Immunoglobulin allotypes, antibody-dependent neutrophil phagocytosis: impact on HIV and influenza

© 2019 Kuangyu FeiAntibodies (Abs) are highly functional immune proteins. Abs can deactivate pathogens through neutralization and mediating various Fc receptor (FcR) functions. Two common FcR-mediated functions are Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent (cellular) phagocytosis (ADCP or ADP). These functions are triggered by binding of the Fc region of Abs to FcRs on innate immune cell surface. Studies including analyses of the moderately protective human phase III RV144 HIV vaccine trial suggest that antibodies with the ability to mediate FcR functions may be important in HIV protection and control. IgG, the most abundant Ab isotype, has different subclasses including IgG1, IgG2, IgG3, and IgG4. The IgG1 and IgG3 subclasses when complexed with antigens, bind efficiently to all Fcγ receptors (FcγRs), thus initiating FcR-mediated functions (the FcR-mediated functions discussed in this thesis are all FcγR-mediated functions). There are several allelic variations (termed allotypes) of IgG1 in the constant region including G1m1 and G1m3. The prevalence of allotypes differs among ethnic groups. There is growing evidence that IgG1 allotypes differentially influence IgG subclass levels, FcR binding, and FcR-mediated functions. However, the understanding of the influence of IgG1 allotype on FcR-mediated functions is still limited at present. This thesis focuses primarily on the investigation of the impact of IgG1 allotypes on vaccine-induced immune responses. The first chapter provides the literature review of the whole study, ranging from the general humoral immunity to specific FcR-mediated functions. It also introduces IgG1 allotypes and the importance of FcR functions in infectious diseases including Human Immunodeficiency Virus (HIV) and Influenza. The second chapter focuses on the material and methods utilized in these studies. Information on the subjects who provided samples are also described in chapter two. The third chapter addresses two approaches used to determine the IgG1 allotypes. Normally the IgG1 allotypes are determined by sequencing as the DNA sequence difference is the intrinsic difference among different IgG1 allotype carriers. This study optimized another ELISA-based approach of IgG1 allotyping on protein levels utilizing only purified IgG or plasma to identify allotypes. This approach makes it possible to allotyp subjects when genomic DNA is unavailable. Furthermore, this study validates that sequencing allotyping and ELISA allotyping for the G1m1 and G1m3 allotypes are consistent with each other. In the fourth chapter, a neutrophil-like HL-60 cell line was developed as a model to study neutrophil responses to Fc-functional HIV-specific antibodies. Neutrophils are innate immune cells that express a range of FcRs and can trigger various FcR-mediated functions including Ab-dependent cellular phagocytosis (ADCP). However, the role of neutrophil FcR-mediated functions in HIV control is not well explored, largely due to the difficulty in working with primary neutrophils as they die rapidly (half-life 6-8 hours) ex vivo. In this chapter we cultured HL-60 cells for 5 days with DMSO such that they expressed a range of neutrophil-like cell markers. We then successfully used these neutrophil-like HL-60 cells to measure neutrophil FcR-mediated ADCP against HIV. The fifth chapter explored the influence of IgG1 allotypes in response to Influenza vaccination. IgG subclass levels, IgG-FcR binding, ADCC and ADCP (using the HL-60 method generated above) were all evaluated. We found that IgG1 allotypes are associated with antigen-specific IgG subclass levels upon Influenza vaccination. However, no functional assay difference regarding ADCC and ADCP was found, implying substantial complexity in allotypic effects on FcR-mediated functions. The final chapter covers the overall discussion and conclusion of the study. In summary, this project developed useful methods for IgG1 allotyping and Fc-functional assays of a neutrophil-like cell line. We utilized these methodologies to provide insight into the role of IgG1 allotypes upon vaccine-induced immune responses. This work could ultimately have implications for the development of global vaccinations against infectious diseases including HIV, where antigen-specific IgG subclasses contribute to protection