Fluorophore-conjugated 4-1BB antibody enables early detection of T-cell responses in inflammatory arthritis via NIRF imaging

Abstract

Purpose We first developed 4-1BB targeting optical probe---IRDye-680RD-4-1BB mAb and evaluated its capacity for the detection of activated T cells (4-1BB positive) in vivo , as well as the diagnosis of rheumatoid arthritis (RA) in an adjuvant-induced arthritis (AIA) mouse model. Methods 4-1BB expression pattern was validated and analyzed by flow cytometry and immunofluorescence staining. 4-1BB monoclonal antibody (mAb) was conjugated with IRDye-680RD NHS ester, and characterized via the fluorescent spectrum. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal NIRF(Near-Infrared fluorescence) imaging of probe was acquired at 24, 48, 72 and 96 hours post probe administration. Results 4-1BB expression was highly upregulated during RA pathogenesis. Good co-localization was also observed between CD3 and 4-1BB immunofluorescence staining and tSNE analysis, which indicates that 4-1BB was mainly expressed on T cells. Compared to the control group, a significantly higher signal could be detected in the right hind paw from mice with AIA at all time points. Ex vivo biodistribution study was in line with in vivo NIRF imaging, which validated the accuracy of ROI measurements. The sensitivity against 100% specificity observed in receiver operator curves (ROC) analysis could distinguish AIA models from the control group at all time points, indicating the capacity of IRDye680RD-4-1BB mAb in RA diagnosis. Conclusion We successfully developed a novel optical imaging probe IRDye-680RD-4-1BB mAb for capturing 4-1BB + activated T cells in vivo , and 4-1BB NIRF imaging is a promising strategy for noninvasively detecting RA pathogenesis.

Comparison of immune cell profiles associated with heatstroke, sepsis, or cardiopulmonary bypass: Study protocol for an exploratory, case-control study trial

“…Flow cytometry data will be analyzed using FlowJo software (Treestar, Ashland, OR, United States), which will be gated to exclude debris, dead cells and doublets but retain single live cells for subsequent analysis. Immune cell subsets will be visualized in two dimensions map using t-distributed stochastic neighbor embedding (t-SNE) (34) and the "uniform manifold approximation and projection" algorithm (UMAP) (35). Then cells will be grouped into phenotypically homogeneous clusters using PhenoGraph (36) or FlowSOM (37), and the clusters will be characterized using Marker Enrichment Modeling (38).…”