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




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Journal of Translational Medicine

, 11:108

Immunobiology and immunotherapy

Abstract

BackgroundCTLA-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.

MethodsCTLA-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.

ResultsAll 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.

ConclusionsOur 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.

KeywordsCTLA-4 Melanoma Ipilimumab ADCC NK-γδ T cell activation AbbreviationsADCCAntibody-dependent cellular cytotoxicity

APCAntigen presenting cells

CTLA-4Cytotoxic T Lymphocyte-Associated antigen 4

ELISAEnzyme-linked immunosorbent assay

FFPEFormalin-fixed, paraffin-embedded

IHCImmunohistochemistry

IRSImmune Reactive Score

mAbsMonoclonal antibodies

MDAMelanocyte differentiation antigens

MFIMean fluorescence intensity

MRFIMean ratio of relative fluorescence intensity

NCANeural crest antigens

PBMCPeripheral blood mononuclear cells

SCAStem cell-related antigens

sCTLA-4Soluble CTLA-4

SNSupernatant

TMATissue microarray

TNF-αTumor Necrosis Factor-α

TregsRegulatory T cells.

Electronic supplementary materialThe online version of this article doi:10.1186-1479-5876-11-108 contains supplementary material, which is available to authorized users.

Stefania Laurent, Paola Queirolo, Silvia Boero, Alessandro Poggi contributed equally to this work.

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Author: Stefania Laurent - Paola Queirolo - Silvia Boero - Sandra Salvi - Patrizia Piccioli - Simona Boccardo - Simona Minghelli -

Source: https://link.springer.com/



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