Background/Purpose: Mechanisms underlying the striking association of SpA with the MHC class I molecule HLA-B27 remain poorly understood. SpA-like disease develops spontaneously in B*2705 transgenic rats in correlation with high HLA-B27 expression levels. This study was undertaken to examine the consequences of expressing HLA-B27 alleles which are differently associated with SpA on their intracellular distribution. It has previously been observed that high expression levels of HLA-B in HeLa cells induced cytoplasmic vesicles formation containing HLA-B molecules and that the density of vesicles was more important for HLA-B27 subtypes associated with SpA than for the non-associated HLA-B*2706 and HLA-B*0702 alleles. Here, we further examined the nature and composition of thoses vesicles and the putative differences between associated and non-associated HLA-B alleles.

Methods: HeLa cells were transfected with complementary DNA encoding for HLA–B proteins fused to yellow fluorescent protein. We studied the composition and nature of HLA-B-containing intra-cellular vesicles by antibodies staining and live-cell imaging.

Results: With increased expression, all HLA-B proteins accumulated in cytoplasmic vesicles. This phenomenon was more pronounced for the SpA-associated HLA-B*2702, -B*2704 and -B*2705 than for the non-associated -B*2706 and -B*0702. We observed comparable staining of those vesicles with HC10 antibody (anti-class I heavy chain) for all HLA-B alleles. In contrast, we observed differential staining with BBM1 antibody that binds to β2m: the SpA-associated HLA-B27 subtypes formed vesicles that were stained significantly more with BBM1 than the non-associated HLA-B*2706 and HLA-B*0702 alleles. On the other hand, we observed no staining of thoses vesicles for EEA1 (early endosome marker), Rab7 (late endosomes marker), LC3 (autophagosomes marker) and Rab6 (Golgi marker) antibodies. However, we found positive staining for ER chaperones (BiP, calreticulin and ERp57) indicating that the HLA-B-containing vesicles belong to the ER. Consistent with such interpretation, those vesicles were still observed using live-cell imaging of HeLa cells transfected with HLA-B after treatment with nocodazole or brefeldin-A that inhibit ER exit. Finally, a lack of staining for tapasin indicated that the peptide-loading complex (PLC) does not localize to those vesicles.

Conclusion: Under conditions of high expression, HLA-B molecules accumulate in vesicles that belong to the ER where they co-localize with chaperones but not with the PLC. Moreover, our data indicate differences between HLA-B-containing cytoplasmic vesicles, depending on the allele: vesicles formed with HLA-B27 subtypes associated with SpA were more abundant and contained significantly more β2m than those formed with non-associated alleles. This report establishes a correlation between the level of predisposition to SpA conferred by HLA-B alleles and their biochemical behaviors that may contribute to their pathogenicity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/spa-associated-hla-b27-subtypes-accumulate-more-in-endoplasmic-reticulum-er-compartment-and-in-association-with-%ce%b22-microglobulin-%ce%b22m-than-non-associated-hla-b-alleles/