Abstract
Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.
Highlights
Neuroblastoma (NB) is a childhood solid tumor derived from precursor cells of the sympathetic nervous system which accounts for 15% of pediatric cancer deaths [1]
T cell-based immunotherapy is an attractive option for treatment of chemotherapy-refractory/high-risk NB patients, since NB expresses known T-cell tumor antigens from the MAGE family, GAGE, NY-ESO-1, PRAME, tyrosine hydroxylase, survivin, MYCN, and Anaplastic Lymphoma Kinase (ALK) [8,9,10,11,12,13,14,15,16,17,18,19,20]
Flow cytometry and Western blotting of NB cell lines revealed that the 3 cell lines expressing higher levels of cell surface MHC class I (MHC-I) (SH-EP, SK-N-AS and SK-N-SH) expressed higher levels of Interferon Regulatory Factor 1 (IRF1) and IRF2, whereas the 5 cell lines expressing lower levels of cell surface MHC-I displayed lower levels of IRF1 and IRF2 (Figs. 1A, 1B and S1)
Summary
Neuroblastoma (NB) is a childhood solid tumor derived from precursor cells of the sympathetic nervous system which accounts for 15% of pediatric cancer deaths [1]. Clinical features of the tumor are very heterogeneous, ranging from spontaneous regression to rapid progression The latter has been shown to be associated with several genomic and functional abnormalities, including MYCN gene amplification and overexpression, allelic loss of chromosome 1p, 3p, 11q and 14q, gains of 1q and 17q, as well as deficient MHC class I (MHC-I) expression and antigen presentation [2,3,4,5,6]. NB poorly presents these antigens due to a complex, coordinated low expression of MHC-I and many gene products collaborating to build functional MHC-I molecules and their antigen cargo [21,22,23] These include b2-microglobulin (b2m), the Transporter Associated with Antigen Processing (TAP1 and TAP2) subunits, the Endoplasmic Reticulum Aminopeptidases (ERAP) 1 and ERAP2, and the peptide antigen editor tapasin (TPN) [24]. Low expression of these genes, often collectively referred to as members of the antigen processing machinery (APM), drastically limits the applicability of T-cell immunotherapy in NB
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