PurposeThe goal of this study was to develop an imaging probe—IRDye-680RD-OX40 mAb—that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA.MethodsOX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups.ResultsNIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring.ConclusionThe results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.Graphical Abstract