其它（051124～051144）

多次小剂量pEgr-IFN-γ基因-放射治疗的抑瘤效应及其机制的研究；Hsp70-肿瘤抗原肽复合物防治小鼠黑色素瘤B16肺转移的作用；CA72-4在10种恶性肿瘤患者血清中的临床界值；肿瘤库(用于分子生物学研究)的建立及管理；围手术期营养支持对外科病人预后的影响；     

4-1BBL联合Hsp70-肿瘤抗原肽抑制小鼠黑色素瘤肺转移

背景与目的正调节性共刺激分子表达偏低是肿瘤逃避机体免疫系统攻击的重要原因之一,增加这些分子的表达是促进抗肿瘤免疫的研究热点。小鼠黑色素瘤B16-F1细胞株是弱免疫原性癌细胞株。本研究旨在观察4-1BBL联合Hsp70-抗原肽对小鼠黑色素瘤肺转移的抑制作用,并探讨其机制。方法建立小鼠黑色素瘤肺转移模型,Hsp70-B16抗原肽小鼠皮下免疫,同时从尾静脉注射4-1BBL表达质粒p4-1BBL。于接种后第17天,解剖小鼠取肺组织,解剖显微镜下计数黑色素瘤肺转移结节的数目,并检测小鼠部分免疫学指标及肝、肾功能。结果4-1BBL联合Hsp70-B16抗原肽治疗组小鼠黑色素瘤肺转移结节数降至50±8,明显少于二者单独治疗组的500±80和450±40;联合治疗组小鼠血清IL-2和IFN-γ的分泌水平分别增加了4倍和3倍,与对照组相比均存在显著性差异(P<0.01);单独应用4-1BBL基因或与Hsp70-B16抗原肽联合应用对小鼠肝、肾的功能均无明显影响。结论表达4-1BBL联合应用Hsp70-B16抗原肽主要通过增强外周T淋巴细胞功能活性而有效抑制小鼠黑色素瘤肺转移。     

HIFU治疗后肿瘤抗原对树突状细胞的活化及其抗肿瘤效应

目的：探讨HIFU治疗小鼠H22抑制性肝癌后产生的肿瘤抗原对机体抗肿瘤免疫功能增强的机制。方法：正常小鼠骨髓中提取骨髓细胞，在rmIL-4、rmGM-CSF奈件下培养7d，制备小鼠骨髓树突状细胞，用HIFU治疗小鼠移植性肝癌后产生的肿瘤抗原活化树突状细胞，再用活化后的树突状细胞激活T淋巴细胞为细胞毒性T细胞，用MTT法检测CTL在体外特异性杀伤肿瘤靶细胞的能力。结果：B16肿瘤HSP70-肽复合物组和H22肿瘤HSP70-肽复合物组的脾淋巴细胞的增殖率均高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组，P〈0．001；但两组之间差异无统计学意义，P〉0．05。H22肿瘤HSP70-肽复合物组CTL对H22肿瘤细胞的杀伤率为70．0％，明显高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组（P〈0．001），但对非靶细胞B16肿瘤的杀伤率与上述各组的差异无统计学意义；B16肿瘤HSP70-肽复合物组CTL对B16肿瘤细胞的杀伤率为78．5％，对H22细胞的杀伤率为21．4％，表明CTL对肿瘤细胞的杀伤作用具有特异性。结论：HIFU治疗后坏死肿瘤组织中的HSP70-肽复合物作为肿瘤疫苗，通过活化DC和刺激T淋巴细胞增殖为CTL，发挥特异性抗肿瘤免疫功能。     

Hsp70-H22肿瘤抗原肽复合物激活树突状细胞诱导抗肿瘤免疫的研究

目的　探讨通过树突状细胞 (DC)提呈途径降低肿瘤抗原肽用量的可行性 ,了解热休克蛋白 70 (Hsp70 )和抗原肽修饰DC作用的特点。方法　以Hsp70于体外结合抗原肽 ,并于体外修饰DC ,检测修饰后DC的代谢活性及分泌的细胞因子 ;比较修饰后DC和Hsp70 H2 2肽对淋巴细胞的激活作用 ;检测激活的淋巴细胞对H2 2瘤细胞的细胞毒作用以及注射DC和Hsp70 H2 2肽对小鼠肿瘤的抑制作用。结果　Hsp70结合 0 .15 μgH2 2肽可使 2× 10 5DC成熟 ,4× 10 3 成熟DC可激活 2× 10 6淋巴细胞 ;Hsp70结合 0 .0 0 3μg肽修饰的DC或Hsp70结合 0 .15 μg肽直接刺激 ,可激活相同数量的淋巴细胞 ,产生同样的杀瘤效果。激活DC后再回输的治疗方式与直接注射Hsp70 肽复合物的治疗方式相比 ,抗原肽的用量可以降低 5 0倍。正常肝组织来源的混合肽结合Hsp70后 ,不能使DC成熟 ,亦不能通过DC途径活化脾淋巴细胞。结论　DC提呈抗原肽激活淋巴细胞的途径能够有效降低Hsp70 肿瘤抗原肽复合物使用剂量。正常细胞的混合肽不能通过Hsp70和DC提呈激活淋巴细胞 ,其诱发自身免疫应答的可能性极低     

抑制整合素α9基因的表达对黑色素瘤细胞B16F1的生长和肺转移的影响

目的 观察shRNA干扰整合素α9（ITGA9）的表达对黑色素瘤细胞B16F1的生长和肺转移的影响。方法 用RNA干扰技术下调B16F1中ITGA9的表达,建立小鼠皮下成瘤和肺转移模型,观察肿瘤生长情况,计数肺转移灶数量。结果 ITGA9-shRNA转染组的肿瘤生长速度减慢（P＜0.05）,实验终点,该组肿瘤平均体积与scramble-shRNA组相比下降36%;肺转移灶数量显著减少（P＜0.05）。结论 下调ITGA9的表达可抑制黑色素瘤细胞B16F1在小鼠体内的生长和肺转移。ITGA9可能成为黑色素瘤的治疗靶点。     

人T淋巴细胞白血病不同细胞株的混合抗原肽诱导特异性CTLs细胞毒效应及其交叉反应性分析

目的探讨T淋巴细胞白血病不同细胞株的混合抗原肽诱导特异性抗瘤免疫的作用及不同细胞株混合抗原肽之间的交叉反应性。方法从不同白血病细胞株中分别制备混合抗原肽,与Hsp70体外结合,观察Hsp70-抗原肽复合物对人外周血单个核细胞(PBMC)的激活增殖作用;以激活增殖的PBMC为效应细胞,对不同的靶细胞进行体外杀伤试验。结果不同白血病细胞株的抗原肽为混合肽,经Hsp70提呈均能够有效激活PBMC,并能使激活的PBMC增殖,增殖活化的PBMC可以特异性杀伤与抗原肽对应的白血病细胞;Hut78-肽和Molt-4-肽激活的PBMC对Hut78细胞、Molt-4细胞和Jurkat细胞的细胞毒作用均显著高于HL-60-肽激活的PBMC(P<0.05)。而Hut78/Molt-4-肽激活的PBMC对Jurkat细胞的杀伤作用则显著高于Hut78-肽和Molt-4-肽单独激活的PBMC(P<0.05)。结论不同T淋巴细胞白血病细胞株来源的混合抗原肽均能够诱导特异性抗肿瘤免疫,其所含的抗原肽之间存在交叉性,通过多株来源的抗原肽混合,这种交叉性可以被放大。     

人参皂甙Rg3和核糖核酸酶抑制因子转基因对小鼠黑色素瘤的抑制作用研究

目的观察人参皂甙Rg3和核糖核酸酶抑制因子(RI)转基因对小鼠B16黑色素瘤肺转移的抑制作用和影响,探讨人参皂甙Rg3和RI抗肿瘤生长和转移作用的分子机制。方法制备转RI基因的B16黑色素瘤肺转移小鼠模型,对野生型对照组(W组)、空质粒转染组(B组)和RI转基因组(RI组)以及给予Rg3的野生型对照组(Rg3/W组)、空质粒转染组(Rg3/B组)和RI转基因组(Rg3/RI组)中,荷瘤小鼠肺重量、肿瘤转移灶数目、生存期和肿瘤组织微血管密度进行检测和分析。结果Rg3和RI转基因使荷瘤小鼠肺重量降低,肿瘤转移灶数目减少,其肺重量降低和肿瘤转移灶数目减少的程度以Rg3/RI组最明显,Rg3/B组、Rg3/W组和RI组次之,与W组和B组差异有显著性(P<0.01),Rg3和RI有一定的协同性。Rg3和RI可延长荷瘤小鼠的生存期,Rg3/RI组小鼠在观察期(1.5个月)内均存活,W组和B组小鼠全部死亡(至26d),且出现死亡的时间较早。经HE染色和第Ⅷ因子相关抗原的免疫组化分析显示,Rg3和RI使肺内瘤组织的微血管密度降低,降低的程度为Rg3/RI组>Rg3/B组>Rg3/W组>RI组>B组>W组。结论人参皂甙Rg3可增强RI转基因对小鼠黑色素瘤肺转移的抑制作用,人参皂甙Rg3和RI基因在抗肿瘤生长、转移及血管生成方面有协同作用。     

蛇葡萄素抗黑色素瘤侵袭和转移的作用

背景与目的：我们先前的研究发现蛇葡萄素在体外对几种人肿瘤细胞以及在体内对小鼠移植性B16黑色素瘤具有抑制作用。为了进一步研究其抑制肿瘤转移的作用。我们研究了蛇葡萄素在体内外抗B16小鼠黑色素瘤侵袭和转移的作用。方法：B16细胞由尾静脉注入C57BL／6小鼠体内,建立实验性肺转移模型,蛇葡萄素以3个剂量从接种瘤细胞前一天腹腔给药,连续18天,于停药后第2天观察肺转移瘤灶数,B16细胞经蛇葡萄素处理3天,用具有聚碳酸酯和重建基底膜（基质胶Matrigel)的Transwell小室侵袭模型,研究药物处理后细胞侵袭,趋化运动,粘附能力的改变。结果：150、200,250mg/kg的蛇葡萄素可抑制小鼠肺转移瘤灶形成,与对照组比,抑制率分别为30．97％,40．58％,61．16％（P＜0．05）,经20,40,80μmol/L蛇葡萄素处理后,对B16细胞侵袭人工基底膜的抑制率分别为36．06％,59．58％和79．09％（P＜0．01）;对细胞的趋化运动抑制率分别为51．59％,56．51％和66．75％（P＜0．01）;显著降低B16细胞与基质成分层粘连蛋白,纤维粘连蛋白及Matrigel的粘附作用,结论：蛇葡萄素具有抑制黑色素瘤的侵袭和转移的作用。     

热休克蛋白72-甲胎蛋白抗原表位肽复合物抗小鼠肝癌Hepal-6细胞免疫的实验研究

目的:以热休克蛋白72(HSP72)-甲胎蛋白(AFP)抗原表位肽复合物免疫小鼠,研究该复合物针对AFP肿瘤是否具有特异性抗肿瘤免疫.方法:以HSP72-AFP多肽复合物皮下注射免疫昆明小鼠,并分别以AFP多肽和HSP72单独免疫小鼠为对照组.ELISA法检测免疫后小鼠血清IFN-γ水平、MTT法检测各免疫组小鼠淋巴细胞对肝癌Hepal-6细胞的杀伤作用、以小鼠体内瘤负荷实验评价蛋白复合物的免疫效应.结果:HSP72-AFP多肽复合物组小鼠血清IFN-γ水平、淋巴细胞对Hepal-6细胞的杀伤作用均显著高于AFP多肽和HSP72组(P＜0.01).HSP72-AFP多肽复合物组瘤体积也明显小于AFP多肽和HSP72免疫组(P＜0.01).结论:HSP72-AFP多肽复合物疫苗可诱导荷瘤小鼠产生针对AFP肿瘤的特异性细胞免疫,其对瘤细胞的杀伤效应明显优于两者单一的多肽纯化疫苗.表明HSP72-AFP多肽复合物可诱导小鼠产生有效的抗肿瘤免疫.     

黑色素瘤B16细胞热休克蛋白 抗原肽复合物的体内外抑瘤效应 *

黑色素瘤B16细胞胞浆HSP-抗原肽复合物（HAC）的制备、免疫原性的诱导及其抑瘤作用的研究。方法:采用Tris-HCl提取和Sephacryl S-200凝胶过滤制备B16细胞HAC,通过C57BL/6J小鼠体内诱导特异性CTL,再经体内、体外实验检测其抑瘤作用。结果:凝胶过滤获得的含60～97 kD蛋白的41,47和53管的HAC可以降低肿瘤发生率、延长肿瘤出现时间及降低小鼠死亡率。结论: B16 细胞胞浆中的60～97 kD的HAC具有免疫原性及抑瘤作用,为制备肿瘤疫苗提供了实验依据。     

Antigenic differences among B16 melanoma variants selected for their differing abilities to metastasize: a possible mechanism for effective adjuvant immunotherapy

Most C57BL/6J mice will develop pulmonary metastases four to six weeks after excision of B16 melanoma isografts and begin to die within 40 days. When applied in an adjuvant setting after isograft excision, specific B16 immune RNA (I-RNA) therapy prevents pulmonary metastases and prolongs survival in 50% of treated animals. Since increased in vitro cell-mediated cytotoxicity (CMC) against B16 targets has been demonstrated in animals whose survival is improved by B16 I-RNA therapy, we have proposed that the treatment functioned, at least in part, by specifically altering host CMC in vivo. In this study, C57BL/6J splenocytes were specifically sensitized in vitro by B16 I-RNA exposure and examined for cytotoxic effect on variants of B16 melanoma selected for their differing metastatic potential in vivo. F10 tumor targets (explanted from a B16 melanoma vairant producing frequent pulmonary metastases in vivo) were consistently more sensitive to specific in vitro cytotoxic effect than F1 target cells (from a B16 melanoma variant with low metastatic potential in vivo). Lung metastases (F1 mets) were explanted after intravenous (IV) injection of F1 and used as target cells in the in vitro CMC assays. F1 mets demonstrated greater cytotoxic effect than F1 targets after exposure to B16 I-RNA-treated syngeneic splenocytes. C57BL/6J mice were sacrificed at weekly intervals following adjuvant in vivo B16 I-RNA therapy (after B16 isograft excision), and their splenocytes were shown to be consistently more cytotoxic in vitro to B16 and F10 than to F1 target cells. Splenocytes harvested from mice treated with I-RNA specific to antigenically distinct Lewis lung carcinoma (3LL) after 3LL isograft excision had no cytotoxic effect on any of the B16 variants. When nonsensitized C57BL/6J splenocytes were examined for cytotoxic effect in natural killer (NK) assays or in NK inhibition assays, differences in cytotoxic sensitivity of B16, F10, F1, and F1 mets could not be demonstrated. We conclude, therefore, that specifically sensitized effector cells could distinguish antigenic differences among B16 variants selected for differing in vivo metastatic potiential. These differences were tumor specific and could be demonstrated by cytotoxic splenocytes after either in vivo B16 I-RNA treatment or in vitro B16 I-RNA exposure. The relationship of these antigenic differences to in vivo metastatic potential and to the effectiveness of adjuvant B16 I-RNA therapy is discussed.     

黑色素瘤B16细胞热休克蛋白—抗原肽复合物的体内外抑？…

目的：黑色素瘤Ｂ１６细胞胞浆ＨＳＰ－抗原肽复合物（ＨＡＣ）的制备、免疫原性的诱导及其抑瘤作用的研究。方法：采用Ｔｒｉｓ－ＨＣＩ提取和Ｓｅｐｈａｃｒｙｌ　Ｓ－２００凝胶过滤制备Ｂ１６细胞ＨＡＣ,通过Ｃ５７ＢＬ／６Ｊ小鼠体内诱导特异性ＣＴＬ,再经体内、体外实验检测其抑癌作用。结果：凝胶过滤获得的含６０～９７ｋＤ蛋白的４１,４７和５３管的ＨＡＣ可以降低肿瘤发生率、延长肿瘤出现时间及降低小鼠死亡率。结论：Ｂ１６细胞胞浆中的６０～９７ｋＤ的ＨＡＣ具有免疫原性及抑瘤作用,为制备肿瘤疫苗提供了实验依据。     

乳腺癌腋窝淋巴结转移血管生成的免疫组化研究

目的　研究乳腺癌腋窝淋巴结转移的血管生成情况。方法　采用内皮细胞ⅧFRAg 免疫组化染色技术,对37 例乳腺癌根治术或改良根治术切除的乳腺癌组织和121 枚腋窝转移淋巴结进行免疫组化染色。在100 倍视野下通过显微电视系统计数微血管密度( MVD) ,并用显微测量器测量转移灶的直径。结果　在121 个淋巴结中找到13 处微转移灶,其平均直径为(210 ±37) μm ,无血管生成。腋窝淋巴结转移瘤的MVD 为89-3 ±18-4 ,与乳腺癌组织MVD(93-8 ±21-8) 差异无显著性,且微血管分布不均,周围高于中央。结论　淋巴结微转移灶无血管生成,转移瘤有血管生成。为抑制微转移灶发展成转移瘤,以及抑制转移瘤的生长,抑制血管生成可能是控制淋巴结转移的有效措施。     

Immunogenicity and cross-protection among b16 melanoma variants with differing proclivities to metastasize

We report here the lack of any relationship between immunogenicity and the potential to metastasize among four B16 melanoma variants. Groups of C57BL/6J mice were immunized with one of four B16 variant lines (maintained by serial IM passage) that were shown in separate experiments to produce the following incidences of pulmonary metastases four to six weeks after isograft excision: M1, 10%–15%; M2, 10%–20%; M3, 75%–80%; and M4, 75%–90%. After immunization, mice were challenged with one of the four variant lines in a crisscross fashion. Two challenge doses for each line were chosen on the basis of earlier experiments showing 100% and 50% challenge tumor take in nonimmunized animals. Despite earlier work published from this laboratory showing that splenocytes harvested from immunized mice could distinguish antigenic differences among the B16 variants, we could find no significant difference in immunogenicity or cross-protection among the lines regardless of their metastatic potential. We conclude that no correlation exists between the antigenic differences shown by in vitro splenocyte-mediated cytotoxicity assays among the B16 variants and in vivo immunogenicity or cross-protection experiments. Furthermore, the relationship (if any) between immunogenicity and the ability to metastasize among these B16 variants is not straightforward.     

小剂量X线全身照射对不同荷瘤时间小鼠的抗肿瘤转移效应

采用肿瘤血道转移模型，选用Ｂ１６黑色素瘤和Ｌｅｗｉｓ肺癌（ＬＬＣ）两种类型细胞，就小剂量Ｘ线全身照射对不同荷瘤时间小鼠的抗肿瘤转移效应进行研究。结果发现，于静脉注射Ｂ１６黑色素瘤或ＬＬＣ细胞后２４ｈ（Ｂ组）或第１３天（Ｃ组）接受７５ｍＧｙＸ线全身照射小鼠的肿瘤肺转移结节数明显低于未照射组（Ａ组）（Ｐ＜０．０５～０．００２）。比较不同照射时间的抗肿瘤转移效果时发现，静注Ｂ１６黑色素瘤的Ｂ组肺转移结节数仅为Ｃ组的５９％，而ＬＬＣ的Ｂ组则为其Ｃ组的７１％。检测注射Ｂ１６黑色素瘤第１４天各组小鼠的免疫功能时发现，Ｂ、Ｃ两组ＮＫ、ＬＡＫ细胞活性及脾细胞对ＩＬ－２反应性明显高于Ａ组（Ｐ＜０．０５～０．００１）。上述结果提示：小剂量Ｘ线全身照射提高荷瘤小鼠的免疫功能可能是其抗肿瘤转移作用的主要机制之一。     

Immunization with tumor-derived ER chaperone grp170 elicits tumor-specific CD8+ T-cell responses and reduces pulmonary metastatic disease

Glucose-regulated protein (grp) 170 is a molecular chaperone localized in endoplasmic reticulum (ER), which has been demonstrated to interact with the peptides translocated by transporter associated with antigen presentation (TAP). In our study, we have evaluated the therapeutic efficacy of tumor-derived grp170 against the highly metastatic and poorly immunogenic murine melanoma B16F10. Immunization of mice with grp170 preparations from autologous tumor significantly delayed progression of the primary cancer and reduced established pulmonary metastases, which was associated with the prolonged survival of metastases-bearing mice. However, grp170 from normal liver or antigenically distinct tumor failed to exhibit therapeutic effect. In addition, tumor-derived grp170 elicited a potent cytotoxic T-lymphocyte response specific for B16F10 tumor, which correlates with in vivo protective effects. Adoptive transfer of splenocytes obtained from B16F10-grp170-primed animals remarkably suppressed pulmonary metastases. Depletion of either CD4(+) or CD8(+) T cells in priming phase significantly abrogated the tumor immunity induced by the B16F10-grp170. However, the vaccine activity was intact when CD4(+), not CD8(+), T cells were depleted in effector phase. It suggests that CD4(+) T helper cells play an important role in the generation of effective antitumor response, whereas CD8(+) T cells are predominantly involved in direct killing of tumor cells. These observations have strong clinical implications for using tumor-derived grp170 as a therapeutic vaccine against metastatic diseases.     

Extraction of immunogenic and suppressogenic antigens from variants of B16 melanoma exhibiting low or high metastatic potentials

This investigation examined the effect of soluble antigen prophylaxis on s.c. and metastatic growth of B16 variants which demonstrated either a low or high propensity to colonize the lungs (B16-F1 and B16-F10, respectively) of syngeneic C57BL/6J mice. The two variants share a tumor-associated antigen, since immunization with crude butanol extracts (CBEs) of B16-F1 cells protected hosts against s.c. challenge with either B16-F1 or B16-F10 cells. CBE from B16-F10 (CBE-F10) were unable to engender a measurable immune response against s.c. challenge with either tumor variant. Pretreatment with 100 to 300 micrograms CBE from F1 cells was also effective in reducing the outgrowth of experimentally induced B16-F1 or B16-F10 pulmonary foci. However, mice immunized with 100 micrograms CBE-F10 bore significantly more pulmonary tumors than did phosphate-buffered saline-treated controls. The enhancing and protective activities were specific for the B16 tumor and could be adoptively transferred 24 hr prior to tumor challenge by i.p. injection of 5 X 10(7) spleen cells from CBE-immunized mice. The enhancing activity in the CBE-F10 immune spleen cell population was abolished by depletion of adherent cells onto plastic. Adoptive transfer of the CBE-F10-immune adherent cell population did not affect metastatic growth, suggesting that, in this experimental system, the adherent population was not an efferent suppressor and could not recruit host elements to effect suppression. Indeed, spleen cell-mixing experiments demonstrated that only immune adherent cells combined with immune nonadherent cells could partially reconstitute the tumor growth-enhancing potential of the unfractionated CBE-F10-immune spleen cell population.     

ANTITUMOR EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR(GM-CSF)-GENE ENCODED VACCINIA MELANOMA ONCOLYSATE AND ITS IMMUNOLOGICAL MECHANISMS

ＡＮＴＩＴＵＭＯＲＥＦＦＥＣＴＯＦＧＲＡＮＵＬＯＣＹＴＥＭＡＣＲＯＰＨＡＧＥＣＯＬＯＮＹＳＴＩＭＵＬＡＴＩＮＧＦＡＣＴＯＲ（ＧＭＣＳＦ）ＧＥＮＥＥＮＣＯＤＥＤＶＡＣＣＩＮＩＡＭＥＬＡＮＯＭＡＯＮＣＯＬＹＳＡＴＥＡＮＤＩＴＳＩＭＭＵＮＯＬＯＧＩ...     

A3 adenosine receptor agonist potentiates natural killer cell activity

Activation of the Gi-protein-coupled A3 adenosine receptor (A3AR) has been reported to be involved in the inhibition of tumor cell growth. A3AR is highly expressed in tumor cells whereas lower expression was noted in a variety of normal cells. Recently we showed that A3AR activation in melanoma cells resulted in growth inhibition via a direct anti-proliferative effect which entailed cell cycle arrest in the G0/G1 and down-regulation of cyclin D1 and c-Myc. In the present study we present an additional mechanism demonstrating that A3AR agonists activate natural killer (NK) cells which further enhance the anti-tumor effect of this group of molecules. NK cells mediate the natural cytotoxicity and their number and function is reduced in cancer patients. We show Cl-IB-MECA to inhibit tumor development via the activation of NK cells is an additional mechanism which accounts for the anti-tumor effect of A3AR agonists. This effect was noted at a low dose of 10 micro g/kg, demonstrating that the response is exclusively A3AR mediated. Treatment of na?ve mice for four days yielded the highest effect on NK cell potentiation. In mice inoculated with B16-F10 melanoma cells and treated each orally with Cl-IB-MECA, melanoma growth inhibition correlated with higher serum level of IL-12 and potentiation of NK cells. Moreover, in adoptive transfer experiments in melanoma bearing mice, marked inhibition of lung metastatic foci was noted upon engraftment with splenocytes derived from Cl-IB-MECA treated mice. Taken together, the ability of Cl-IB-MECA to inhibit tumor development via the activation of NK cells is an additional mechanism which accounts for the anti-tumor effect of A3AR agonists.