Anti-human Interleukin(IL)-4 Clone 8D4-8 Cross-Reacts With Myosin-9 Associated With Apoptotic Cells and Should Not Be Used for Flow Cytometry Applications Querying IL-4 Expression

Abstract

Interleukin(IL)-4 is produced by T cells and other leukocytes and is a critical mediator of monocyte and B cell responses. During routine flow cytometry panel validation for the investigation of intracellular cytokines, we observed unique IL-4 expression patterns associated with the widely available monoclonal antibody 8D4-8. Namely, IL-4 (8D4-8) expression was observed in the absence of cellular activation and enhanced following staurosporine exposure. Mass spectrometry analysis of immunoprecipitates from peripheral blood lymphocytes (PBL) revealed that 8D4-8 cross-reacts with the ubiquitous cytoskeletal protein myosin-9. We confirmed these results by western blotting immunoprecipitates, using immunofluorescence among staurosporine-treated Caco-2 cells, and by surface-labeling PBL for 8D4-8 and myosin-9 and analyzing by flow cytometry. Although previously reported from several independent groups, we found no evidence to support the hypothesis that IL-4 is produced by apoptotic cells. Rather, this appears to have been myosin-9. Our data indicate clone 8D4-8 should not be used in the flow cytometric study of IL-4. Furthermore, our work calls for a reevaluation of previous flow cytometric studies that have used this clone for IL-4 analysis and highlights the importance of validation in antibody-based assays.

Design, optimisation and standardisation of a high‐dimensional spectral flow cytometry workflow assessing T‐cell immunophenotype in patients with melanoma

“…Where possible, previously validated clones were selected. 53 In the case of IL‐4, the initially tested clone 8D4‐8 was exchanged for MP4‐25D2, which resulted in improved binding specificity, 54 and more reliable performance. UltraComp eBeads (Invitrogen, Waltham, MA, USA) were used in reference controls for IL‐2 AF488, IL‐4 APC, IL‐10 BV421 and IL‐17A PE‐Cy7.…”

A relação entre polimorfismos de IL-4 e asma: uma revisão sistemática

“…IL-4 is a lymphokine that acts as a growth factor (CSF) that induces the Th2 response while inhibiting the Th1 response, further increasing the expression of low-affinity IgE receptors (FcεRII) in non-activated B cells and macrophages. The cited cytokine also plays a role in the activation of B cells by increasing the expression of MHC class II molecules, as well as increasing the expression of CD23, CD40 and the alpha chain of the IL-2 receptor (IL-2RA) 15 .…”