Functional Analysis of Anti-cytokine Autoantibodies Using Flow Cytometry

Abstract

Autoantibodies to cytokines are increasingly being detected in association with immunodeficient, autoimmune and immune dysregulated states. Presence of these autoantibodies in an otherwise healthy individual may result in a unique phenotype characterized by predisposition to infection with specific organisms. The ability to detect these autoantibodies is of importance as it may direct treatment toward a combination of anti-microbial agents and immunomodulatory therapies that decrease autoantibody levels, thereby releasing the immune system from autoantibody-mediated inhibition. Ligand binding assays such as ELISA or bead multiplex assays have been used to detect these antibodies. However, not all anti-cytokine autoantibodies have demonstrable function in vitro and therefore their clinical significance is unclear. Assays that evaluate the functionality of anti-cytokine autoantibodies can supplement such ligand binding assays and add valuable functional information that, when viewed in the context of the clinical phenotype, may guide the use of adjunctive immunomodulatory therapy. This mini review provides an overview of anti-cytokine autoantibodies identified to date and their clinical associations. It also describes the use of flow cytometry for the functional analysis of anti-IFNγ and anti-GM-CSF autoantibodies.

Keywords: GM-CSF; autoantibodies; cytokines; flow cytometry; interferon gamma; non-tuberculous mycobacteria; phosphorylation.

Figure 1

Utility of the IFNγ and GM-CSF signaling pathways for analysis of anti-IFNγ or anti-GM-CSF AAbs. (A) IFNγ or GM-CSF bind to their cognate receptors causing Jak1 or 2 to be phosphorylated, leading to phosphorylation and dimerization of STAT1 or STAT5, respectively. Phosphorylated STAT1 and STAT5 dimers translocate to the nucleus and initiate transcription of IFNγ or GM-CSF responsive genes respectively; (B) Recombinant GM-CSF induces phosphorylation of STAT5 in human monocytes (pink: unstimulated cells; blue: stimulated cells); (C) Human PBMCs were incubated with recombinant IFNγ and control serum (CS) or serum from a patient with disseminated NTM (PS). PS strongly inhibited IFNγ-induced phosphorylation of STAT1; (D) Specificity of the flow cytometry assay for detection of anti-IFNγ AAbs. Human PBMC were stimulated with IFNα alone, or with IFNα and control serum or patient serum. Patient serum did not inhibit IFNα-induced phosphorylation of STAT-1 indicating that p-STAT1 inhibition was specific to IFNγ AAbs.