CD16b: Primary Structure of Human Neutrophil Antigen Epitopes and Their Functional Consequences
Authors: Sachs Ulrich J , Reil Angelika , Simtong Piyapong , Santoso Sentot 
Published: October 2019

Abstract
Neutrophil specific Fcg receptor IIIb (CD16b) is a glycosylphosphatidylinositol-anchored low-affinity glycoprotein that plays a significant role in phagocytosis and the clearance of immune complexes. CD16b has numerous polymorphic variants; the most relevant variants are associated with human neutrophil antigens (HNA) -1a, -1b, and -1c. HNA-1a and HNA-1b differ in four amino acids (aa) (positions 36, 65, 82 and 106). One additional aa mutation on HNA-1b at position 78 results in HNA-1c.
Immunization against CD16b variants can lead to the production of alloantibodies (aabs) responsible for neonatal alloimmune neutropenia (NIN), autoimmune neutropenia of infancy (AIN) and transfusion-related acute lung injury (TRALI). The exact contribution of five aa mutations for the formation of HNA-1a, -1b and -1c and function of CD16b is currently unknown.
In this study, permutation of each polymorphic aa from wild-type CD16b cDNA constructs was performed and stably expressed on HEK293 cells. In total, 3 cell lines expressing wild-type (HNA-1a, -1b and -1c) and 18 cell lines expressing mutant variants were produced; 14 derived from HNA-1a or HNA-1b (panel 1) and 4 derived from HNA-1c (panel 2). When panel 1 was tested with well-defined aabs by antigen capture assay, 4 cell lines reacted with anti-HNA-1a only, 4 with anti-HNA-1b only, 4 with both aabs, and 4 did not react with both aabs. These results indicate that aa36 and aa65 are responsible for the formation of the HNA-1a and HPA-1b epitope(s), respectively. Analysis of panel 2 showed that 2/4 mutant variants reacted with anti-HNA-1c exclusively, suggesting that the HNA-1c epitope is formed by Ala78Asp irrespective of the other polymorphic aa.
To analyze the binding affinity of CD16b variants, adhesion assay onto immobilized IgG was performed. In comparison to HNA-1a, HNA-1b cells bound slightly weaker to IgG (p<0.002). In contrast, HNA-1c cells interacted significantly stronger with IgG in comparison to both, HNA-1a and -1b cells (p<0.0001). Similar results were obtained with HNA-1a, -1b and -1c recombinant proteins by direct protein-binding analysis (solid phase ELISA and Surface Plasmon Resonance). Interestingly, all mutant variants carrying HNA-1c bound also stronger to IgG than HNA-1a and -1b transfected cells. Similar results were observed with HNA-1c phenotyped neutrophils.
In conclusion, although HNA-1a and HNA-1b differ in four aa positions, only two (aa36, aa65) are necessary for the complete formation of the respective epitopes. In contrast, the HNA-1c epitope is created by only one aa mutation (Ala78Asp). Furthermore, our results show that HNA-1c exhibits higher affinity to IgG compared to HNA-1a and HNA-1b forms. Accordingly, the Ala78Asp mutation is not only responsible for epitope formation, but is also involved in the regulation of CD16 affinity. Characterization of HNA-1 epitopes by tailored recombinant tools will help us to improve our diagnostic work-up in patients with suspected NIN, AIN and TRALI and our understanding on the role of CD16 polymorphism in other diseases such as, immune complex-mediated disorders.

Disclosures
No relevant conflicts of interest to declare.