The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.
The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.
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DOI：
10.1186/1479-5876-11-108
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发表时间：
2013-05-01
影响因子：
7.4
通讯作者：
Pistillo MP
中科院分区：
文献类型：
--
作者：
Laurent S;Queirolo P;Boero S;Salvi S;Piccioli P;Boccardo S;Minghelli S;Morabito A;Fontana V;Pietra G;Carrega P;Ferrari N;Tosetti F;Chang LJ;Mingari MC;Ferlazzo G;Poggi A;Pistillo MP
CTLA-4(细胞毒性T淋巴细胞抗原-4)传统上被认为是T细胞活化的负性调节因子。在转移性黑色素瘤中，用人的单抗如伊匹单抗阻断CTLA-4目前被用来解除CTLA-4介导的抗肿瘤免疫反应的抑制。在此，我们分析了CTLA-4在皮肤黑色素瘤患者黑色素瘤细胞系和肿瘤组织中的表达和Ipiimumab的反应性。然后，我们从抗体依赖的细胞毒作用和肿瘤坏死因子-α的释放两个方面研究了伊匹单抗是否能够激发天然免疫。最后，建立了异种移植小鼠模型，在体内检测了Ipiimumab和NK细胞对黑色素瘤的作用。用细胞荧光法检测17株黑色素瘤细胞株(14株原代细胞和3株长期细胞株)和33例黑色素瘤组织(免疫组织化学)中CTLA-4的表达和Ipiimumab反应性。对CTLA-4转录本进行定量RT-PCR分析。用酶联免疫吸附试验检测血清中可溶性CTLA4和肿瘤坏死因子α。用艾氏单抗和黑色素瘤细胞株检测外周血单个核细胞、NK细胞和γδT细胞。用单抗检测NK黑色素瘤细胞中肿瘤坏死因子-α的释放。在NOD/SCID小鼠体内进行异种移植实验。结果用非配对学生t检验进行分析。所有黑色素瘤细胞株均表达信使核糖核酸和胞浆CTLA-4，但与伊匹单抗的表面反应性差异很大。相应地，大约2/3的黑色素瘤标本不同程度地表达CTLA-4。Ipilimumab通过FcγReceptor IIIA(CD16Receptor IIIA)在体外激活NK细胞和PBMC，IL-2激活的NK细胞和γδT细胞向CTLA4+黑色素瘤细胞的ADCC转化。与CTLA-4-FO-1黑色素瘤细胞株相互作用未检测到ADCC。NK细胞与CTLA4+黑色素瘤细胞株相互作用后释放肿瘤坏死因子α。值得注意的是，伊匹单抗既不影响CTLA-4+T淋巴细胞的增殖和活性，也不触发ADCC。在嵌合的小鼠异种移植模型中，与接受对照异种移植的小鼠相比，经Ipiimumab处理的黑色素瘤细胞与人类同种异体NK细胞共移植延迟并显著减少了肿瘤的生长。我们的研究表明，伊匹单抗可触发效应淋巴细胞的细胞毒作用和肿瘤坏死因子-α的释放。这些发现表明，伊匹单抗除了阻断CTLA-4外，还可以直接激活CTLA-4+黑色素瘤的消除。
CTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma. CTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student’s t-test. All melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity. Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts. Our studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.
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DOI：
10.1056/nejmoa1003466
发表时间：
2010-08-19
期刊：
The New England journal of medicine
影响因子：
--
作者：
Hodi FS;O'Day SJ;McDermott DF;Weber RW;Sosman JA;Haanen JB;Gonzalez R;Robert C;Schadendorf D;Hassel JC;Akerley W;van den Eertwegh AJ;Lutzky J;Lorigan P;Vaubel JM;Linette GP;Hogg D;Ottensmeier CH;Lebbé C;Peschel C;Quirt I;Clark JI;Wolchok JD;Weber JS;Tian J;Yellin MJ;Nichol GM;Hoos A;Urba WJ
通讯作者：
Urba WJ
影响因子：
56.9
作者：
Lee, KM;Chuang, E;Bluestone, JA
通讯作者：
Bluestone, JA
影响因子：
20.3
作者：
Pistillo, MP;Tazzari, PL;Ferrara, GB
通讯作者：
Ferrara, GB
影响因子：
30.5
作者：
Fallarino, F;Grohmann, U;Puccetti, P
通讯作者：
Puccetti, P
影响因子：
5.8
作者：
Sabel, MS;Hess, SD;Bankert, RB
通讯作者：
Bankert, RB