End9: an early-passage cell line defective in master HLA-I transcriptional coordinators

Abstract

Disruption in the HLA class I (HLA-I), β2-microglobulin (β2M) and/or Antigen Processing and Presentation Machinery (APPM) structural genes is believed to favor tumor immune escape, whereas purely regulatory mutants are few and poorly characterized. The endometrial carcinoma End9 was the extreme HLA-I/APPM-low outlier in a panel of >100 early-passage tumor cell lines established in culture by us from essentially all major tumor histotypes. The defect arose in vivo, was retained in culture, and remained stable for >100 passages. End9 was found to express no constitutive or IFN-responsive HLA-A, -B, -F, and-G, little HLA-C and β2m, ‘normal‘ HLA-E, and barely detectable TAP, tapasin, LMP2, LMP7, ERAP1 and ERAP2, as evaluated by protein and nucleic acid biochemistry. Lacking evidence for genomic damage, we used a reporter construct to assess the 3 major upstream HLA-I regulatory boxes (EnhA, ISRE and SXY), and attempted phenotypic rescue by transduction with missing trans-acting function(s). The NF-KB:EnhA interplay was apparently conserved, whereas the ISRE and SXY modules were simultaneously shut off due to (a) a complete JAK1 absence, (b) a physiological lack of CIITA, and (c) an epigenetic (5-Azacitidine-reversible) suppression of an intact (as per sequencing) NLRC5. The near-zero HLA-I/APPM phenotype of End9 highlighted several features of HLA-I regulation, including: (a) SXY-independent HLA-C transcription; (b) subordination of the EnhA and ISRE boxes (IFN and TNFα responsiveness) to promoter opening by CIITA/NLRC5; and (c) marginal HLA-I/APPM transactivation by residual NLRC5 at baseline and even upon >100X overexpression. End9 is the first regulatory mutant defective in a hierarchically high set of transacting functions operating upstream to most HLA-I/APPM members, resulting in a trophoblast-like, presumably immunoevasive phenotype. The defect involves NLRC5 but NLRC5 refractoriness implies a shortage of other transcriptional co-activators, making End9 a suitable recipient for complementation cloning to discover novel HLA-A/-B/APPM master coordinators.