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BACKGROUND: Distinguishing patients with axial spondyloarthritis (axial SpA) from patients with other causes of chronic back pain remains a challenge. The lack of reliable biomarkers contributes to the diagnostic delay in axial SpA. Recently, macrophage migration inhibitory factor (MIF) has been proposed as a candidate diagnostic and prognostic biomarker. MIF is a proinflammatory cytokine that was shown to be upregulated in several autoimmune diseases, including axial SpA. The putative role of CD8+ T cells in the disease process suggests further that serum markers of cytotoxicity might have value as serological biomarkers in axial SpA, and that subpopulations of cytotoxic lymphocytes might deserve attention as candidate cellular biomarkers. OBJECTIVE: The goal of this study was to compare serum levels of MIF and other candidate serum proteins in patients with axial SpA and controls, and to develop a flow cytometry panel to analyze cytotoxic lymphocyte cell subpopulations in these cohorts, including KIR+CD8+ T cells, Granzyme B+ CD8+ T cells, MAIT cells, and InEx cells. METHODS: Study subjects were recruited from the Brigham and Women’s Hospital Orthopedic and Arthritis Center. Four cohorts were compared: healthy controls (HC), patients with chronic low back pain (cLBP), axial SpA patients not on a biologic (axSpA/-), and axial SpA patients treated with a TNF inhibitor (axSpA/TNFi). Study subjects were matched for age, sex, and race, when possible. Serum was evaluated using the LEGENDplex Human CD8/NK panel (BioLegend) for thirteen markers including IL-17A, IL-6, TNF, granzyme B, and perforin. CRP and MIF were evaluated by DuoSet ELISA (R&D Systems). A high-dimensional flow cytometry panel was designed to evaluate 14 cell populations of interest. RESULTS: The severity of back pain in the cLBP controls and axSpA/- patients was comparable (BASDAI Q2 mean 5.0 +/- 1.9 vs. 5.0 +/- 3.0). axSpA/- patients had higher back pain, BASDAI and ASDAS scores than axSpA/TNFi patients consistent with higher disease activity in the biologic naïve group. Serum CRP values were significantly higher in axSpA/- patients compared with HC, cLBP controls, and axSpA/TNFi patients (P= 0.01, P=0.0029, P=0.004 respectively). Serum MIF levels were not statistically different between all four groups (P= 0.8069). Additionally, there were no statistically significant differences between the groups for any of the markers included in the LEGENDplex Human CD8/NK panel. A 32-color staining panel was developed to evaluate cytotoxic cell populations. CONCLUSION: In contrast to a previous study, we did not find differences in serum MIF levels between axial SpA patients and controls. Of the evaluated serum biomarkers, only CRP values correlated with active axial SpA. We have developed a promising flow cytometry panel that will help analyze subpopulations of cytotoxic cells. This ultimately could shed light on a candidate cellular biomarker. Our results underscore the need for more research into diagnostic biomarkers in axial SpA.