HLA-B27 Subtypes Predisposing to Ankylosing Spondylitis Accumulate in an Endoplasmic Reticulum-Derived Compartment Apart From the Peptide-Loading Complex.

OBJECTIVE: It was previously shown that HLA-B27 subtypes predisposing to spondyloarthritis (SpA), i.e., B*27:02, B*27:05, and B*27:07, displayed an increased propensity to form intracellular oligomers and to accumulate at a high density in cytoplasmic vesicles, as compared to the non-SpA-associated HLA-B*07:02 and HLA-B*27:06. This study was undertaken to characterize the nature and content of HLA-B-containing vesicles and to further examine their relevance to SpA predisposition. METHODS: Vesicles containing HLA-B proteins were detected in transfected HeLa cells and in cells from SpA patients or HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats, by microscopy. The nature and content of HLA-B-containing vesicles were characterized in colocalization experiments with appropriate markers. RESULTS: The SpA-associated HLA-B*27:04 subtype accumulated at higher levels (P < 10-5 ) in cytoplasmic vesicles compared to HLA-B*27:06, from which it differs only by 2 substitutions, reinforcing the correlation between vesicle formation and SpA predisposition. Colocalization studies showed that those vesicles contained misfolded HLA-B heavy chain along with ß2 m and endoplasmic reticulum (ER) chaperones (calnexin, calreticulin, BiP, glucose-regulated protein 94-kd) and belonged to the ER but were distinct from the peptide-loading complex (PLC). Similar vesicles were observed in immune cells from HLA-B27+ SpA patients, in greater abundance than in healthy controls (P < 0.01), and in dendritic cells from HLA-B27/hß2 m transgenic rats, correlating with SpA susceptibility. CONCLUSION: Accumulation of misfolded HLA-B heavy chain along with ß2 m and ER chaperones into ER-derived vesicles distinct from the PLC is a characteristic feature of HLA-B27 subtypes predisposing to SpA. This phenomenon could contribute to HLA-B27 pathogenicity, via a noncanonical mechanism.

HLA-B27 subtype oligomerization and intracellular accumulation patterns correlate with predisposition to spondyloarthritis.

OBJECTIVE: Mechanisms underlying the striking association of spondyloarthritis (SpA) with the class I major histocompatibility complex molecule HLA-B27 remain poorly understood. SpA-like disease develops spontaneously in B*2705-transgenic rats, in conjunction with high HLA-B27 expression levels. This study was undertaken to examine the effects of increased expression of HLA-B27 alleles that are differentially associated with SpA on oligomerization and intracellular redistribution. METHODS: HeLa cells were transfected with complementary DNA encoding for HLA-B proteins fused to yellow fluorescent protein and/or Renilla luciferase and harvested at an early phase and a later phase of expression. We monitored HLA-B intracellular trafficking and localization by means of microscopy and live-cell imaging. Bioluminescence resonance energy transfer (BRET) and Western blotting were used to monitor HLA-B oligomerization. RESULTS: At low expression levels, BRET signals were similarly elevated for all SpA-associated HLA-B27 alleles tested, but were lower for the nonassociated B*2706. Of note, at higher expression levels, HLA-B27 signals remained steady while signal for HLA-B7 decreased sharply, reaching the level observed for B*2706. This was due at least in part to a decreased oligomer proportion without unfolded protein response outbreak. Such differential behavior was not abrogated by proteasome inhibition. With increased expression, all HLA-B proteins accumulated to a high density in cytoplasmic vesicles with labile form and size. The extent of this phenomenon was closely correlated with the level of association with predisposition to SpA. CONCLUSION: To our knowledge, this is the first report of a correlation between the level of predisposition to SpA conferred by HLA-B27 alleles and their biochemical behavior. These findings open new perspectives for understanding the pathogenicity of HLA-B27.