Investigating the role of IL-21 in the early stages of a T-dependent B cell response

© 2016 Dr. Ivan Ting Hin FungDuring a T-dependent B cell response, activated B cells undergo a number of critical events to establish a robust antibody response and long-lived memory. These regulated events include migration, T and B cell interaction, proliferation, selection and differentiation. Activated B cells can differentiate into short-lived plasmablasts (PBs), early memory cells and germinal center (GC) B cells. Inside the GC, GC B cells undergo further changes including proliferation, affinity maturation and can differentiate into high affinity long-lived plasma cells and memory cells. A network of factors regulating these differentiation events have been identified, including the cytokine IL-21. IL-21 is the signature cytokine produce by T follicular helper cells. Initial in vitro studies have shown that IL-21 to be a complex regulator of immune response with the capacity to upregulate the transcription factors Bcl6 and Blimp1 in B cells. Subsequent mixed bone chimera studies have established IL-21 acts directly on B cells to regulate plasma cell formation, GC maintenance and memory cell production. However, the role of IL-21 in regulating activated B cell differentiation during the early stages of a TD B cell response (prior to GC formation) is not fully understood. This thesis describes the use of the IL-21 receptor (IL-21R)-deficient SWHEL mouse system in adoptive transfer experiments to track and analyse the response kinetics in the context of IL-21R signalling. While the initial proliferation of the IL-21R-deficient SWHEL B cells was unaffected, the lack of IL-21R signalling severely reduced the overall magnitude of the response. IL-21R-deficient SWHEL B cells exhibited an expanded early memory compartment, reduced PB numbers and Bcl6+ GC B cells. The amount of Bcl6 expressed in the IL-21R-deficient GC SWHEL B cells was also significantly lower than that of the WT. The reduction in GC B cell frequency and numbers in the IL-21R-deficient SWHEL B cells correlated with impaired recruitment of Bcl6+ B cells into GCs in the absence of IL-21R signalling. Bcl6+ IL-21R-deficient SWHEL B cells were localised at the perimeter of GCs which correlated with impaired downregulation of the chemotactic receptor EBI2. Impaired downregulation of EBI2 on IL-21R-deficient SWHEL B cells translated to a higher responsiveness to the EBI2 ligand in directed chemotactic assays. This thesis also described the generation of the FucciRG/IL-21R-deficient mouse system. The Fucci cell cycle reporter system can provide a high resolution read out for the proliferative status of different B cell subsets. Characterisation of these mice by flow cytometry and histology has demonstrated the FucciRG/IL-21R-deificient mouse system will be a versatile tool in future investigations into GC dynamics in the context of IL-21R signalling. Overall, this investigation revealed a novel role for IL-21 in regulating the early phases of a TD B cell response. The results demonstrated IL-21 regulates GC B cell differentiation via upregulating and sustaining Bcl6 expression in pre-GC B cells. This facilitates the downregulation of EBI2 on pre-GC B cells which in turn promotes their recruitment into GCs