Winfried F. Pickl: No financial relationships to disclose
Introduction/Rationale: Impaired immune tolerance triggers aberrant or sometimes overshooting immune responses, contributing to disease manifestations and/or exacerbations. Notably, the prevalence of serum cytokine-specific autoantibodies (c-AAbs) has been documented in ‘healthy’ individuals as well as in association with acute and chronic states of immune disorders. Precise detection of c-AAbs, which can either neutralize, stabilize, or modulate the function of cytokines, requires sensitive assays that present the antigen in its native conformation.
Methods: A novel multiplex flow cytometry-based cellular assay (mFCCA) was devised based on a collection of stable HEK-293T single-cell clones expressing functional interferons and interleukins, for the assessment of c-AAbs in patients’ serum (n = 400), comprising controls, HIV-infected, and those with suspected hyperinflammatory, autoimmune, or malignant disorders characterized by laboratory parameters. The IgG immunoreactivity against 15 cytokine-expressing cell lines was evaluated in a multiplex format, alongside the parental cell line, with specific binding quantified as delta channel geometric mean fluorescence intensity. Subsequently, a cross-validated Random Forest algorithm was applied to discriminate between the five disease phenotypes and the controls. Model performance was gauged by the receiver operating characteristic curves.
Results: Distinct disease-specific c-AAb signatures were identified across patient groups. For instance, HIV patients exhibited significantly higher frequencies of anti-IFN-α2a, IL-1α, IL-2, and IL-5 autoantibodies compared with controls. The classification model demonstrated high accuracy, with area under the curve values ranging from 0.91 to 0.99, and correct assignment of 331 of 400 samples (83%) to their respective groups.
Conclusion: The high-throughput approach for screening c-AAb reactivities against surface-expressed cytokines via multiplexed flow cytometry enables rapid immunopathology classification and may assist targeted therapy.
