I would like to titrate a new antibody for NKp44 staining in PBMCs. After initial attempts with staining, I have noticed that unstimulated PBMCs do not express NKp44. How do I go about titrating this antibody if it is not detected? Could titrations be made after stimulating PBMCs with IL-2? If so, is there a recommended protocol?
To my knowledge, NKp44 is critical in differentiating between ILC1 and ILC3 subtypes, could this be replaced with NKp46? Or another marker?
Many thanks in advance for your advice.
Not expert on NKp-44 but I miss from your information whether there is any other antibody that is able to detect NKp-44 in PBMCs?
You say that unstimulated PBMC do not express NKp-44 but then nobody could detect it with any antibody. In other words if you have used PBMCs for checking the Ab, I assume that it is because somebody else has described that some of the cell types present PBMCs do express NKp-44.
Dear Christina,
NKp44 is not expressed on PB ILC3s, only on those that you find in tissues. To discriminate between PB ILC1s and ILC3s you can use c-kit as ILC1s as c-kit- and ILC3s are c-kit+.
Hope this helps.
Thank you both for your responses.
From my understanding, peripheral blood ILCs are not activated and detection of NKp44 on ILC3s are next to nothing. However, most ILC panels use NKp44 and c-kit to differentiate between ILC1 and 3, therefore I assume it is a critical marker?
Rafael - I believe that NKp44 can be detected on PBMCs when they are activated/primed by IL-2. I was wondering if this should be done for the antibody titration step?
I have attached a figure for the normal gating pathway for ILCs and in this publication we can see that NKp44 is not expressed in the peripheral blood but the marker was used anyway.
Dear Christina,
You're right in that PB ILC3s are not activated and that is probably why these cells do not express NKp44, as they do upregulate it in culture. It depends on your research question whether you have to include it in your panel.
If you want to titrate the antibody you can always try to do so on beads or on tissue cells.
Hi Christina,
NKp44 on IL-15 cultured PBMC
-CD3 (or CD4/CD8) depleted PBMC
-Cultured for 5-6 days with 10 ng/ml IL-15
they will express NKp44
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