C组 47．9士
5．3Pg／ml，均显著低于A组70．3土4．5 Pg加1中值分另为0．013和0．008）。
说明荷红白血病小鼠syn于见后，CpG仆CSF免疫治疗诱导了W 型免
疫反应。
4、荷红白血病小鼠syn－BMT及免疫治疗后，长期生存的小鼠的脾细
胞对 EL961靶细胞的细胞毒活力显著高于其对 CAB靶细胞的细胞毒活力
（P＝0．000）。用CPG＋GM－CSF免疫治疗的小鼠脾细胞对EL9611细胞的特
异性杀伤活力明显增强，C组：83．5 ig．l％，B组：72．8f5．0％均显著
高于 A组：52．6土 8．4％…值分别为 0．000和 0．005）。对 CAB靶细胞的
杀伤活力，三组间比较无显著差异。
5、荷红白血病小鼠syn－BMT及免疫治疗后，外周血T细胞亚群三组
之间比较无显著差异。但经 EL961细胞二次攻击后 14天，A组小鼠 T
细胞亚群已有明显改变，CD3＋T细胞％，CD4＋T细胞％明显下降h值分
别为：o．0站和0．024），ms八细胞明显上升（p0．022）：而此时8和C
组T细胞亚群尚无明显变化；同时，三组CD4＋／CD45RA十和＊＋／CD45RA＋T
细胞在免疫治疗后和EL96ll细胞二次攻击后均未见明显的变化。
小结
1、荷红白血病小鼠syn－BMT后，使用CpG＋GM－CSF免疫治疗不仅可以明
显改善移植后生存率及生存时间，而且可以保护大于 50％小鼠在致
死剂量 EL961细胞H次攻击时获得长期生存。
2、荷红白血病小鼠syn－BMT后，使用CpG＋GM－CSF兔疫治疗可以明显增
强小鼠脾细胞的细胞毒活力，明显增加血清IFN－Y的水平及降低
IL－4的水平，说明其通过诱导Thl型免疫反应而增强了特异性抗白
血病免疫能力。
．3－
3、荷红白血病小鼠syn－BMT及使用CpG＋GM－CSF免疫治疗后，虽然外
周血T细胞亚群的比例与对照组长期生存的小鼠比较无显著差异，但
致死剂量EL9611细胞攻击后，对照组的T细胞亚群则出现变化，而
免疫治疗组的T细胞亚群却无明显变化。syn－BMT后早期免疫治疗的
机理可能未涉及到初始T细胞的变化。
由此说明，syn－BMT不仅可以清除绝大部分的肿瘤细胞，而且可以
激发一定程度的抗白血病免疫效应；syn亿MT后的 CpG川M｛SF免疫治疗，
可以诱导仆 免疫反应，从而显著增强抗白血病免疫能力及具有一定的
持续性。本研究为CpG＋GM－CSF用于auto－SCT后白血病的免疫治疗提供
了实验依据。
Objective:
To study the effects of immunotherapy with synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODNs) and GM-CSF (CpG+GM-CSF) in the erythroleukemia-bearing mice after syngeneic bone marrow transplantation (syn-BMT).
Methods:
1. The erythroleukemia-bearing mice (Balb/c) were immunized with PBS (A group),CpG+GM-CSF (B group),CpG+GM-CSF+tumor vaccines (C group) after syn-BMT.
2.Peripheral blood serums were harvested for the measurement of IFN-Y and IL-4 and spleen cells were harvested to measure the cytotoxicity from the mice after immunotherapy.
3. Subsets of CDS,CD4,CDS and CD45RA in peripheral blood cells were assessed after the immunotherapy and at the 14d after the mice were rechalleged with EL9611 cells at a lethal dose.
4. After syn-BMT and immunotherapy,all of the survival transplant mice with syn-BM cells and the normal mice were rechallenged with EL9611 cells at a lethal dose. The survival time and long-term survival rate were observed.
5.Peripheral blood cell smears and pathology determined whether the cause of death was due to erythroleukemia. The mice survived without erythroleukemia sixty days after EL9611 rechallenging were considered as long-term survivors.
Results:
1. All the mice died of erythroleukemia.
2.1mmunotherapy could prolong the survival time of erythroleukemia-bearing mice receiving syn-BMT. Within 60 days,in the
control group 11/20 mice died,B group 4/20 died,C group 3/20 died (p=0.011),the average survival time was as follows:the control 32.9+3.7 days,B group 40.8+4.9 days,C group 42.6+3.2 days (p=0.000). Then the long-term survivors were rechallenged with EL9611 cells at a lethal dose. The control group 5/5 died,B group 6/12 died,C group 4/13 died (p=0.009) .The average survival time was as follows:the control 21.8+1.9 days,B group 30.8+2.1 days,C group 34.5+3.1 days (p=0.000).
3.The average serum level of IFN- Y of the B and C group was significantly higher than the level of the control group (B group:224.1 + 15.6 pg/ml,C group:274.6 + 19. Ipg/ml vs A group:134.4+28.3 pg/ml,p= 0.011 and 0.001,respectively). The average serum level of IL-4 from B group and C group was significantly less than the level of the A group (B group:49.8+ 6.7 pg/ml and C group:47.9+5.3pg/ml,p= 0.013 and 0.008,respectively).
4. The cytotoxic activity of spleen cells to EL9611 cells targets from the erythroleukemia-bearing mice were significantly higher than the Concanavalin A (ConA) lymphoblast (CAB) targets after the transplantation. After immunotherapy the spleen cells from B and C group showed higher cytotoxic activity to the EL9611 target cells than that from the control group. The average cytotoxic activity of spleen cells was as follows:B group:72.8 +5.0 %,C group:83.5 + 9.1% vs E group:52.6+8.4%,(p= 0.005 and 0.000,respectively. The cytotoxicity to CAB target cells of spleen cells from all groups were similar.
5.There was no significantly change in the CD3+%,CD3+CD4+%,CD3+CD8+%,CD4+/CD45RA+,CD8+/CD45RA+ among the three groups after immunotherapy. But the CD3+%,CD3+CD4+% decreased significantly (p=0.048 and 0.024,respectively)and the CD3+CD8+% increased significantly(p=0.022) hi A group at the 14d after the EL9611 cells rechallege,while no significant change was seen in the B and C group. There was no significant difference of the CD4+/CD45RA+ and CD8+/CD45RA+ among the three groups.
Conclusion
1. After syn-BMT,imunotherapy with CpG+GM-CSF not only prolong the survival time and improve the long-term survival rate of the erythroleukemia-bearing mice,but also protect more than 50% of the mice from the rechallege with EL9611 cells at a lethal dose.
2. Immunotherapy with CpG+GM-CSF could significantly increase the cytotoxicity activity of spleen cells and significantly increase the level of IFN- y and reduce IL-4 after the erythroleukemia-bearing mice were transplantated with syngeneic bone marrow.
3. There was no significantly change in the T cell subsets among the three groups after immunotherapy. But the T cell subs
引文
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9.Nagahama M, Nomura S, Katsura K, et al. Serum levels of soluble CD30 in autologous peripheral blood stem cell transplantation. J Cancer Res Clin Oncol, 2000, 126(2): 101-6.
10.Nordoy T, Kolstad A, Endresen P, et al. Persistent changes in the immune system 4-10 years after ABMT. Bone-Marrow-Transplant, 1999, 24(8): 873-8.
11.Borrello. I,. Sotomayor. EM, Pattis. FM,et al. Sustaining the graft-versus-tumor effect through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing tumor vaccines. Blood, 2000,95 (10): 3011-3019
12.Bohle. B; Jahn-SB; Maurer, et al. Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-gamma production in cells from allergic individuals and inhibit IgE synthesis in vitro. Eur-J-Immunol. 1999,29(7): 2344-53
13.Scharton. K, Ersten, T, Krieg AN et al. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12-and IFN-gamma-dependent mechanisms. Proc-Natl-Acad-Sci-U-S-A. 1999,8; 96(12): 6970-5
14.Bohle. B, Orel L, Kraft D,et al. Oligodeoxynucleotides containing CpG motifs induce low levels of TNF-alpha in human B lymphocytes: possible adjuvants for Th1 responses. J-Immunol, 2001, 166(6): 3743-8.
15.Kranzer K, Bauer M, Lipford GB,et al. CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-gamma production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12. Immunology, 2000, 99(2): 170-8.
16.Bendigs S,Salzer U, Lipford GB, et al. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells. Eur-J-Immunol, 1999,29(4): 1209-18.
17.Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol, 1996, 157(5):1840-5.
18.Sweet MJ, Stacey KJ, Kakuda DK, et, al. IFN-gamma primes macrophage responses to bacterial DNA. J Interferon Cytokine Res, 1998,18 (4):263-71.
19.Blazar BR, Krieg AM, Taylor PA. Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in na(?)ve and bone marrow transplant recipients.Blood, 2001,98:1217-1225.
20.Sparwasser T, Koch ES, Vabulas RM, et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol, 1998, 28(6): 2045-54.
21.Dunussi-Joannopoulos K, Dranoff G, Weinstein HJ, et al. Gene Immunotherapy in Murine Acute Myeloid Leukemia: Granulocyte-Macrophage Colony-Stimulating Factor Tumor Cell Vaccines Elicit More Potent Antitumor Immunity Compared With B7 Family and Other Cytokine Vaccines Blood, 1998, 91 (1): 222-230.
22.Liu HM, Newbrough SE, Bhatia SK, et al. Immunostimulatory CpG Oligodeoxynucleotides Enhance the Immune Response to Vaccine Strategies Involving Granulocyte-Macrophage Colony-Stimulating Factor, Blood, 1998,92(10):3730-3736.
23．唐华，银平章，曹雪涛等．冻融抗原冲击致敏的树突状细胞对结肠癌小鼠的治疗作用．中国肿瘤生物治疗杂志，2000，7(3)，199-202．
24.Krieg AM, Homan LL, Yi Ak, et al. CpG DNA Induces Sustained IL-12 Expression In Vivo and Resistance to Listeria monocytogenes Challenge. The Journal of Immunology, 1998, 161: 2428-2434.。
25.Anderson LD, Savary CA, Mullen CA. Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus-host disease. Blood, 2000, 95 (7), 2426-2433。
26．段连宁，郭坤元，袁进等．同种异基因Th2细胞移植对GVHD和GVL效应的作用．中国实验血液学杂志，2000；8(1)：57-60。
27.Boughton BJ, Simpson AW. Acute myeloblastic leukaemia: graft-versus-host and graft-versus-leukaemia responses to autologous IL-2 activated lymphocytes in rapid and slow disease. Cytokines-Cell-Mol-Ther, 1999, 5(1): 1-6.
28.Patterson AE, Korngold R. Infusion of select leukemia-reactive TCR Vbeta+ T cells provides graft-versus-leukemia responses with minimization of graft-versus-host disease following murine hematopoietic stem cell transplantation. Biol-Blood-Marrow-Transplant, 2001,7(4): 187-96.
29.Hsieh MH, Patterson AE, Korngold R.T-cell subsets mediate graft-versus-myeloid leukemia responses via different cytotoxic mechanisms. Biol-Blood-Marrow-Transplant, 2000, 6(3): 23140.
30.Austyn JM. Death, destruction, danger and dendritic cells. Nat Med, 1999, 5(11):1232-3.Appelbaum FR, Rowe JM, Radich J, et al. Acute myeloid leukemia. Hematology, 2001, Jan, 62-86.
31.Pawlowska AB, Hashino S, Kenna HM, et al. In vitro tumor-pulsed or in vivo Flt3 ligand-generated dendritic cells provide protection against acute myelogenous leukemia in nontransplanted or syngeneic bone marrow-transplanted mice. Blood, 2001, 97(5): 1474-1482.
32.Mitra DK, Singh HP, Singh M,et al. Reconstitution of naive T cells and type 1 function after autologous peripheral stem cell transplantation: impact on the relapse of original cancer. Transplantation, 2002,73(8):1336-9.
33.64.Sparwasser T, Koch ES, Vabulas RM, et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol, 1998, 28(6): 2045-54.
34.Jakob T, Walker PS, Krieg AM, et al. Activation of Cutaneous Dendritic Cells by CpG-Containing Oligodeoxynucleotides: A Role for Dendritic Cells in the Augmentation of Thl Responses by Immunostimulatory DNA. The Journal of Immunology, 1998, 161 (6):3042-9.
35.Davis HL, Weeranta R, Waldschmidt. TJ, et al. CpG DNA Is a Potent Enhancer of Specific Immunity in Mice Immunized with Recombinant Hepatitis B Surface Antigen. The Journal of Immunology, 1998, 160: 870-876.
36.Halpern MD, Kurlander RJ, Pisetsky DS. Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha. Cell Immunol, 1996, 167(1):72-8.
37.Ballas. ZK, Rasmussen. WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. The Journal of Immunology, 1996, 157(5): 1840-1845.
38.Cowdery JS, Chace JH, Krieg AY, et al. Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides The Journal of Immunology, 1996, 156(12): 4570-4575,
39.Sun SQ, Zhang XH, Tough DF, et al. Type I Interferon-mediated Stimulation of T Cells by CpG DNA J. Exp. Med, 1998, 188(12): 2335-2342.
40.Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. The Journal of Immunology, 1996, 157(5): 2116-2122.
41.Chace JH, Hooker NA, Mildenstein KL, et al. Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12. Clin Immunol Immunopathol, 1997, 84(2):185-93.
42.Dunussi-Joannopoulos K, Dranoff G, Weinstein HJ, et al. Gene Immunotherapy in Murine Acute Myeloid Leukemia: Granulocyte-Macrophage Colony-Stimulating Factor Tumor Cell Vaccines Elicit More Potent Antitumor Immunity Compared With B7 Family and Other Cytokine Vaccines Blood, 1998, 91 (1): 222-230.
43.Matzinger P. The Danger Model: A Renewed Sense of Self. Science, 2002, 296(5566):301-5.
44.Hartmann G, Weeratna RD, Ballas ZK, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol, 2000,164(3): 1617-24.
45.Sugita K, Soiffer RJ, Murray C, et al. The phenotype and reconstitution of immunoregulatory T cell subsets after T cell-depleted allogeneic and autologous bone marrow transplantation. Transplantation 1994 May 27;57(10): 1465-73
46.Soares MV,Borthwick NJ,Maini MK, et al. IL-7-dependent extrathymic expansion of CD45RA+T cells enables preservation of a na(?)ve repertoire. J Immunol, 1998, 161:5909-17.
47.Livak F,Schatz D.T-cell receptor a locus V(D)J recombination by-products are abudant in thymocytes and mature T cell.Moll Cell Biol,1996,16:609-18.
48.Douek DC,Mcfarland RD,Keiser PH,et al.Changes in thymic function with age during the treatment of HIV infection. Nature, 1998,396:690-95.
2.Boyer MW, Vallera DA, Taylor PA, et al. The role of B7 costimulation by murine acute myeloid leukemia in the generation and function of a CD8+ T-cell line with potent in vivo graft-versus-leukemia properties. Blood, 1997, 89(9): 3477-85.
3.Zeis M, Uharek L, Glass B, et al. Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice. Br-J-Haematol,. 1997, 96(4): 757-61.
4.Guillaume T, Rubinstein D.B,Symann M,et al.Immune Reconstitution and Immunotherapy After Autologous Hematopoietic Stem Cell Transplantation. Blood, 1998, 92:1471-1490.
5.Scheid C,Pettengell R,Ghielmini M,et al.Time-course of the recovery of cellular immune function after high-dose chemotherapy and peripheral blood progenitor cell transplantation for high-grade non-Hodgkin's lymphoma. Bone Marrow Transplant,1995,15:901-6.
6.Nolte ABuhmann R, Straka C,et al.Assessment and characterization of the cytolytic T lymphocyte response against Epstein-Barr virus in patients with non-Hodgkin's lymphoma after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant, 1998,21:909-16.
7.Uher F,Puskas E,Torbagyi E, et al. Regeneration of the immune system after bone marrow transplantation. Orv Hetil,2001,142:59-65.
8.Singh RK, Varney ML, Ino K, et al.Immune dysfunction despite high levels of immunoregulatory cytokine gene expression in autologous peripheral blood stem cell transplanted non-Hodgkin's lymphoma patients. Exp Hemato,2000,28:499-07.
9.Nagahama M, Nomura S, Katsura K, et al. Serum levels of soluble CD30 in autologous peripheral blood stem cell transplantation. J Cancer Res Clin Oncol, 2000, 126(2): 101-6.
10.Nordoy T, Kolstad A, Endresen P, et al. Persistent changes in the immune system 4-10 years after ABMT. Bone-Marrow-Transplant, 1999, 24(8): 873-8.
11.Borrello. I,. Sotomayor. EM, Pattis. FM,et al. Sustaining the graft-versus-tumor effect through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing tumor vaccines. Blood, 2000,95 (10): 3011-3019
12.Bohle. B; Jahn-SB; Maurer, et al. Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-gamma production in cells from allergic individuals and inhibit IgE synthesis in vitro. Eur-J-Immunol. 1999,29(7): 2344-53
13.Scharton. K, Ersten, T, Krieg AN et al. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12-and IFN-gamma-dependent mechanisms. Proc-Natl-Acad-Sci-U-S-A. 1999,8; 96(12): 6970-5
14.Bohle. B, Orel L, Kraft D,et al. Oligodeoxynucleotides containing CpG motifs induce low levels of TNF-alpha in human B lymphocytes: possible adjuvants for Th1 responses. J-Immunol, 2001, 166(6): 3743-8.
15.Kranzer K, Bauer M, Lipford GB,et al. CpG-oligodeoxynucleotides enhance T-cell receptor-triggered interferon-gamma production and up-regulation of CD69 via induction of antigen-presenting cell-derived interferon type I and interleukin-12. Immunology, 2000, 99(2): 170-8.
16.Bendigs S,Salzer U, Lipford GB, et al. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells. Eur-J-Immunol, 1999,29(4): 1209-18.
17.Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol, 1996, 157(5):1840-5.
18.Sweet MJ, Stacey KJ, Kakuda DK, et, al. IFN-gamma primes macrophage responses to bacterial DNA. J Interferon Cytokine Res, 1998,18 (4):263-71.
19.Blazar BR, Krieg AM, Taylor PA. Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in na(?)ve and bone marrow transplant recipients.Blood, 2001,98:1217-1225.
20.Sparwasser T, Koch ES, Vabulas RM, et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol, 1998, 28(6): 2045-54.
21.Dunussi-Joannopoulos K, Dranoff G, Weinstein HJ, et al. Gene Immunotherapy in Murine Acute Myeloid Leukemia: Granulocyte-Macrophage Colony-Stimulating Factor Tumor Cell Vaccines Elicit More Potent Antitumor Immunity Compared With B7 Family and Other Cytokine Vaccines Blood, 1998, 91 (1): 222-230.
22.Liu HM, Newbrough SE, Bhatia SK, et al. Immunostimulatory CpG Oligodeoxynucleotides Enhance the Immune Response to Vaccine Strategies Involving Granulocyte-Macrophage Colony-Stimulating Factor, Blood, 1998,92(10):3730-3736.
23．唐华，银平章，曹雪涛等．冻融抗原冲击致敏的树突状细胞对结肠癌小鼠的治疗作用．中国肿瘤生物治疗杂志，2000，7(3)，199-202．
24.Krieg AM, Homan LL, Yi Ak, et al. CpG DNA Induces Sustained IL-12 Expression In Vivo and Resistance to Listeria monocytogenes Challenge. The Journal of Immunology, 1998, 161: 2428-2434.。
25.Anderson LD, Savary CA, Mullen CA. Immunization of allogeneic bone marrow transplant recipients with tumor cell vaccines enhances graft-versus-tumor activity without exacerbating graft-versus-host disease. Blood, 2000, 95 (7), 2426-2433。
26．段连宁，郭坤元，袁进等．同种异基因Th2细胞移植对GVHD和GVL效应的作用．中国实验血液学杂志，2000；8(1)：57-60。
27.Boughton BJ, Simpson AW. Acute myeloblastic leukaemia: graft-versus-host and graft-versus-leukaemia responses to autologous IL-2 activated lymphocytes in rapid and slow disease. Cytokines-Cell-Mol-Ther, 1999, 5(1): 1-6.
28.Patterson AE, Korngold R. Infusion of select leukemia-reactive TCR Vbeta+ T cells provides graft-versus-leukemia responses with minimization of graft-versus-host disease following murine hematopoietic stem cell transplantation. Biol-Blood-Marrow-Transplant, 2001,7(4): 187-96.
29.Hsieh MH, Patterson AE, Korngold R.T-cell subsets mediate graft-versus-myeloid leukemia responses via different cytotoxic mechanisms. Biol-Blood-Marrow-Transplant, 2000, 6(3): 23140.
30.Austyn JM. Death, destruction, danger and dendritic cells. Nat Med, 1999, 5(11):1232-3.Appelbaum FR, Rowe JM, Radich J, et al. Acute myeloid leukemia. Hematology, 2001, Jan, 62-86.
31.Pawlowska AB, Hashino S, Kenna HM, et al. In vitro tumor-pulsed or in vivo Flt3 ligand-generated dendritic cells provide protection against acute myelogenous leukemia in nontransplanted or syngeneic bone marrow-transplanted mice. Blood, 2001, 97(5): 1474-1482.
32.Mitra DK, Singh HP, Singh M,et al. Reconstitution of naive T cells and type 1 function after autologous peripheral stem cell transplantation: impact on the relapse of original cancer. Transplantation, 2002,73(8):1336-9.
33.64.Sparwasser T, Koch ES, Vabulas RM, et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol, 1998, 28(6): 2045-54.
34.Jakob T, Walker PS, Krieg AM, et al. Activation of Cutaneous Dendritic Cells by CpG-Containing Oligodeoxynucleotides: A Role for Dendritic Cells in the Augmentation of Thl Responses by Immunostimulatory DNA. The Journal of Immunology, 1998, 161 (6):3042-9.
35.Davis HL, Weeranta R, Waldschmidt. TJ, et al. CpG DNA Is a Potent Enhancer of Specific Immunity in Mice Immunized with Recombinant Hepatitis B Surface Antigen. The Journal of Immunology, 1998, 160: 870-876.
36.Halpern MD, Kurlander RJ, Pisetsky DS. Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha. Cell Immunol, 1996, 167(1):72-8.
37.Ballas. ZK, Rasmussen. WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. The Journal of Immunology, 1996, 157(5): 1840-1845.
38.Cowdery JS, Chace JH, Krieg AY, et al. Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides The Journal of Immunology, 1996, 156(12): 4570-4575,
39.Sun SQ, Zhang XH, Tough DF, et al. Type I Interferon-mediated Stimulation of T Cells by CpG DNA J. Exp. Med, 1998, 188(12): 2335-2342.
40.Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. The Journal of Immunology, 1996, 157(5): 2116-2122.
41.Chace JH, Hooker NA, Mildenstein KL, et al. Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12. Clin Immunol Immunopathol, 1997, 84(2):185-93.
42.Dunussi-Joannopoulos K, Dranoff G, Weinstein HJ, et al. Gene Immunotherapy in Murine Acute Myeloid Leukemia: Granulocyte-Macrophage Colony-Stimulating Factor Tumor Cell Vaccines Elicit More Potent Antitumor Immunity Compared With B7 Family and Other Cytokine Vaccines Blood, 1998, 91 (1): 222-230.
43.Matzinger P. The Danger Model: A Renewed Sense of Self. Science, 2002, 296(5566):301-5.
44.Hartmann G, Weeratna RD, Ballas ZK, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol, 2000,164(3): 1617-24.
45.Sugita K, Soiffer RJ, Murray C, et al. The phenotype and reconstitution of immunoregulatory T cell subsets after T cell-depleted allogeneic and autologous bone marrow transplantation. Transplantation 1994 May 27;57(10): 1465-73
46.Soares MV,Borthwick NJ,Maini MK, et al. IL-7-dependent extrathymic expansion of CD45RA+T cells enables preservation of a na(?)ve repertoire. J Immunol, 1998, 161:5909-17.
47.Livak F,Schatz D.T-cell receptor a locus V(D)J recombination by-products are abudant in thymocytes and mature T cell.Moll Cell Biol,1996,16:609-18.
48.Douek DC,Mcfarland RD,Keiser PH,et al.Changes in thymic function with age during the treatment of HIV infection. Nature, 1998,396:690-95.