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Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells.

ABSTRACT: Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity. These observations indicate that specific manipulation of CD16a N-glycan composition in CD16a-expressing effector cells including NK cells may improve treatment efficacy. However, it is unclear if modifying the expression of select genes that encode processing enzymes in CD16a-expressing effector cells is sufficient to affect N-glycan composition. We identified substantial processing differences using a glycoproteomics approach by comparing CD16a isolated from two NK cell lines, NK92 and YTS, with CD16a expressed by HEK293F cells and previous reports of CD16a from primary NK cells. Gene expression profiling by RNA-Seq and qRT-PCR revealed expression levels for glycan-modifying genes that correlated with CD16a glycan composition. These results identified a high degree of variability between the processing of the same human protein by different human cell types. N-glycan processing correlated with the expression of glycan-modifying genes and thus explained the substantial differences in CD16a processing by NK cells of different origins.

SUBMITTER: Patel KR

PROVIDER: S-EPMC7948478 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Natural killer (NK) cell effector functions include cytotoxicity and secretion of cytokines such as interferon-γ (IFN-γ). The immature CD56bright subset of human NK cells lacks expression of FcγRIIIa/CD16a, one of the low-affinity immunoglobulin G receptors, or exhibits low-density expression (CD56brightCD16-/dim) and produces IFN-γ in response to cytokine stimulation, whereas the mature CD56dimCD16+ subset is the most cytotoxic one. A further differentiation/maturation of the latter subset according to the gradual loss of NKG2A and/or gain of KIR2DL (CD158a and CD158b) has been demonstrated and the ability to produce IFN-γ in response to activating receptor (AR) co-engagement is gradually acquired during terminal differentiation. In the course of flow cytometry analysis of CD56dim NK cells, we noted a substantial intraindividual heterogeneity of expression of FcγRIIIa. FcγRIIIa is unique among ARs: it does not require the co-engagement of other ARs to induce substantial cytotoxicity or cytokine synthesis in CD56dim cells. We, therefore, investigated whether individual differentiation/maturation of polyclonal CD56dim NK cells defined by expression of NKG2A/KIR2DL is related to FcγRIIIa expression and to the heterogeneity of NK cell responses upon FcγRIIIa engagement. When we analyzed unstimulated CD56dim cells by increasing level of FcγRIIIa expression, we found that the proportion of the more differentiated CD158a,h+ and/or CD158b,j+ cells and that of the less differentiated NKG2A+ cells gradually increased and decreased, respectively. FcγRIIIa engagement by using plate-bound murine anti-CD16 monoclonal antibody (mAb) or rituximab or trastuzumab (two therapeutic mAbs), resulted in donor-dependent partial segregation of IFN-γ-producing and/or degranulating CD56dim cells. Importantly, the proportion of CD158a,h/b,j+ cells and that of NKG2A+ cells was increased and decreased, respectively, IFN-γ-producing cells, whereas these proportions were poorly modified in degranulating cells. Similar results were observed after engagement of ARs by a combination of mAbs targeting NKG2D, NKp30, NKp46, and 2B4. Thus, the gradual increase of FcγRIIIa expression is an important feature of the differentiation/maturation of CD56dim cells and this differentiation/maturation is associated with a shift in functionality toward IFN-γ secretion observed upon both FcγRIIIa-dependent and FcγRIIIa-independent stimulation. The functional heterogeneity related to the differentiation/maturation of CD56dim NK cells could be involved in the variability of the clinical responses observed in patients treated with therapeutic mAbs.

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Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells.

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