Background/Purpose:

The majority of patients with rheumatoid arthritis (RA) harbor anti-citrullinated peptide antibodies, which are correlated with more severe disease. Tetramer based assays have demonstrated that RA patient T cells that recognize citrullinated antigen are more likely to be activated effectors that produce inflammatory cytokines such as IFNg, and less likely to be T regulatory cells.  Measurement of citrullinated autoantigen T cell responses could be clinically useful to help predict prognosis or treatment response in patients with RA.  A T cell assay that is not restricted to certain MHC II alleles would enable testing of large numbers of RA patients. Since NETing neutrophils externalize targets of RA autoantibodies, we hypothesized that neutrophil NETS could be a source of naturally processed citrullinated antigen to elicit RA patient T cell responses in vitro.  

Methods:

All-Trans Retinoic Acid (ATRA) was used to differentiate HL60 cells.  Growth rate, nuclear morphology, cell surface CD11b expression, and PAD2, PAD4 and MPO mRNA expression were measured. Differentiated cells were exposed to UV irradiation, PMA and ionomycin and cell death by necrosis, apoptosis and NETosis were compared by immunohistochemistry as well as FACS staining with TOPRO and Annexin V. Citrullination of cells undergoing different types of cell death was compared by western blot.  ACPA+ RA and matched control PBMC derived DC were fed granulocytes that had died by various mechanisms and used to probe immediate ex vivo T cell responses using the 40 hour IFNg ELISPOT assay and 18 hour FACS based assays for early activation markers such as CD69 and CD25. 

Results:

ATRA treatment of HL60 cells causes them to differentiate into neutrophil like cells as measured by growth curves, CD11b expression and upregulation of PAD2 and PAD4, enzymes that cause citrullination.  When treated with UV, ATRA differentiated cells die by apoptosis, while ionomycin and PMA induce NETosis.  RA patient but not control T cells produced IFNg and upregulated CD69 and CD25 in response to ionomycin treated ATRA HL60 NET lysate but not ionomycin HL60 (PAD2 and PAD4 low expressing) control lysate.

Conclusion:

RA patients may harbor an expanded population of memory T cells, which recognize NET derived antigen. 

This was supported by grants from the Arthritis Foundation and the Rockefeller University CTSA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rheumatoid-arthritis-patient-t-cells-recognize-neutrophil-extracellular-traps/