Investigating the genetic basis of autoimmune disease
Keywords: PEPD
lymphocyte
genetic
autoimmunity
NDRG1
tolerance
anergy
immunophenotyping
immunology

The maintenance of immune tolerance is essential to prevent the initiation of inappropriate and potentially harmful responses by autoreactive lymphocytes. Autoimmune diseases are ultimately caused by a loss of tolerance to self; this can be driven by rare genetic variants; however, the aetiologies are more often complex, covering an incompletely understood combination of environmental, immunological and common genetic risk factors. Since rare monogenic disorders can illuminate mechanisms relevant to common disease, furthering our knowledge of the inherited mechanisms that affect tolerance loss may help to advance treatment options across many immune disorders, beyond current strategies of broad immunosuppression with limited efficacy and significant side effects. Using forward and reverse genetics approaches and engineered in vivo models, this thesis aims to establish whether two genes, N-myc Downstream-Regulated Gene 1 (Ndrg1) and Pepd, with apparent roles in the maintenance of immune tolerance, do alter the threshold for autoimmunity and begin to explore how this is mediated.
NDRG1 is a tumour suppressor that has previously been implicated as a CD4+ T-cell clonal anergy factor, induced by T cell receptor (TCR) signalling and negatively regulated by co-stimulation. By RNA Sequencing (RNA-Seq), the Cornall lab previously identified Ndrg1 as the third highest differentially expressed gene in anergic B cells compared to naïve follicular B cells. In this thesis, I show that Ndrg1 is upregulated by activation of the B cell receptor (BCR) (signal one) and suppressed by positive co-stimulation (signal two), initially suggesting that NDRG1 may also play an important role in B cell tolerance. To analyse the function of NDRG1 in B cells, I describe here the generation of a Ndrg1-/- knockout mouse model using directed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9) mutagenesis. NDRG1-deficient mice have a neurological defect characteristic of Charcot-Marie-Tooth Type 4d (CMTD4d) disease but a normal primary and secondary immune response, and induction of anergy is normal. This work shows that NDRG1 is not required for the maintenance of B cell tolerance and does not play a role in downstream responses during re-stimulation of in vivo antigen-experienced CD4+ T cells.
In contrast to reverse genetics targeting of suspected immune-involved genes, phenotype-driven screening programmes provide an alternative way to discover novel genes and regulatory pathways involved in maintaining immunological tolerance. A novel mouse model with hallmarks of autoimmunity identified within an autoimmune screening programme presents potential to provide insight into the pathogenesis of prolidase deficiency (PD). Patients with PD present with a multi-system disorder including symptoms concurrent with immunodeficiency, though many also have autoimmune characteristics and 6% meet the criteria to be diagnosed with systemic lupus erythematosus (SLE). PD is known to be caused by mutations in the PEPD gene, but is unclear whether prolidase directly regulates immune tolerance or whether autoimmunity is a secondary complication, in addition to a primary immunodeficiency. A murine Pepd null mouse provides a tool to explore the immunological basis of this disease and more general adaptive tolerance mechanisms.
Deep characterisation of the mouse model of PD in this thesis demonstrates that Pepd loss is associated with autoimmunity, with some elevation of anti-nuclear antibodies (ANAs), increased serum IgA and a histological phenotype reminiscent of human immune complex nephropathy in SLE patients, with glomerular IgA, IgG and C1q deposition. Deep immunophenotyping using flow cytometry and multimodal single cell RNA sequencing (scRNA-Seq) demonstrates that prolidase modulates CD8+ T cell fate, characterised by accumulation of CD8+ effectors expressing a transcriptional signature concurrent with chronic exhaustion, whilst resting populations are reduced. Mixed chimeras confirm that this phenotype is intrinsic, and in vivo labelling suggests that more proliferating cells adopt this exhausted phenotype independent of TCR specificity or antigenic stimulation, which results in the accumulation of these populations in sites of likely breakdown. These mechanistic insights shed light how prolidase regulates self-tolerance as well as pathways involved in CD8+ T cell differentiation, highlighting the potential of studying rare genetic variants implicated in human disease.