荷EL4肿瘤小鼠模型的建立及美法仑抑瘤作用免疫机制的探讨

目的:建立荷小鼠淋巴瘤EL4的野生型C57BL/6小鼠及其裸鼠模型,探讨美法仑(melphalan)抑瘤作用的免疫机制。方法:给正常野生型C57BL/6小鼠皮下接种小鼠淋巴瘤EL4细胞,建立荷EL4肿瘤的小鼠模型。于野生型C57BL/6小鼠皮下接种瘤细胞后12d,经腹腔给荷瘤小鼠单次注射不同剂量的美法仑,找出美法仑可发挥最大的抑瘤作用,并能致使肿瘤消退、不再复发的最小使用剂量。然后再给野生型C57BL/6小鼠及其裸鼠(遗传背景相同)皮下同时接种小鼠淋巴瘤EL4细胞建立两种荷瘤小鼠模型。同样于接种瘤细胞后12d,经腹腔给两种荷瘤小鼠模型均注射可使野生型C57BL/6小鼠肿瘤消退、不再复发的最低剂量的美法仑,以正常野生型C57BL/6小鼠为对照,观察在T淋巴细胞缺陷的裸鼠体内美法仑的抑瘤作用。结果:注射7.5mg/kg美法仑治疗后,免疫功能正常的野生型C57BL/6荷瘤小鼠的肿瘤消退;而荷瘤C57BL/6裸鼠的肿瘤仍继续生长。结论:单一剂量的美法仑对荷淋巴瘤EL4小鼠具有明显的治疗作用,其作用的发挥需要T淋巴细胞的参与,可能与T细胞的杀伤作用有关。     

美法仑治愈荷瘤小鼠的过程与TNFα的关系

目的探讨单一剂量的美法仑治愈荷瘤野生型C57BL/6小鼠的过程与肿瘤坏死因子α(TNFα)的关系。方法以3种遗传背景相同、肿瘤坏死因子受体1(TNFR1)基因型不同的TNFR1 / 、TNFR1 /-和TNFR1-/-C57BL/6小鼠为实验动物,皮下接种数量相同的小鼠淋巴瘤EL4细胞。接种瘤细胞后12d,给基因型不同的各组荷瘤小鼠腹腔内单次注射7.5mg/kg的美法仑。以荷瘤野生型C57BL/6小鼠(TNFR1 / )为对照,观察美法仑对荷瘤TNFR1 /-C57BL/6小鼠和荷瘤TNFR1-/-C57BL/6小鼠的治疗效应。结果在美法仑(7.5mg/kg)治疗后的1周内,基因型不同的各组荷瘤小鼠肿瘤消退的速度基本相同。在随后的2月内,荷瘤TNFR1 / 和TNFR1 /-C57BL/6小鼠的肿瘤结节逐渐消退、肿瘤治愈;而多数荷瘤TNFR1-/-C57BL/6小鼠的肿瘤结节缩小后又再次出现并逐渐长大、肿瘤复发。结论TNFα与美法仑治愈肿瘤的过程密切相关,其中美法仑的抗肿瘤作用与荷瘤小鼠TNFR1的表达无关,但在美法仑治疗后,机体预防或避免肿瘤复发方面需要TNFR1在机体细胞的表达,而不是在肿瘤细胞的表达。     

美法仑体内的抗肿瘤作用与IFN-γ的关系

目的:探讨单一剂量的美法仑对荷瘤野生型C57BL/6小鼠的疗效与IFNγ的关系。方法:以3种遗传背景相同、基因型不同的IFNγ / 、IFNγ /-和IFNγ-/-C57BL/6小鼠为模型,皮下接种数量相同的小鼠淋巴瘤EL4细胞。接种瘤细胞后12d,给基因型不同的各组荷瘤小鼠腹腔内单次注射7.5mg/kg的美法仑。以野生型C57BL/6荷瘤小鼠(IFNγ / )为对照,观察美法仑对IFNγ /-C57BL/6荷瘤小鼠和IFNγ-/-C57BL/6荷瘤小鼠的治疗效应。结果:7.5mg/kg的美法仑单次腹腔内注射治疗后,可使IFNγ / 和IFNγ /-C57BL/6小鼠的肿瘤完全消退并治愈,而对IFNγ-/-荷瘤C57BL/6小鼠无治疗作用。结论:IFNγ与美法仑化疗的效果密切相关,即美法仑的抗肿瘤作用需要IFNγ参与。     

B7.1、GM-CSF基因修饰的EL- 4细胞激发小鼠抗淋巴瘤免疫反应

目的 研究B7.1，GM-CSF基因修饰的淋巴瘤瘤苗进行恶性淋巴瘤免疫基因治疗的有效性。方法 用逆转录病毒载体分别将B7.1和GM-CSF基因导入小鼠淋巴瘤细胞EL-4中，得到EL-4/B7和EL-4/GM转基因瘤苗细胞，观察EL-4/B7和EL-4/GM瘤苗细胞对荷淋巴瘤小鼠的免疫治疗作用，以及预先接种过EL-4/B7或/和EL-4/GM瘤苗的小鼠，对野生型EL-4细胞致瘤性作用的影响，并检测接种EL-4/B7或/和EL-4/GM瘤苗的小鼠血浆IL-2及TNF-α的水平，及其脾淋巴细胞对野生型EL-4的细胞毒效应，另外，检测经EL-4/B7，EL-4/GM刺激后的小鼠脾淋巴细胞的增殖程度，结果 （1）经EL-4/B7、EL-4/GM细胞注射后，荷瘤小鼠的淋巴瘤生长速度变慢，生存时间延长，淋巴瘤组织中出现大量的炎症细胞；（2）接种一定数量的EL-4/B7，EL-4/GM细胞的小鼠，能抵抗野生型EL-4细胞的攻击；（3）接种瘤苗的小鼠血浆IL-2的水平明显升高，而TNF-α的水平无明显变化；（4）接种瘤苗的小鼠脾淋巴细胞，对野生型EL-4细胞有明显的细胞毒效应；EL-4/B7、EL-4/GM细胞能刺激小鼠脾淋巴细胞明显增殖。结论 B7.1,GM-CSF基因修饰的EL-4细胞，能有效地激发C57BL/6小鼠的抗淋巴瘤免疫反应，且B7.1基因与GM-CSF基因具有协同作用。     

不同种鼠对实验脑型疟发生的影响研究

目的比较昆明小鼠和C57BL/6小鼠作为种鼠对实验脑型疟模型的影响。方法分别以伯氏疟原虫ANKA株感染C57BL/6小鼠和昆明小鼠作为传代用种鼠,当种鼠原虫率为5%～15%时接种子代C57BL/6小鼠,观察两组小鼠原虫率、脑型疟发生率以及死亡率。同时,通过脑组织切片和脑部淋巴细胞的流式检测,观察两组发生脑型疟小鼠的脑部微血管中感染疟原虫红细胞和CD8＋T细胞的粘附情况,另外,通过感染CD8＋TKO小鼠,证实CD8＋T细胞在两组小鼠发生脑型疟中的作用。结果用昆明小鼠作为种鼠的实验组的原虫率和脑型疟发生率均明显高于用C57BL/6小鼠作为种鼠的实验组,发生脑型疟小鼠的脑部组织切片发现,脑部微血管可见明显的感染疟原虫红细胞的粘附和CD8＋T淋巴细胞浸润;而用昆明小鼠作为种鼠感染CD8＋T细胞缺失的C57BL/6小鼠并不能诱导实验脑型疟的发生。结论与C57BL/6小鼠相比,昆明小鼠作为种鼠的实验组的脑型疟发病率更高,而且感染疟原虫红细胞和CD8＋T细胞在脑部微血管内的粘附也是该脑型疟发生的主要因素,因此,更适合用于实验脑型疟模型的建立及其机制的探讨。     

小鼠白介素-12基因转染对EL细胞在小鼠体内成瘤性的抑制作用

目的探讨逆转录病毒(RV)载体介导的小鼠白介素12(mIL-12)融合基因转染,对低免疫原性的小鼠T细胞淋巴瘤细胞EL4体内生长的影响.方法用共培养法导入基因,以PCR法检测基因的导入.以脾细胞增殖法测定mIL-12的生物学活性.观察导入mIL-12基因的肿瘤细胞EL4/12于小鼠皮下接种后的成瘤性.对预先接种野生型肿瘤细胞EL4的小鼠,用60Co照射的肿瘤疫苗EL4/12接种于瘤周,观察瘤苗的抗肿瘤作用等.结果在EL4/12细胞中,用PCR可特异地检测到mIL-12p35和P40亚基的cDNA片段.5×105EL4/12细胞48h表达的mIL-12达(10.2±3.2)ng.EL4/12细胞在小鼠体内的成瘤性明显下降.EL4/12瘤苗治疗组约50%的小鼠可长期无瘤生存,而对照组小鼠则在短期内全部成瘤死亡.部分长期生存的小鼠产生了有效的抗肿瘤免疫,能耐受野生型肿瘤细胞的二次攻击.与对照组相比较,疫苗治疗组中其余小鼠的成瘤时间延迟(P＜0.01),小鼠存活时间延长(P＜0.01);死亡时肿瘤体积小(P＜0.05),形成的肿瘤有完整包膜,瘤周和肿瘤内部有较多的淋巴细胞和浆细胞浸润等.结论转染mIL-12基因能抑制EL4肿瘤细胞的成瘤性.mIL-12基因修饰的肿瘤疫苗对野生型肿瘤细胞具有治疗作用.     

小鼠白介素—12基因转染对EL4细胞在小鼠体内成瘤性的抑制作用

目的探讨逆转录病毒(RV)载体介导的小鼠白介素12(mIL-12)融合基因转染,对低免疫原性的小鼠T细胞淋巴瘤细胞EL4体内生长的影响。方法用共培养法导入基因,以PCR法检测基因的导入。以脾细胞增殖法测定mIL-12的生物学活性。观察导入mIL-12基因的肿瘤细胞EL4/12于小鼠皮下接种后的成瘤性。对预先接种野生型肿瘤细胞EL4的小鼠,用60Co照射的肿瘤疫苗EL4/12接种于瘤周,观察瘤苗的抗肿瘤作用等。结果在EL4/12细胞中,用PCR可特异地检测到mIL-12p35和p40亚基的cDNA片段。5×105EL4/12细胞48h表达的mIL-12达(10.2±3.2)ng。EL4/12细胞在小鼠体内的成瘤性明显下降。EL4/12瘤苗治疗组约50%的小鼠可长期无瘤生存,而对照组小鼠则在短期内全部成瘤死亡。部分长期生存的小鼠产生了有效的抗肿瘤免疫,能耐受野生型肿瘤细胞的二次攻击。与对照组相比较,疫苗治疗组中其余小鼠的成瘤时间延迟(P∨0.01),小鼠存活时间延长(P∨0.01);死亡时肿瘤体积小(P∨0.05),形成的肿瘤有完整包膜,瘤周和肿瘤内部有较多的淋巴细胞和浆细胞浸润等。结论转染mIL-12基因能抑制EL4肿瘤细胞的成瘤性。mIL-12基因修饰的肿瘤疫苗对野生型肿瘤细胞具有治疗作用。     

在大剂量5-氟尿嘧啶治愈荷瘤小鼠过程中外周血细胞的变化与肿瘤消退之间的相关性分析

目的:观察在单一剂量5-氟尿嘧啶(5-FU)治愈荷瘤小鼠过程中外周血细胞数量的变化与肿瘤结节缩小之间的关系,为进一步研究化疗治愈肿瘤的作用机制奠定基础.方法:利用单一剂量5-FU治愈荷瘤野生型C57BL/6小鼠模型,荷瘤小鼠腹腔内分别注入25、75、135 mg/kg的5-FU,等量生理盐水对照,确定5-FU治愈肿瘤的量效关系.然后,再取6只荷瘤小鼠,分别在75 mg/kg 5-FU治疗前3 d、治疗后第1、4、7、11、15、28天内眦静脉取血,进行血常规检测,分析外周血白细胞、血红蛋白和血小板的变化与荷瘤小鼠肿瘤结节缩小之间的相关性.结果:75、135 mg/kg的5-FU均可使荷瘤小鼠的肿瘤结节在治疗后1周内逐渐缩小、直至消退,因此5-FU治愈荷瘤小鼠的最低有效量为75 mg/kg.75 mg/kg 5-FU治疗后第1天外周血白细胞降为(5.0±1.1)×109/L,并持续到化疗后第4天,与治疗前(9.5±1.58)×109/L比较差异有统计学意义(P<0.01);5-FU治疗后第7 ～15天外周血白细胞的数量升至化疗前水平,随后逐渐下降,在治疗后第28天降至(5.9±1.96)×109/L,低于化疗前水平(P<0.01).Hb含量在5-FU治疗后第1天也出现降低,由化疗前的(133±7) g/L降为(112±9) g/L,差异有统计学意义(P<0.01),随后Hb含量持续在低水平,治疗后15 d 逐渐恢复至化疗前水平.75 mg/kg的5-FU对荷瘤小鼠血小板的抑制作用不明显,反而在治疗后第1、11天出现2次血小板一过性增高(P<0.01).结论:75 mg/kg 5-FU可引起外周血白细胞数和血红蛋白浓度的降低,但对血小板的抑制作用不明显.5-FU发挥抗肿瘤作用使荷瘤小鼠的肿瘤结节缩小与化疗后外周血白细胞和Hb的降低无关,而与化疗后第1天血小板计数的增加有关.     

氟尿嘧啶联合CIK细胞在小鼠结肠癌模型中抗肿瘤效应的研究

探讨氟尿嘧啶(5-FU)、CIK细胞以及5-FU联合CIK细胞对小鼠结肠癌移植瘤生长的作用,为临床上联合应用5-FU和CIK细胞治疗结肠癌提供理论依据。建立BALB/C小鼠结肠癌模型,在体外诱导培养小鼠CIK细胞,5-FU(12.5mg/kg,d0)联合CIK细胞(1×107,d3)处理小鼠结肠癌荷瘤模型,并绘制肿瘤生长曲线,生存分析采用Kaplan-Meier法及logrank检验,应用流式细胞仪分析荷瘤鼠肿瘤组织中淋巴细胞CTLA-4和非淋巴细胞CD80、CD86表达;RT-PCR法检测肿瘤组织中IFN-γ和TNF-α表达量。5-FU联合CIK细胞治疗组小鼠肿瘤生长速度明显慢于单治疗组,差异有统计学意义(P<0.05);5-FU处理后小鼠移植瘤组织中CD45+细胞表面CTLA-4表达量提高,且CD45-细胞CD80和CD86表达量增加;5-FU联合CIK细胞治疗可提高肿瘤组织IFN-γ与TNF-α表达量,且联合治疗组与单治疗组中IFN-γ的表达量之间的差异有统计学意义(P<0.05)。5-FU联合CIK细胞治疗对结肠癌移植瘤生长的抑制作用大于单独应用5-FU或CIK细胞,且可提高BALB/C小鼠结肠癌模型免疫功能。     

Hsp70L1增强肿瘤细胞疫苗免疫原性的研究

目的:考察Hsp70L1对肿瘤细胞免疫原性的增强作用。方法:用RT-PCR的方法,从小鼠黑色素瘤B16细胞和C57BL/6小鼠脾脏中获得TRP2153-243及Hsp70L1基因。分别插入pcDNA3.1/V5-His真核表达载体,构建pHSP70L1、pTRP和pTRP2-Hsp3种表达载体。分别转染B16肿瘤细胞并制备坏死或凋亡瘤苗,免疫C57BL/6小鼠后移植B16肿瘤细胞,观察肿瘤生长曲线,采用流式细胞术(FCM)或微量细胞毒的方法检测荷瘤小鼠细胞因子INF-γ和CTL活性。结果:将经过HSP70L1、TRP2及TRP2-HSP基因修饰的坏死或凋亡的B16肿瘤细胞免疫正常小鼠后,观察到Hsp70L1及TRP2-Hsp基因修饰的坏死瘤苗可显著抑制荷瘤鼠肿瘤的生长,并显著促进荷瘤鼠脾脏淋巴细胞CTL活性和IFN-γ产生(P0.05,P0.01);HSP70L1免疫刺激作用在坏死瘤苗中更明显。结论:Hsp70L1可明显提高B16瘤苗的免疫原性,且对坏死细胞瘤苗的作用更显著。     

Decreased “natural killer” effect in tumor-bearing mice and its relation to the immunity against oncorna virus-determined cell surface antigens

In comparison to control animals the natural killer (NK) efficiency of spleen and blood lymphocyte population was reduced in three systems of tumor-bearing animals: transplanted methylcholanthrene (MC) and primary Moloney sarcoma virus (MSV) induced sarcomas in CBA mice, and athymic “nude” mice carrying grafts of human lymphoblastoid cell lines. No evidence for suppressor cells was found. After regression of the MSV tumors the activity was high again. The effect was measured on YAC - a culture line of a Moloney leukemia virus-induced lymphoma in an A mouse - which is highly sensitive to the NK effect. Against another lymphoma, RBL-5, (Rauscher leukemia virus induced in C57BL mice), the effect of control spleen cells was low while that of the MSV tumor-bearing spleen cells was high, indicative of immunization effect. With regard to the virus-induced antigens, YAC and RBL-5 cross-react. Analysis of the NK effector population indicated that anti-YAC killer cells are enriched after passage through nylon wool. This would indicate a cytotoxic role of T cells. However, treatment with anti-RBL-l.2 serum did not markedly influence the effect. On the other hand, anti-RBL-5 activity of spleens from tumor-bearing (CBA × C57BL)F₁ mice decreased after removal of Thy-1.2-positive cells. YAC thus seems to be more sensitive to the NK effect, while RBL-5 to the T cell-mediated immune effect. Cytotoxic lymphocytes for YAC and RBL-5 could be isolated from MSV tumors of CBA but not of A mice in spite of the regularly occurring regression also in the latter strain.     

Experimental Yersinia enterocolitica infection in euthymic and T-cell-deficient athymic nude C57BL/6 mice: comparison of time course, histomorphology, and immune response.

To elucidate the role of T lymphocytes in primary infection with Yersinia enterocolitica, we investigated the elimination rate of this pathogen, the histomorphology of tissue lesions, and the immune responses of athymic T-cell-deficient C57BL/6 nude mice and their euthymic littermates after parenteral infection with Y. enterocolitica of serotype O:8. While a low inoculum of 3 x 10(2) Y. enterocolitica cells (about 0.01 times the median lethal dose for normal C57BL/6 mice) was cleared by normal C57BL/6 mice within 7 to 10 days, athymic nude C57BL/6 mice developed progressive infections after this inoculum, leading to death on days 20 to 25 postinfection (p.i.). While normal C57BL/6 mice experienced short-term transient infections, nude mice exhibited a biphasic, progressive infectious process. Thus, in the early phase (days 1 to 7 p.i.), a rapid influx of CD11b/18-positive cells (Mac-1 antigen) and natural killer cells was evident in the spleens and livers of the nude mice. The late phase (from day 8 p.i. onward) was characterized by a rapid progression of the infection and a further influx of CD11b/18-positive cells into the liver accompanied by an increase in bacterial counts and development of tissue lesions particularly in the liver and spleen. In normal mice, granuloma-like lesions composed of CD11b/18-, CD4-, and CD8-positive cells could be observed. However, granulomata were not found in nude mice. Yersinia-specific immunoglobulin G antibodies appeared on day 15 p.i. in the sera of normal mice, while nude mice failed to develop significant antibody titers. Adoptive transfer of Yersinia-specific T cells into athymic nude mice mediated resistance to Y. enterocolitica infection and restored both the ability of granuloma formation and the production of specific antibodies. In summary, the data presented herein strongly suggest that T lymphocytes play an essential role in the defense of C57BL/6 mice against Y. enterocolitica.     

Damage in B2m genes and DNA methylation of H-2 genes are involved in loss of expression of class I MHC products on the membrane of LR.4, a cell line derivative of the T-cell lymphoma L5178Y.

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.     

DAMAGE IN B2m GENES AND DNA METHYLATION OF H-2 GENES ARE INVOLVED IN LOSS OF EXPRESSION OF CLASS I MHC PRODUCTS ON THE MEMBRANE OF LR.4, A CELL LINE DERIVATIVE OF THE T-CELL LYMPHOMA L5178Y

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.     

Bacteriophages support anti-tumor response initiated by DC-based vaccine against murine transplantable colon carcinoma

Bacteriophages in eukaryotic hosts may behave as particulate antigens able to activate the innate immune system and generate adaptive immunity. Dendritic cells (DCs) play a key role in the initiation of the immune response, mainly by priming T cell-mediated immunity. For this reason, they are increasingly applied as an adjuvant for effective anti-tumor therapies in animal models as well as in a few clinical trials. The presented study focused on the application of mouse DCs which were activated with T4 bacteriophages (T4 phages, T4) and further loaded with tumor antigens (TAg) in inducing an anti-tumor response. The activation of bone marrow-derived DCs with T4 phages and TAg resulted in augmentation of their differentiation marker expression accompanied by an enhanced ability to prime T cells for IFN-gamma production. These activated DCs (BM-DC/T4+TAg) were used in experimental immunotherapy of C57BL/6 mice bearing advanced MC38 colon carcinoma tumors. As a result of their triple application, a significant tumor growth delay, up to 19 days, was observed compared with the controls - treated with BM-DCs activated only with T4 phages, TAg, or lipopolysaccharide solution ["solvent"], where the tumor growth delay did not exceed 7 days. The percentage of tumor growth inhibition estimated 10 days after the third cell injection ranged from 32% (for animals treated with BM-DC/TAg cells) to 76% (for animals treated with BM-DC/T4+TAg cells) over the tumor-bearing untreated control mice. The obtained data indicate that in vitro interactions between T4 phages and BM-DCs followed by TAg activation caused augmentation of the anti-tumor effect when DCs were used as a vaccine for tumor-bearing mice treatment. Therefore, pretreatment of DCs with the phages may be considered as a beneficial element of a novel strategy in anti-tumor immunotherapy.     

A single gene controls resistance to Japanese encephalitis virus in mice

Summary The inheritance of resistance to Japanese encephalitis virus (JEV) was investigated using inbred strains of mice to study genetic resistance against JEV infection. C57BL/6 mice immunized intraperitoneally (i.p.) with an infective dose of JEV were resistant to intracerebral (i.c.) challenge with JEV, whereas most C3H/He mice treated in the same manner died. C57BL/6 mice developed this resistance 2 weeks earlier than C3H/He after intraperitoneal (i.p.) immunization. Passive transfer of spleen cells from immunized C57BL/6 protected the recipient mice from i.c. challenge, while transfer from immunized C3H/He was less effective. Since immunized athymic nude mice were not resistant to i.c. challenge with JEV, T lymphocytes were considered to be necessary for protection. When F₁, F₂ and backcross mice derived from C57BL/6 and C3H/He were challenged i.c. with JEV after i.p. immunizations, the number of resistant and susceptible mice were consistent with Mendelian ratios. Thus it can be concluded that resistance to JEV in mice was controlled by a single, dominant autosomal gene which was not linked toa (non agouti)-locus (chromosome 2).     

Trichosanthin enhances anti-tumor immune response in a murine Lewis lung cancer model by boosting the interaction between TSLC1 and CRTAM

Trichosanthin (TCS), extracted from the Chinese medicinal herb Trichosanthes kirilowi, has shown promise for the inhibition of tumor growth. However, its immunomodulatory effect on tumor-host interaction remains unknown. In this study, we focused on the effect of TCS on murine anti-tumor immune response in the 3LL Lewis lung carcinoma tumor model and explored the possible molecular pathways involved. In addition to inhibiting cell proliferation and inducing apoptosis in the 3LL tumor, TCS retarded tumor growth and prolonged mouse survival more significantly in C57BL/6 immunocompetent mice than in nude mice. This reflected the fact that the host immune system was involved in tumor eradication. Using FACS analysis, we found that TCS increased the percentage of effector T cells, particularly Interferon-gamma (IFN-γ) producing CD4(+) and CD8(+) T cells from tumor-bearing mice. TCS also promoted the vigorous proliferation of antigen-specific effector T cells, markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice, consequently enhancing the anti-tumor response and inducing immune protection. Furthermore, we found that TCS upregulated the expression of tumor suppressor in lung cancer 1 (TSLC1) in 3LL tumor cells and the expression of its ligand, class I-restricted T cell-associated molecule (CRTAM), in effector T cells. Blocking TSLC1 expression with small interfering RNA (siRNA) significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells, suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM. Collectively, our data demonstrate that TCS not only affects tumor cells directly, but also enhances anti-tumor immunity via the interaction between TSLC1 and CRTAM. These findings may lead to the development of a novel approach for tumor regression.