Abstract
The HLA-G 5’URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5′URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5′URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5′URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5′URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-β and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5′URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5′URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.
Highlights
HLA-G is an immune checkpoint molecule that inhibits the function of immunocompetent cells such as the cytotoxic activity of NK and T CD8+ lymphocytes, through the binding to ILT-2/LIRB1, ILT-4/LIRB2 inhibitory receptors[1]
Other elements are repressors of the HLA-G expression: (i) the Ras responsive element binding protein 1 (RREB-1) that acts through three Ras response elements (RRE) (−1378 bp to −1358 bp, −157 bp to −143 bp, and −59 bp to −54 bp)[37]; and (ii) the glioma-associated oncogene-3 (GLI3) (−1116 bp to −1108 bp), a signal transducer of the Hedgehog pathway (HH) that is induced by cyclopamine treatment
Choriocarcinoma JEG-3 and melanoma FON+ cell lines were transfected, as previously described[19], with pGL3-Basic vector constructions containing one of the ten most frequent HLA-G 5′URR haplotypes known as G0104a; G0104b; G010102a; G010101a; G010101b; G010101c; G010101d; G010101f; G0103a and G0103e
Summary
HLA-G is an immune checkpoint molecule that inhibits the function of immunocompetent cells such as the cytotoxic activity of NK and T CD8+ lymphocytes, through the binding to ILT-2/LIRB1, ILT-4/LIRB2 inhibitory receptors[1]. Considering that: (i) The HLA-G 5′URR presents peculiar response to transcription factors that may differ from classical HLA class I genes; (ii) several variable sites have been described at the HLA-G promoter segment, some of them coincide with or are close to transcription factor binding sites; (iii) little is known about the influence of HLA-G 5′URR variability on gene expression[24], in this study we: (i) cloned the ten most frequent HLA-G 5′URR haplotypes observed worldwide and analysed their activity on a luciferase reporter gene in two HLA-G positive cell lines (choriocarcinoma JEG-3 and melanoma FON+) in the presence or absence of modulators (inducers: interferon-β and progesterone; repressor: cyclopamine) of the HLA-G expression, and (ii) determined HLA-G 5′URR alleles, genotypes, haplotypes, diplotypes and haplotype groups in healthy individuals to associate them with their plasma sHLA-G levels
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