Background/Purpose: Distinguishing true antigen (Ag) stimulated lymphocytes from bystanders activated by the inflammatory milieu has been difficult. Infiltrating immune cells at sites of inflammation become activated not only through direct Ag stimulation, but also indirectly by other inflammatory mediators. Nur77 is an immediate early gene whose expression is rapidly up regulated by T cell receptor (TCR) signaling. Nur77-GFP transgenes serve as specific TCR signaling reporters in murine transgenic models. In this study, we demonstrate that Nur77 protein expression serves as a reporter of TCR and B cell receptor (BCR) specific signaling in human PBMCs. This can be used as a tool to identify Ag experienced lymphocytes in human disease.  

Methods: Intracellular Nur77 protein amounts were assessed by immunofluorescence and flow cytometry in T and B cells isolated from human PBMCs stimulated through their Ag receptors (anti-CD3 or anti-IgM Abs) or with immunostimulants: CpG oligodeoxynucleotides (CpG), IFN-α, or LPS.  

Results: We demonstrate that in vitro TCR stimulation of human PBMCs from healthy donors rapidly induced Nur77 protein expression and reflected Ag receptor signaling strength, much like CD69, a commonly used marker of lymphocyte activation (Figure 1). In a more physiologic approach, PBMCs stimulated with Staphylococcus superantigen (SEE), which polyclonally activates Vβ5.1 expressing T cells through the TCR, resulted in enrichment of Nur77 positive cells in the population of responding cells with the appropriate Vβ (Figure 2). However, stimulation with various immunostimulants that do not signal via the TCR (i.e., IFN-α, CpG, LPS) did not induce Nur77 in human PBMCs, in contrast to CD69 (Figure 3), reflective of differences in their upstream specific signaling events. Similarly, Nur77 induction in B cells reflected the strength of BCR signaling after crosslinking with anti-IgM. Likewise, non-BCR specific immunostimulants did not induce Nur77 in human peripheral B cells in contrast to CD69.  

Conclusion: Nur77 is a reporter of Ag receptor signalling in peripheral human T and B cells. Furthermore, we demonstrate that Nur77 is a more specific reporter of Ag specific signaling than CD69 in both human T and B cells. This reporter strategy has great potential to identify Ag activated lymphocytes in human disease.          

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/endogenous-nur77-is-a-specific-indicator-of-antigen-receptor-signaling-in-human-t-and-b-cells/