AbstractMajor histocompatibility complex class II (MHC II) proteins display peptides to CD4+ T cells. Their regulation of expression is important for normal immune function and autoimmunity. Two aspects of this were explored here. Expression of HLA-DR, the major human MHCII isoform, was manipulated in immortalised antigen-presenting cells (APCs), and characterised by qRT-PCR, flow cytometry and Western blot. Mass spectrometry was used to quantify expression changes of polymorphic HLA-DR beta chain variants. First, the mechanism of DR proteolysis was elucidated. MHCII degradation had been shown to be regulated by ubiquitylation and peptide loading. This study showed that cathepsin D (Cat D), a lysosomal aspartyl protease, degrades DR molecules not associated with peptide or invariant chain. Interestingly, when Cat D activity was ablated, APCs eventually restored wild-type DR levels, using feedback mechanisms that remain to be explored. Secondly, the effects of vitamin D and retinoic acid (RA) on DR expression were explored. Previous work had shown both indirect effects on DR levels and transactivation of some DRB allelic variants by vitamin D. This study revealed synergistic actions of RA and vitamin D on DR expression by myeloid model APCs but mutual antagonism in lymphoblastoid cells, and suggested that DR beta-chain copy number variants may be regulated discordantly. This work suggests that in physiological DR protein turnover, Cat D attacks the groove of DR molecules that lost bound peptide, mediating quality control of antigen presentation. This could mitigate against autoimmunity by limiting unconventional presentation pathways independent of invariant chain or HLA-DM. The small size of the vitamin D and RA effects on DR argues against a major role in autoimmunity, with the possible exception of the unusual DRB1*15-DRB5*01 MS risk haplotype. Novel tools were generated for the quantification of codominant expression of DR allelic and copy number variants, which will be useful in extending this work to primary APCs.
|Date of Award||10 Jan 2019|
|Sponsors||Multiple Sclerosis Society UK & Multiple Sclerosis International Federation|
|Supervisor||Jolanta Opacka Juffry (Supervisor) & Robert Busch (Supervisor)|
Regulation of MHC II protein expression by lysosomal degradation and vitamin D
Shah, N. (Author). 10 Jan 2019
Student thesis: Doctoral Thesis