p100 abundance as a regulator of T and B cell development
Creators: name: Davies, Ainsley
Publisher: name: The Australian National University
Year: 2022

description: NF-kB transcription factors are crucial for normal development of the immune system. They promote lymphocyte development and lymphoid organogenesis. Our group identified a novel mutation in NFKB2 in a patient with complete deficiency of mature B cells, antibody deficiency, alopecia, and recurrent infections. More than 50 patients have now been described with this syndrome, although immune deficiency and autoimmunity vary in severity and the reason for this variation is unknown. NFKB2 encodes p100. Upon activation of the noncanonical NF-kB signalling pathway, p100 is processed by the proteasome into the active transcription factor p52. By contrast, unprocessed p100 inhibits other NF-kB transcription factors. The majority of pathological human NFKB2 mutations encode residues found in the region of p100 that is responsible for activating processing. In this thesis, I describe experiments aimed at elucidating how subtle differences in sequence variation in NFKB2 result in substantial differences in immune deficiency and autoimmunity. Remarkably, most of this variation is accounted for by changes in abundance of p100 rather than deficiency of p52. To model the human genotypes, we used CRISPR-Cas9 to generate an Nfkb2 allelic series of mice including truncating, missense and frameshift mutations. Our allelic series includes the mutation orthologous to the NFKB2 variant identified in our index patient, providing in vivo evidence of the pathogenic nature of this mutation. Throughout the thesis, we compared cellular, histological, and biochemical phenotypes to determine the contribution of p100 to immunological development and pathology. To enable comparison of disease progression in the context of p100/p52 abundance, we first performed biochemical analysis of the Nfkb2 allelic series (Chapter 3). Different Nfkb2 alleles result in substantial differences in p100 processing and degradation. Interestingly, only mice with p100 processing resistance have shortened lifespans. These mice develop T cell-dependent autoimmunity caused by a thymic defect resulting in impaired central tolerance (Chapter 4). Comparison of our allelic series with established models of defective thymic tolerance revealed that autoimmunity in mice with Nfkb2 mutations cannot be attributed to p52 or Aire deficiency. Instead, we established that p100 regulates the threshold of T cell selection in the thymus, which appears to be a new monogenic mechanism of human autoimmunity. Immune deficiency in Nfkb2 mutant mice is characterised by splenic B cell deficiency (Chapter 5). The stage of B cell ontogeny at which deficiency arises is determined by p100 abundance. We found that mutant p100 inhibits nuclear translocation of canonical NF-kB factor p65 within primary B cells (Chapter 6). Surprisingly, we found that p100 abundance controls RelB abundance, and p52 deficiency results in BAFF receptor deficiency. This places p100 and p52 at the centre of a cybernetic loop required for mature B cell homeostasis. In this thesis, I establish the molecular mechanism that underlies complex autoimmune disease and B cell deficiency caused by Nfkb2 mutations using unique animal models. Our findings provide strong support for the notion that p100 is a crucial regulator of T and B cell development. Mutations that confer pathological abundance of p100 result in autoimmunity through defective thymic selection, and B cell deficiency through defective maturation beyond the transitional stage of B cell ontogeny. We expect that these findings will be relevant to understanding mechanisms of autoimmunity more generally, and could find application in new therapeutic approaches for B cell reduction.