Background/Purpose:

Axial Spondyloarthritis (AxSpA) is a chronic inflammatory rheumatic disease of axial skeleton. Our group recently identified a novel rare mutation of the SEC16A gene that strongly tracked with AxSpA in a multiplex family. Remarkably, all individuals with HLA-B27 and the SEC16A mutation had or went on to develop AxSpA. Sec16a is an important player in the ER-Golgi transport pathway with potential effects on antigen presentation and the pathogenesis of AxSpA. We have previously shown that the SEC16A mutation hinders the assembly of COPII vesicles budding from the ER that leads to abnormal MHC I trafficking and ER stress. In this study we explore the functional impact of Sec16A abnormalities on CD8+ T cell immune response. In addition, protein-protein interaction assays followed by pathway analysis help us understand the impact of SEC16A mutation on different cellular processes.

Methods:

AxSpA patients from the multiplex family (N=9) had both SEC16A mutation and HLA-B27. Controls (N=5) were family members with no disease and had HLA-B27 and wild-type (WT) SEC16A. B cell lines were generated from patients and controls by EBV transformation. For validation, SEC16A was silenced in B-lymphoblastoid cells with stable HLA-B27 expression (C1R-B27) and assays repeated. Total protein was extracted from B cells and the formation of HLA free heavy chain homodimer (FHC) was studied using HC-10 western blot under non-reducing and reducing conditions. HLA-B27 mediated immune response was assessed by cytotoxic T-lymphocyte assay. NP383-391 specific CTL clones were generated from HLA-B27 positive donor. The GFP-ubiquitin-NP383-391 construct was introduced by DNA transfection in B cells. Two days after transfection, B cells were incubated with NP383-391 specific CTL at a ratio of 1:10 for 4 hours. CTL activity was evaluated using caspase 3 cleavage in target cells. Pathway analysis assessed by protein-protein interaction was carried out to investigate the interactome of SEC16A variants.

Results:

Both SEC16A mutation in patients’ B cells and Sec16a deficiency (90% suppression) in C1R-B27 significantly (P<0.01 and P<0.001 respectively) increased the level of HLA-B27 FHC homodimers. This is likely due to a general disruption of MHC I trafficking by Sec16A abnormalities as we previously reported. GFP-ubiquitin-NP383-391 constructs were stably expressed in EBV-B cells at a similar level without having an effect on HLA-B27 surface expression. However, the cytotoxicity of NP 383-391 specific T cells were significantly lower against EBV-B cells with mutant SEC16A compared to EBV-B cells with WT SEC16A (p<0.01). T cell cytotoxicity was almost completely abolished in SEC16A-suppressed C1R-B27 cells. Using protein-protein interaction and pathway analysis, we found that mutant Sec16a had lower affinity to interactors involved in ER-to-Golgi vesicle mediated transport, membrane budding, vesicle organization, and antigen processing/presentation.

Conclusion:

Sec16a abnormalities can affect HLA-B27 intracellular transport, dimerization, antigen presentation and cytotoxic T cell responses. These changes can potentially play a role in AS pathogenesis.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/sec16a-and-antigen-presentation-abnormalities-in-the-pathogenesis-of-axial-spondyloarthritis/