TNF is a key cytokine in autoimmune diseases like rheumatoid arthritis, and TNF antagonists are commonly prescribed therapeutics. Although anti-TNF drugs have enabled a very significant progress in this field, disease heterogeneity remains and causes diversity in patient response. These challenges increase the need for anti-TNF characterization tools that may open perspectives toward the development of personalized medicine. In this study, we present a novel whole blood-based flow cytometry functional assay that allows, within a given whole blood sample, the characterization of an anti-TNF molecule mechanisms of action. Whole blood from healthy human donors was employed to mimic the physiological state but also to streamline experimental workflows. Samples were incubated with LPS alone or in combination with various anti-TNF molecules such as adalimumab (ADA), etanercept (ETA), and infliximab. A 10-color flow cytometry panel including CD69, transmembrane TNF, CD16, CD62L, CD66b, CD11b, and CD54 as activation markers was used following a centrifugation-free protocol. CD69 expression decreased on NK, NKT, and T cells upon treatment with ADA, ETA, and IFX as a direct indication of forward signaling neutralization. Percentages of transmembrane TNF + monocytes increased after incubation when using ADA or IFX but not ETA, revealing the potential of the two first molecules to trigger reverse signaling. Ab-dependent cell cytotoxicity was informed by CD16 and CD69 expressions in some donors that showed increasing levels of CD16 2 CD69 + NK cells when incubated with anti-TNFs. This study proposes a novel approach to assess anti-TNF mechanisms of action and provides a path toward capturing donor heterogeneity.

Despite introduction of biological disease modifying anti-rheumatic drugs (DMARDs) for Rheumatoid arthritis (RA) treatment, therapeutic strategies do not always lead to disease control and remission. Hence, a more efficient patient stratification and monitoring biomarkers and tools are needed to enable a more personalized medicine. We used a whole blood based functional flow cytometry assay to characterize immune cells from RA patients (treated or not), healthy donors and psoriatic arthritis (PsA) patients according to their responses to LPS and/or anti-TNFα (infliximab, IFX). Activation marker expression was measured using a 10-color flow cytometry panel following a no-wash protocol. Naïve-to-treatment RA patients had a stronger inflammatory profile in comparison to healthy donors at basal level. Higher expression of activation markers (CD69 and/or CD11b) on NK, B cells and granulocytes and lower expression of the adhesion molecule CD62L were measured on monocytes, granulocytes and B cells. After LPS, naïve RA patients’ cells were less capable of regulating CD69, CD11b, CD16 or CD62L showing impaired activation capabilities. Upon LPS and IFX co-incubation, hierarchical clustering analysis showed different profiles between cohorts. We believe that this whole blood-based approach should further be assessed for RA patient characterization as it provides new perspectives for stratification and/or monitoring.