Background/Purpose:

 

Ankylosing Spondylitis (AS) is an inflammatory arthritis affecting the axial and peripheral joints. Human Leukocyte Antigen (HLA)-B27 and Endoplasmic Reticulum Aminopeptidase (ERAP) 1 genes are strongly associated with AS. An AS pathogenesis mechanism proposes that variant forms of HLA-B27 including B27 free heavy chains (FHC) can be recognized by Natural Killer (NK) cells through receptors like KIR3DL1and KIR3DL2, and could be pathogenic. ERAP1 variants K528R and Q730E with decreased function are protective in AS. Decreased function results in higher HLA-B27 (FHC) expression and can bind to the inhibitory receptors KIR3DL1 and KIR3DL2 altering NK cell activation.

Methods:

C1R cells stably expressing HLA-B27 were transfected with ERAP1shRNA to silence the endogenous ERAP1 expression. These cells were transfected with either the common or one of the rare AS associated ERAP1 variants K528R / Q730E. The target cell line was co-cultured with both a KIR3DL1 expressing as well as a non-KIR3DL1 expressing YTS NK cell line for 9 hours. Supernatants were analyzed for 64 cytokines and chemokines using a bead based multiplex assay. Separately, co-cultured cells were stained for intracellular Macrophage Inflammatory Protein (MIP)-1α. NK cells and target cells were each labeled with separate surface staining fluorescent dyes before co-culture. Co-cultured cells were analyzed by flow cytometry at baseline and after 5 and 10 minutes of co-incubation, for NK cell-target cell conjugate formation. Activation of NK cells was also analyzed by flow cytometry after staining for the degranulation marker CD107a. 

Results:

Duplicate results of the multiplexing assay show MIP-1α is reduced when C1R-B2705 + wild type ERAP1, C1RB2705 + ERAP1 K528R, and C1RB2705 + ERAP1 Q730E cells were co-cultured with 3DL1 NK cells in comparison to YTS NK cells (60%, 45% and 40% reduction respectively). Conjugate formation with the passage of co-incubation time is also reduced in the variant co-cultures as a result of KIR3DL1 interaction (K528R: 27%, Q730E: 18% reduction). Intracellular staining for NK cell MIP-1α production in the above co-cultures revealed a corresponding inhibition of MIP-1α (K528R: 4.6%, Q730E: 28% reduction). YTS-3DL1 NK cell CD107a expression trends lower in the ERAP1 variants in comparison to wild type ERAP1.

Conclusion:

Our results are a logical extension of our previous results that showed protective ERAP1 variants lead to increased surface HLA-B27 FHC in monocytes. Here we show that the protective ERAP1 variants, when expressed in C1R cells, result in decreased conjugate formation with KIR3DL1 expressing NK cells, in comparison to wild type ERAP1. A corresponding decrease in MIP-1α chemokine expression and NK cell activation was observed. Reduced function ERAP1 variants may be protective in AS because increased surface HLA-B27 FHC can interact with inhibitory NK cell receptors like KIR3DL1 and cause reduced conjugate formation and NK cell suppression. 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ankylosing-spondylitis-protective-endoplasmic-reticulum-aminopeptidase-1-variants-lead-to-decreased-natural-killer-cell-conjugation-and-activation/