IL-17AF Gets its Close-up!
IL-17AF, the molecule no one is talking about - but should be!
We’ve known of the existence of this molecule for some time now, but no one is really talking about it! IL-17A and IL-17F are members of the IL-17 family and are signature Th17 cytokines. Although both of these were originally found to be produced as disulfide-linked homodimers (we should probably be calling them IL-17AA and IL-17FF), studies using ELISA and IP/WB have confirmed the production of IL-17AF heterodimers in CD4+ T cells.
These heterodimers were first reported several years ago and there have been many papers documenting expression and function of IL-17A and IL-17F. But why hasn’t IL-17AF received more attention? Perhaps it’s because good reagents for flow cytometry have not been available. This problem exists generally for any dimer molecule that can exist in multiple configurations. In the case of IL-17, all existing antibodies for IL-17A only detect one monomer of the dimer. Therefore, they cannot distinguish between IL-17AA homodimer and IL-17AF heterodimer. The same is true for IL-17F reagents. So in most cases, when someone uses existing antibodies to identify an IL-17A producer by flow cytometry, they are actually identifying an IL-17AA and/or IL-17AF producer. With the appropriate reagents, when someone co-stains IL-17A and/or IL-17F, they could potentially identify three populations: IL-17A single positive, IL-17F single positive, and IL-17A/IL-17F double positive. It is then clear which cells are producing only IL-17AA (below left, green) and only IL-17FF (below left, blue). However, with the double positives (below left, orange) one cannot determine whether both IL-17AA and IL-17FF are present or just IL-17AF or all three. We can now tell you that IL-17AF is definitely present!
IL-17AF's time in the spotlight is now!
Here we have used flow cytometry to identify the in vitro-polarized producers of IL-17AF heterodimers for the first time. Under our polarization conditions, it turns out that the majority of the IL-17A and IL-17F double-positive population is expressing IL-17AF heterodimers (below right, orange). We have also proven specificity in that only the IL-17A and IL-17F double positives stain with the IL-17AF antibody. In the next few weeks, we will be releasing this new antibody that specifically detects human IL-17AF heterodimers in flow cytometry and non-reduced western blot.
Th17-polarized CD4+ PBMC were treated with Cell Stimulation Cocktail plus protein transport inhibitors and then stained for IL-17A, IL-17F and IL-17AF. IL-17A was plotted vs IL-17F (left) and the resulting quadrants were analyzed for staining of IL-17AF (right).