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

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.

NK cells and cancer: you can teach innate cells new tricks

“…For example, cytokines produced by activated T cells by CTLA4 blockade therapy might activate NK cells and initiate their antitumour effector functions. CTLA4 can also be expressed on some tumour cells 105 and thus induce ADCC after treatment with CTLA4 anti bodies 106 . Moreover, tumour cells infected with oncolytic viruses, such as Newcastle Disease virus 107 and H-1PV rat parvovirus 108 , have been shown to stimulate NK cells directly by inducing activating ligands on the virus-infected cells.…”

Immune checkpoints programmed death 1 ligand 1 and cytotoxic T lymphocyte associated molecule 4 in gastric adenocarcinoma

“…17,24 Although the prevailing notion is that ipilimumab targets CTLA-4 on T cells, its exact mode of action is still under discussion, as CTLA-4 can be expressed on regulatory lymphocytes as well as on malignant cells. [25][26][27][28][29] In an early trial investigating the CLTA-4 antibody tremelimumab in metastatic gastric adenocarcinoma, tumor shrinkage with some long-lasting responses have been described. Of note, this study did not report on CTLA-4 expression on primary tumor cells.…”

Potential function of CTLA‑4 in the tumourigenic capacity of melanoma stem cells

“…Prior studies indicated that CTLA-4 expression in tumour cells is associated with poor prognosis (12,17). CTLA-4 engagement with B7 ligands induces tumour cell death through apoptosis (18), and ipilimumab triggers effector lymphocytes and tumour necrosis factor release to directly eliminate CTLA-4 + melanomas (14). These studies have indicated the potential function of CTLA-4 in tumour cells.…”

“…CTLA-4 has also been implicated to serve functions in neoplastic cells, including B cell chronic lymphocytic leukaemia (10), non-Hodgkin's lymphoma (11), breast cancer (12), lung cancer (13), melanoma (14), gastric cancer (15), colorectal cancer (15), cervical cancer (16) and oesophageal carcinoma (17). Prior studies indicated that CTLA-4 expression in tumour cells is associated with poor prognosis (12,17).…”