乳腺癌细胞抗原负载的自体树突状细胞体内诱导特异性T细胞应答

目的 以乳腺癌细胞抗原体外负载自体树突状细胞(DCs)对24例乳腺癌患者进行自身免疫,探讨其体内诱导特异性T细胞免疫应答的能力.方法 新鲜癌组织制备成热休克凋亡细胞抗原负载外周单核细胞来源DC,分别在采血后第1、2、4、6周于患者腹股沟淋巴结富集区进行皮内注射,细胞剂量为4×10+～6×106/次.结果 DC免疫治疗后患者血清抗瘤免疫因子水平明显上升:白细胞介素(IL)-2治疗前为(33.8±7.2)ng/L,治疗后为(68.5±12.4)ng/L;IL-12治疗前为(48.5±10.9)ng/L,治疗后为(118.2±31.5)ng/L;肿瘤坏死因子(TNF)-α治疗前为(18.7±5.3)ng/L,治疗后为(78.3±11.5)ng/L;干扰素(IFN)-γ治疗前为(20.5±6.3)ng/L,治疗后为(92.6±14.9)ng/L,治疗前后比较差异有统计学意义(P<0.01);DTH试验阳性率为7/10;4/10例IFN-γ+CD8+T细胞频率较治疗前明显增加.随访发现:除1例患者在治疗后3个月内疾病进展(PD),其余患者病情稳定无复发与转移症状(SD).结论 以乳腺癌细胞为抗原负载自体DC免疫患者,能够有效提高患者非特异免疫水平,激发体内特异性T细胞免疫应答,是一种安全、副反应较小、有效的乳腺癌辅助治疗手段.     

乳腺癌细胞抗原负载的自体树突状细胞体内诱导特异性T细胞应答

Objective To explore the potential of autologous dendritic cells (DCs) loaded with breast tumor cells antigen in inducing tumor-specific T cells response in vivo. Methods Fresh tumor samples from patients with breast cancer were collected and made into apoptotic heat shock tumor cells. Peripheral blood mononuclear cells (PBMCs) were isolated. DCs were induced from monocytes with granulocytemacrophage colony-stimulating factor (rhGM-CSF) and interleukin-4 (rhIL-4) combination for 5 days. DCs were co-cultured with autologous apoptotic heat shock tumor cells for 48 h. Twenty-four patients with breast cancers received four vaccinations of auto-DCs at 1 st, 2nd, 4th, and 6th week with cell number from 4×106 to 6×106. Results The vaccinations were well tolerated, and most patients experienced improved quality of life after DC immunization. Cytokine levels including IL-2, IL-12, TNF-α and IFN-γ in sera were obviously increased after DC vaccination [(68. 5±12. 4), (118. 2±31. 5), (78. 3±11.5) and (92. 6±14. 9) ng/L respectively] as compared with those before vaccinations [(33. 8±7. 2), (48. 5±10. 9), (18. 7±5. 3 ) and (20. 5±6. 3) ng/L respectively]. Both antigen specific delayed type hypersensitivity (DTH) reaction and the frequency of antigen specific IFN-gamma+CD8+T lymphocytes were examined after 4 times of DC vaccinations. The DTH tests were positive in 7 out of 10 patients. The frequency of IFN-γ+CD8+T cells was enhanced in 4 out of 10 patients. One out of 24 patients experienced a progressive disease, while the others showed no progress during a follow-up period of 34 months. Conclusion Autologous DCs loaded with heat-shocked apoptotic breast cancer cells antigen elicit specific anti-tumor T cell immune responses companying with high levels of Thl type cytokine secretion. DCs vaccination after surgery may be a novel and effective combination tool for tumor treatment.     

以树突状细胞为基础的乳腺癌免疫治疗

树突状细胞是目前已知功能最强的抗原提呈细胞,在启动细胞免疫中起关键作用。本文介绍以树突状细胞为基础的肿瘤免疫治疗,尤其在乳腺癌中应用的概况。     

抗原负载的树突状细胞体外诱导抗肿瘤免疫的研究

目的 观察抗原负载的树突状细胞(DCs)体外诱导抗肿瘤免疫效应,探讨树突状细胞肿瘤疫苗的制备方法 .方法 以细胞因子粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4体外诱导人单核细胞来源的树突状细胞,第6天加入肝癌细胞BeL-7402的冻融抗原并以联合细胞因子鸡尾酒法[肿瘤坏死因子(TNF-α)+IL-6+IL-1β+前列腺素E2(PGE2)]诱导成熟,对照组仅以鸡尾酒法诱导成熟.24 h后收获DCs以流式细胞仪检测其成熟表型CD80、CD83、CD86和LHA-DR,酶联免疫吸附试验(ELISA)法检测其IL-12的分泌,噻唑蓝(MTT)比色法检测其刺激淋巴细胞增殖活性,乳酸脱氢酶释放实验检测其诱导的免疫效应细胞对肝癌细胞的特异性细胞毒作用.结果 联合细胞因子可诱导的DCs成熟和IL-12的分泌(P＜0.05),成熟的DCs有较强的刺激淋巴细胞增殖能力;抗原负载组DCs可诱导效应细胞对肝癌细胞BeL-7402的特异性杀伤作用(P＜0.05).结论 抗原负载的DCs可体外诱导特异性抗肿瘤免疫效应,提示以抗原负载的树突状细胞作为肿瘤免疫治疗的方法 是可行的.     

负载肾癌肿瘤裂解物的树突状细胞诱导产生抗原特异性细胞毒T淋巴细胞

目的　利用树突状细胞呈递肿瘤抗原的特性提高细胞毒T淋巴细胞 (CTLs)对肾癌细胞的杀伤活性。　方法　肾细胞癌患者骨髓来源的有核细胞体外经GM CSF和IL 4诱导产生树突状细胞 ,负载肿瘤裂解物后诱导自体CTLs产生。用细胞毒试验和ELISA测定CTLs杀伤活性和细胞因子的分泌。　结果　肾癌患者自体来源的DC Tuly能 (1)增加CTLs增殖 ,16d时使T细胞增殖达 4 3倍 ;(2 )上调CTLs中CD3 和CD8 T细胞群 ;(3)诱导产生的CTLs对自体肾癌细胞具有高杀伤率 ,显著高于异体肾癌和异种肿瘤细胞 (P <0 .0 5 ) ;(4)上调TNF α分泌。　结论　DCs能呈递Tuly抗原。诱导产生抗原特异性CTLs,提示DC Tuly具有为肾癌患者制作疫苗和进行特异性CTLs过继免疫治疗的临床应用前景     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

目的 观察自体负载肿瘤抗原树突状细胞(DC)对大鼠甲状腺癌的治疗作用.方法 建立Wistar荷瘤大鼠动物模型,随机分为治疗组和对照组,治疗组静脉输入负载肿瘤抗原DC、对照组输入生理盐水,检测分析2组血清中自细胞介素(IL)-12、IL-8表达水平,以及瘤重、瘤体体积及大鼠存活.结果 治疗组与对照组瘤重、瘤体体积、存活期、IL-12及IL-8表达水平分别为:(3.56±0.79)g比(4.97±0.56)g、(3.98±0.84)cm3比(5.06±0.58)cm3、(36.0±3.0)d比(28.0±3.2)d、(368.9±21.7)ng/L比(227.7±13.4)ng/L和(218.9±19.3)ng/L比(371.2±24.1)ng/L(P值分别<0.01、<0.01、<0.05、<0.01、<0.01).结论 自体负载肿瘤抗原DC对甲状腺癌具有较好的抑瘤效应,可延长荷瘤鼠的生存期.     

负载抗原的树突状细胞诱导特异性抗膀胱癌细胞的效应

树突状细胞(DCs)是体内“专职的”抗原提呈细胞，与机体抗瘤免疫关系密切。本研究旨在用DCs负载膀胱癌细胞冻融抗原，探讨其在体外对膀胱癌细胞的杀伤效应。     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

Objective To investigate the immunotherapeutic effects of auto dendritic cells (DC) loaded with tumor lysates on rat transitional thyroid carcinoma. Methods After the thyroid tumor models in Wistar rats were successfully established, all rats were divided randomly into the treatment group and the control group. DC loaded with tumor lysates were injected into the tail vein of the rats in the treatment group, and the rats in control group, normal saline was injected into the tail vein. The levels of interleukin (IL)-12 and IL-8 in serum, the tumor weight and volume, and the life span of the rats were measured. Results The average tumor weight and volume, the life span, the levels of IL-12 and IL-8 in the treatment group and the control group were respectively (3. 56±0. 79) g vs (4. 97±0. 56) g, (3. 98±0. 84) cm3 vs (5.06±0.58) cm3, (36.0±3.0) days vs (28.0±3.2) days, (368.9±21.7) ng/L vs (227.7±13.4) ng/L, (218.9±19.3) ng/L vs (371.2±24. 1) ng/L. P values <0.01, <0.01, <0.05,<0.01, <0.01, respectively. Conclusion Auto DC loaded with tumor lysates can inhibit tumor growth and prolong the survival time of Wistar rats with thyroid carcinoma.     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

Objective To investigate the immunotherapeutic effects of auto dendritic cells (DC) loaded with tumor lysates on rat transitional thyroid carcinoma. Methods After the thyroid tumor models in Wistar rats were successfully established, all rats were divided randomly into the treatment group and the control group. DC loaded with tumor lysates were injected into the tail vein of the rats in the treatment group, and the rats in control group, normal saline was injected into the tail vein. The levels of interleukin (IL)-12 and IL-8 in serum, the tumor weight and volume, and the life span of the rats were measured. Results The average tumor weight and volume, the life span, the levels of IL-12 and IL-8 in the treatment group and the control group were respectively (3. 56±0. 79) g vs (4. 97±0. 56) g, (3. 98±0. 84) cm3 vs (5.06±0.58) cm3, (36.0±3.0) days vs (28.0±3.2) days, (368.9±21.7) ng/L vs (227.7±13.4) ng/L, (218.9±19.3) ng/L vs (371.2±24. 1) ng/L. P values <0.01, <0.01, <0.05,<0.01, <0.01, respectively. Conclusion Auto DC loaded with tumor lysates can inhibit tumor growth and prolong the survival time of Wistar rats with thyroid carcinoma.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

Objective To study the effect of pancreatic cancer microenvironment on maturation of dendritic cells ( DCs), and to investigate the mechanism by which tumors escape from immune recognition. Methods DCs were cultured with rmGM (40μg/L) ,rmIL-4 (40μg/L) ,and the supernatant of cell line AR42J cells was added on the day 6, then a large number of DCs was gained. DCs cultured and in-duced by routine methods were used as controls. The expression of surface molecules CD86, and CD80 in DCs was detected to observe whether this intervention could delay or block maturation of DCs and this effect could be reversed by stimulation with lipopolysaccharide (LPS). T cell stimulatory effects were also determined. Results Compared to normal DCs, CD86 and CD80 expression positive rate was significantly decreased in DCs induced by AR42J supernatant from (70.88±3.60)% to (7.15±0.71 )%. After stimulation with LPS, the expression of CD86, and CD80 in DCs was still low, suggesting the AR42J super-natant could block the maturation of DCs. The allostimulatory capacity of the immature DCs stimulated with the AR42J superoatant was significantly lower than that of the normal immature DCs. Conclusion The supernatant from pancreatic cancer ceils in rats could inhibit the maturation of DCs, which was not easily reversed.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

Objective To study the effect of pancreatic cancer microenvironment on maturation of dendritic cells ( DCs), and to investigate the mechanism by which tumors escape from immune recognition. Methods DCs were cultured with rmGM (40μg/L) ,rmIL-4 (40μg/L) ,and the supernatant of cell line AR42J cells was added on the day 6, then a large number of DCs was gained. DCs cultured and in-duced by routine methods were used as controls. The expression of surface molecules CD86, and CD80 in DCs was detected to observe whether this intervention could delay or block maturation of DCs and this effect could be reversed by stimulation with lipopolysaccharide (LPS). T cell stimulatory effects were also determined. Results Compared to normal DCs, CD86 and CD80 expression positive rate was significantly decreased in DCs induced by AR42J supernatant from (70.88±3.60)% to (7.15±0.71 )%. After stimulation with LPS, the expression of CD86, and CD80 in DCs was still low, suggesting the AR42J super-natant could block the maturation of DCs. The allostimulatory capacity of the immature DCs stimulated with the AR42J superoatant was significantly lower than that of the normal immature DCs. Conclusion The supernatant from pancreatic cancer ceils in rats could inhibit the maturation of DCs, which was not easily reversed.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

Objective To study the effect of pancreatic cancer microenvironment on maturation of dendritic cells ( DCs), and to investigate the mechanism by which tumors escape from immune recognition. Methods DCs were cultured with rmGM (40μg/L) ,rmIL-4 (40μg/L) ,and the supernatant of cell line AR42J cells was added on the day 6, then a large number of DCs was gained. DCs cultured and in-duced by routine methods were used as controls. The expression of surface molecules CD86, and CD80 in DCs was detected to observe whether this intervention could delay or block maturation of DCs and this effect could be reversed by stimulation with lipopolysaccharide (LPS). T cell stimulatory effects were also determined. Results Compared to normal DCs, CD86 and CD80 expression positive rate was significantly decreased in DCs induced by AR42J supernatant from (70.88±3.60)% to (7.15±0.71 )%. After stimulation with LPS, the expression of CD86, and CD80 in DCs was still low, suggesting the AR42J super-natant could block the maturation of DCs. The allostimulatory capacity of the immature DCs stimulated with the AR42J superoatant was significantly lower than that of the normal immature DCs. Conclusion The supernatant from pancreatic cancer ceils in rats could inhibit the maturation of DCs, which was not easily reversed.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

Objective To study the effect of pancreatic cancer microenvironment on maturation of dendritic cells ( DCs), and to investigate the mechanism by which tumors escape from immune recognition. Methods DCs were cultured with rmGM (40μg/L) ,rmIL-4 (40μg/L) ,and the supernatant of cell line AR42J cells was added on the day 6, then a large number of DCs was gained. DCs cultured and in-duced by routine methods were used as controls. The expression of surface molecules CD86, and CD80 in DCs was detected to observe whether this intervention could delay or block maturation of DCs and this effect could be reversed by stimulation with lipopolysaccharide (LPS). T cell stimulatory effects were also determined. Results Compared to normal DCs, CD86 and CD80 expression positive rate was significantly decreased in DCs induced by AR42J supernatant from (70.88±3.60)% to (7.15±0.71 )%. After stimulation with LPS, the expression of CD86, and CD80 in DCs was still low, suggesting the AR42J super-natant could block the maturation of DCs. The allostimulatory capacity of the immature DCs stimulated with the AR42J superoatant was significantly lower than that of the normal immature DCs. Conclusion The supernatant from pancreatic cancer ceils in rats could inhibit the maturation of DCs, which was not easily reversed.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

Objective To study the effect of pancreatic cancer microenvironment on maturation of dendritic cells ( DCs), and to investigate the mechanism by which tumors escape from immune recognition. Methods DCs were cultured with rmGM (40μg/L) ,rmIL-4 (40μg/L) ,and the supernatant of cell line AR42J cells was added on the day 6, then a large number of DCs was gained. DCs cultured and in-duced by routine methods were used as controls. The expression of surface molecules CD86, and CD80 in DCs was detected to observe whether this intervention could delay or block maturation of DCs and this effect could be reversed by stimulation with lipopolysaccharide (LPS). T cell stimulatory effects were also determined. Results Compared to normal DCs, CD86 and CD80 expression positive rate was significantly decreased in DCs induced by AR42J supernatant from (70.88±3.60)% to (7.15±0.71 )%. After stimulation with LPS, the expression of CD86, and CD80 in DCs was still low, suggesting the AR42J super-natant could block the maturation of DCs. The allostimulatory capacity of the immature DCs stimulated with the AR42J superoatant was significantly lower than that of the normal immature DCs. Conclusion The supernatant from pancreatic cancer ceils in rats could inhibit the maturation of DCs, which was not easily reversed.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

Objective To study the effect of pancreatic cancer microenvironment on maturation of dendritic cells ( DCs), and to investigate the mechanism by which tumors escape from immune recognition. Methods DCs were cultured with rmGM (40μg/L) ,rmIL-4 (40μg/L) ,and the supernatant of cell line AR42J cells was added on the day 6, then a large number of DCs was gained. DCs cultured and in-duced by routine methods were used as controls. The expression of surface molecules CD86, and CD80 in DCs was detected to observe whether this intervention could delay or block maturation of DCs and this effect could be reversed by stimulation with lipopolysaccharide (LPS). T cell stimulatory effects were also determined. Results Compared to normal DCs, CD86 and CD80 expression positive rate was significantly decreased in DCs induced by AR42J supernatant from (70.88±3.60)% to (7.15±0.71 )%. After stimulation with LPS, the expression of CD86, and CD80 in DCs was still low, suggesting the AR42J super-natant could block the maturation of DCs. The allostimulatory capacity of the immature DCs stimulated with the AR42J superoatant was significantly lower than that of the normal immature DCs. Conclusion The supernatant from pancreatic cancer ceils in rats could inhibit the maturation of DCs, which was not easily reversed.