Biology subjects

Cocker, A. T. H.

Publications and source records attributed to Cocker, A. T. H..

2 recordsLinked to original sources

bioRxiv2026

Extravillous trophoblast model shows generation of bioequivalent N-glycans can maintain immunological protection against natural killer cell cytotoxicity

Extravillous trophoblasts (EVTs) are a trophoblast subpopulation critical for feto-maternal tolerance during early pregnancy, primarily using HLA-G to exert immunomodulatory effect, and possessing N-glycomic profiles distinct from other trophoblast subpopulations. However, whether the N-glycosylation confers distinct immunological properties to EVTs remains poorly understood. To investigate this, we employed JEG-3, a human choriocarcinoma cell line having the capacity to produce pregnancy-related hormones and expressing both HLA-C and HLA-G resembling placental EVTs, as an in vitro EVT model, alongside cell line JAR which exhibits villous trophoblast phenotypes distinct from JEG-3. Both cell lines were treated with kifunensine or swainsonine, inhibitors of -mannosidases, to remodel their N-glycosylation patterns. This led to significant remodelling of their N-glycomic profiles, with JEG-3 cells showing an increased level of polylactosamine chains and decreased levels of -2,6-sialylation and core -1,6-fucosylation. Western blot analysis showed that inhibiting -mannosidases altered only the composition of N-glycans on cell-surface HLA-G, without affecting the overall abundance of cell-surface HLA-G. In kifunensine-treated JEG-3 cells that predominantly express oligomannose type N-glycans, an intracellular accumulation of unfolded HLA-G fragments, increased hCG secretion, and down-regulations of EVT markers GATA3 and KRT7 were observed compared to untreated control, while swainsonine treatment did not impact N-glycan expression. Cytotoxicity assays using NK-92 as effector cells showed that the de-sialylation of JEG-3 by neuraminidase treatment led to increased NK-92 mediated killing. JEG-3 cell sustained its EVT immunological properties through generating bioequivalent N-glycans, exemplified by NK-92 cells pre-conditioned with used culture media of kifunensine-treated JEG-3 cells displaying reduced cytotoxicity toward NK-sensitive lymphoblast cell line K562, an effect not observed with swainsonine-treated JEG-3 cells. This model suggests that EVTs immunological properties are dependent on specific N-glycomic profiles that are maintained by unique N-glycosylation homeostasis, and overall improves our understanding of how EVTs maintain their immunomodulatory effect at the maternal-fetal interface.

immunology↗

bioRxiv2026

Cell-surface N-linked glycans manipulation of K562 cells for augmented susceptibility to natural killer cell killing

Glycosylation of proteins is arguably the most diverse post-translational modification that is altered in almost all cancer types, which has been demonstrated to play a crucial role in creating an immunosuppressive microenvironment that promotes immune tolerance and evasion. However, the biosynthesis of N-linked glycan is mediated by a series of enzymatic reactions catalysed by glycosyltransferases and glycosidases in a template-independent manner, hindering our understanding of specific structure-function relationships and roles of specific glycans on specific proteins. Here we use HLA class I-negative cell line K562, a known reference target for NK-mediated cytolysis, to establish a model investigating how cell surface glycan dynamics influence its susceptibility to cytolysis mediated by NK-92 cells. Treatment of K562 cells with kifunensine, swainsonine, 2F-peracetyl-fucose, or 3Fax-peracetyl Neu5Ac, inhibitors of N-linked glycan processing, resulted in drastic alterations in cell surface carbohydrate phenotype, as could be shown by flow cytometric analysis of the lectiNbinding properties of the cells. Despite these clear changes in carbohydrate phenotype, only K562 cells treated with either kifunensine or 3Fax-peracetyl Neu5Ac exhibited higher susceptibility to the cytolysis medidated by NK-92 cells accompanied with an increased CD107a expression by NK-92 cells. Although K562 cells overexpressing gene MGAT3 exhibited a decreased NK-susceptibility, we further found that this decrease was not exclusively determined by the overexpression of gene MGAT3 product bisecting {beta}1,4-GlcNAc, because the treatment of 3Fax-peracetyl Neu5Ac reversed the resistance of K562 cell against NK-92 cell in despite of expressing higher levels of bisecting {beta}1,4-GlcNAc. Expressing HLA-G on cell surface as extravillous trophoblast did not change the NK-susceptibility of K562 cells, despite evidence that HLA-G molecules expressed by K562 cells can bind to inhibitory receptor ILT2 expressed on NK-92 cell surface. These findings suggest that the level of terminal sialylation, outweighing other components in N-linked glycan, determines the NK-susceptibility of K562 cell, offering a new strategy to weaken the resistance of cancer cells so that the immune system can maximise the elimination.

immunology↗