title: The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.
abstract: 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.
authors: Department of Internal Medicine, University of Genoa, Genoa, Italy.
authors: Stefania
authors: S
authors: Laurent
authors: Paola
authors: P
authors: Queirolo
authors: Silvia
authors: S
authors: Boero
authors: Sandra
authors: S
authors: Salvi
authors: Patrizia
authors: P
authors: Piccioli
authors: Simona
authors: S
authors: Boccardo
authors: Simona
authors: S
authors: Minghelli
authors: Anna
authors: A
authors: Morabito
authors: Vincenzo
authors: V
authors: Fontana
authors: Gabriella
authors: G
authors: Pietra
authors: Paolo
authors: P
authors: Carrega
authors: Nicoletta
authors: N
authors: Ferrari
authors: Francesca
authors: F
authors: Tosetti
authors: Lung-Ji
authors: LJ
authors: Chang
authors: Maria Cristina
authors: MC
authors: Mingari
authors: Guido
authors: G
authors: Ferlazzo
authors: Alessandro
authors: A
authors: Poggi
authors: Maria Pia
authors: MP
authors: Pistillo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000494:pharmacology*; D060908:CTLA-4 Antigen / Q000378:metabolism*; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D000074324:Ipilimumab; D007694:Killer Cells, Natural / Q000166:cytology; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D017452:Receptors, IgG / Q000378:metabolism; D012878:Skin Neoplasms / Q000188:drug therapy / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C496124:FCGR3A protein, human; D000074324:Ipilimumab; D017452:Receptors, IgG; D014409:Tumor Necrosis Factor-alpha
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