EXPRESSION OF IMMUNECHECKPOINT HUMAN LEUKOCYTE ANTIGEN G IN A CHORIOCARCINOMA CELL LINE CULTURED IN HYPOXIC MICROENVIRONMENT

ABAL, LUCIANA; PALMA, MARÍA BELÉN; IRIBARNE, AILEN; RICCILLO, FERNANDO; MIRIUKA, SANTIAGO G.; GARCÍA, MARCELA N.

Capital Federal

Congreso; Congreso Anual de Fisiología 2023; 2023

Sociedad Argentina de Fisiología

Introduction: The immunecheckpoint HLA-G is a non-classical MHC class I molecule that modules negatively the local immune response. This protein has restricted tissue expression in physiological conditions. However, its expression can be ectopically induced under malignant cell transformation in several tumors. The quick growth of tumor cells creates a hypoxic environment where low oxygen levels lead to the accumulation of hypoxia inducible factors (HIF), which modulate gene expression of angiogenesis, cell proliferation and migration further increasing the tumor development. In this context, HIF could positively modulate the HLA-G expression in tumor cells to evade the immune system. Objective: To evaluate the effect of hypoxia on HLA-G expression in a choriocarcinoma cell line called JEG-3. Methods: JEG-3 were incubated with deferrioxamine 200µM (DFX an iron chelator mimicking hypoxia) for 6, 24, 48 and 72 h in DMEM + 10%FBS. HIF and HLA-G expression was measured by western blot (WB), flow cytometry (FC) and RT-qPCR. Results: The effect of DFX was analyzed by WB determining HIF-1 and -2 isoforms at different incubation times. HIF-1 levels increased gradually in a time-dependent manner, but HIF-2 only showed increased values at 6 and 24 h of incubation. Then, HLA-G expression was analyzed. RT-qPCR showed an increment of HLA-G level after 6 and 48 h with DFX (1.8-fold change compared to control cells), however after 24 and 72 h with DFX, the expression levels decreased. By FC, 63% of control JEG-3 were positive for membrane-bound HLA-G, while 64, 75, 78 and 70% of JEG-3 incubated for 6, 24, 48 or 72 h respectively, were positive. Total HLA-G protein levels measured by WB increased gradually in a time-dependent manner. Conclusion: HLA-G protein levels increased during hypoxia culture. As HIF-1 levels also increased gradually, it could be considered that this factor is one of the positive modulators of HLA-G expression. No correlation with HIF-2 levels was observed. Furthermore, RT-qPCR and FC results showed that when HLA-G reaches high levels of mRNA and membrane-bound protein, these start to decline, which could suggest a negative autoregulation of HLA-G.