The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production

Journal of Translational Medicine

Maria Pia Pistillo²  Alessandro Poggi⁴  Guido Ferlazzo⁸  Maria Cristina Mingari⁶  Lung-Ji Chang^1,10  Francesca Tosetti⁴  Nicoletta Ferrari⁴  Paolo Carrega⁹  Gabriella Pietra⁶  Vincenzo Fontana⁵  Anna Morabito²  Simona Minghelli⁹  Simona Boccardo¹  Patrizia Piccioli³  Sandra Salvi¹  Silvia Boero⁴  Paola Queirolo³  Stefania Laurent⁷ 

[1] Department of Pathology, IRCCS AOU San Martino-IST, Genoa, Italy;Unit of Tumor Genetics and Epigenetics, IRCCS AOU San Martino-IST, Genoa, Italy;Department of Medical Oncology A, IRCCS AOU San Martino-IST, Genoa, Italy;Unit of Molecular Oncology and Angiogenesis, IRCCS AOU San Martino-IST, Genoa, Italy;Unit of Epidemiology and Biostatistics, IRCCS AOU San Martino-IST, Genoa, Italy;Department of Experimental Medicine, University of Genoa, Genoa, Italy;Department of Internal Medicine (DIMI), University of Genoa, Genoa, Italy;Department of Human Pathology, School of Medicine, University of Messina, Messina, Italy;G. Gaslini Institute, Genoa, Italy;Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA

关键词: NK/γδ T cell activation;    ADCC;    Ipilimumab;    Melanoma;    CTLA-4;   

Others  :  827613
DOI  :  10.1186/1479-5876-11-108

 received in 2012-12-19, accepted in 2013-04-23,  发布年份 2013

【 摘 要 】

Background

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.

Methods

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.

Results

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.

Conclusions

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.

【 授权许可】

   
2013 Laurent et al.; licensee BioMed Central Ltd.

【 图 表 】

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