title: Characterisation of a Novel Human IL-21R Mutation
Creators: name: Kwong, Kristy
Publisher: name: The Australian National University
Year: 2022

description: Inborn errors of immunity (IEI) encompass a group of inherited disorders that manifest in various forms, including increased susceptibility to infection, allergy, autoinflammatory or autoimmune disease, and malignancy. Rapid advances in genomics have enabled the identification of more causative mutations that can confer either loss- or gain-of-function upon the affected translated protein. Identification of new inborn errors of immunity can provide explanations for patients without a clear diagnosis. Functional characterisation of variants that confer altered function can provide important biological insights. Inborn errors of immunity affecting different genes can present with similar clinical and cellular phenotypes, and this can provide insight into interactions within biochemical pathways that regulate those phenotypes. IL-21 is a pleiotropic cytokine important for immune regulation and T and B cell effector functions. Several reports describing germline loss-of-function mutations in either IL-21 or IL21R revealed the significance of IL-21 signaling in T cell-dependent B cell activation, germinal centre reactions, and humoral immunity. However, there are currently no reports on hypermorphic mutations affecting IL-21 signaling. In this thesis, I describe the immunological consequences of a novel IL21R mutation identified in a kindred with common variable immunodeficiency (CVID) characterised by hypogammaglobulinemia and susceptibility to fungal and mycobacterial infections. The novel variant is a missense mutation substituting a serine 492 for arginine in the translated protein. Using a combination of ex vivo phenotyping and in vitro functional assays, I identified a distinct expansion in the circulating follicular helper T cells of the patients and a robust response to B cell proliferative cues antithetically accompanied by a functional impairment in plasmablast formation. I employed a bespoke mouse model bearing the orthologous Il21r mutation to demonstrate and test the effect of this variant in primary and secondary lymphoid organs. Extensive phenotyping of cellular subsets from the various lymphoid organs revealed an expansion in the germinal centre B cells and follicular helper T cell subsets. Additionally, in vitro assays of naive CD4+ T cells from IL21R mutant mice responded more robustly to Th1 and Th17 polarising stimuli. However, introducing recombinant IL-21 to these conditions resulted in reduced differentiation of Th1 cells but not Th17. Overall, these phenotypic features recapitulate and extend the immunological phenotype identified in the patients. Using biochemical analysis of splenocytes from the IL21R mouse model, I demonstrated a delay in STAT1 and STAT3 dephosphorylation in cells carrying the novel IL21R variant, consistent with a gain-of-function mutation. Having established that PBMCs from STAT1 and STAT3 GoF patients have impaired plasmablast formation similar to our IL21R mutant patients, I induced plasmablasts from PBMCs from healthy donors in the presence of cytokines predominantly signalling through either STAT1 or STAT3. Surprisingly, our findings indicate that additional STAT1 and STAT3 signals diminish plasmablast formation in response to IL-21. In summary, my thesis describes and demonstrates the effect of a missense mutation in IL21R on T and B cell subsets in both humans and mice. The characterisation of this IL21RS492R mutation will be the first description of an IEI caused by a hypermorphic IL21R variant. Furthermore, my thesis highlights the importance of maintaining the fragile balance between STAT1 and STAT3 signaling for IL-21-induced B cell differentiation, which has broad implications for immunity and immune modulating therapy.