Upregulation of Fcγ Receptor IV on Activated Monocytes and Macrophages Causes Nonspecific Binding of the PK136 Anti-NK1.1 Antibody in Murine Models of Toll-Like Receptor-Induced Inflammation.

Abstract

Nonspecific binding of monoclonal antibodies to Fcγ receptors (FcγRs) is a well-known root cause of unreliable results in flow cytometry. Over the past decade, liver Group 1 innate lymphoid cells (ILCs), including conventional natural killer (cNK) cells and type 1 ILCs (ILC1s), have been extensively studied by flow cytometry in various inflammatory liver disorders. In our previous work, we specifically evaluated changes in liver ILC1 numbers in two murine models of Toll-like receptor (TLR)-induced macrophage activation syndrome, a hyperinflammatory disorder with liver inflammation that is classified as a secondary form of hemophagocytic lymphohistiocytosis. Here, we follow up on a cell population that significantly expands during TLR triggering and resembles ILC1s, as they express CD49a and NK1.1 but lack expression of CD49b, a marker for cNK cells. However, detailed analysis revealed that these are CD49a⁺ monocytes/macrophages instead of ILC1s. During TLR triggering, their expression of FcγRIV increases significantly, leading to nonspecific binding of the frequently used PK136 anti-NK1.1 antibody, which cannot be blocked by standard Fcγ receptor blocking protocols. Instead, preincubation with anti-FcγRIV antibody or additional rat or mouse serum during antibody staining is necessary to prevent nonspecific anti-NK1.1 binding. Although we observed nonspecific binding of the anti-NK1.1 antibody in ex vivo applications, we confirmed that in vivo anti-NK1.1 only depletes truly NK1.1⁺ populations. In conclusion, we emphasise that studying NK1.1⁺ ILCs during inflammation by flow cytometry requires additional FcγRIV blocking reagents and careful exclusion of myeloid cells.