Background/Purpose:

ERAP1 and HLA-B27 are strongly associated with ankylosing spondylitis (AS). Using well-controlled tissue culture systems, we studied the effects of AS-associated ERAP1 variants on HLA-B27 expression and interaction with KIR receptors

Methods:

C1R cells stably expressing HLA-B27 were transfected with ERAP1shRNA to silence the endogenous ERAP1 expression (C1R^ERAP1sh). The stable cells were confirmed by flow cytometry with GFP and western blot for ERAP1. We then transfected either the common variant ERAP1 (ERAP1^WT) or one of the two AS-associated ERAP1 variants, K528R or Q730E into the C1R^ERAP1shcells. Lentivirus expression vector alone was used as control and the ERAP1 variants were tracked with the HA-tag.

The stable cell lines were tested by flow cytometry for intact HLA-B27 (ME1 antibody), MHC-I free heavy chain (FHC) expression (HC-10), MARB4 staining and binding to KIR-3DL1 and KIR-3DL2-Fc. Anti-Mouse IgG-PE was used as secondary antibody. 

Results:

Following antibiotic selection, nearly all cells were GFP positive indicating successful stable ERAP1 shRNA expression. We achieved more than 80% suppression of ERAP1. After introduction of ERAP1 into C1R^ERAP1sh cells, Western Blot with anti-HA antibodies showed similar expression of ERAP1^WT. K528R and Q730E variants respectively.  

There was significant increase in surface FHC, intracellular FHC and MARB4 staining with ERAP1 shRNA and subsequent normalization of levels with introduction of ERAP1^WT. C1R cells with AS-associated ERAP1 variants failed to completely normalize these levels.

KIR3DL1 and KIR3DL2 binding to target cells were assessed using the Fc-chimeric molecules of these receptors. As expected, KIR-3DL1 and KIR-3DL2 binding was increased in C1R^ERAP1sh cells and C1R cells with ERAP1 variants known to have less peptide trimming function compared to C1R-ERAP1^WT Cells. The percentage of cells with KIR3DL1-Fc binding increased from 40.1% of cells to 64.9% with ERAP1 suppression. KIR-3DL1-Fc binding dropped to 55.9% with introduction of ERAP1^WTbut was higher at 70.4% and 57.3% in C1R cells with K528R and Q730E ERAP1 variants respectively.

Conclusion:

Cells with stable ERAP1 knock down had higher intracellular FHC, surface FHC expression and MARB4 staining as well as higher KIR-3DL1 and KIR-3DL2 receptor binding. These changes were corrected with the introduction of the common ERAP1 variant but not with AS-associated K528R or Q730E variants. Decreased ERAP1 activity could be protective because higher surface FHC can interact with inhibitory receptors on NK cells. On the other hand, higher intracellular FHC seen with decreased ERAP1 activity could be pathogenic by triggering the unfolded protein response. Thus the effect of ERAP1 variants on AS pathogenesis may vary depending on the relative balance of these effects.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ankylosing-spondylitis-associated-endoplasmic-reticulum-aminopaptidase-1-variants-in-antigen-presentaing-cells-affect-hla-b27-free-heavy-chain-expression-and-binding-to-nk-cell-receptors/