FluoroSpot for NK cells

[Guest Jane]

Hi Mabtech,

 

I am wondering if we can use fluorospot assay to determine cytokine secreting NK cells. 

Also, Nk cell also produce IFN-g, in that case how can we know that the IFN-g spot we got in the fluorospot plate are from T cell and not from NK cell since they also get activated and produce IFN-g when encounter with pathogen. 

[Esther Schoutrop]

Hej Jane,

Interesting question! You can most definitely use FluoroSpot to identify IFNy (as well as GzB and TNF) producing NK cells. 

As you already mentioned, activated T cells produce the same cytokines. In case you want to avoid a T cell depletion or NK cell selection procedure prior to FluoroSpot you can design your experiment in a certain way to avoid T cell activation and thus cytokine production. You could choose for setting up a co-culture in the FluoroSpot plate using HLA-devoid target cell lines such as K562 or 721.221. Another way could be to include anti-CD16 to boost cytokine production by NK cells and limit T cell cytokine production (PMID: 23287865, Figure 6). Of course, an NK purification step prior to FluoroSpot will take away the risk of background IFNy production by T cells.

[Guest Jane]

Hi Esther,

 

Thank you for your answer. This is a really helpful information.

I got a similar question, let's say we want to stimulate PBMC with peptide and  measure the cytokine response from antigen-specific T cells, how can we make sure that the IFN-g secreting cells are from the antigen-specific T cell and not from NK cells. We cannot do T cell purification method from the PBMC since T cells require another cell from PBMC to present the antigen to them. So, in this case, what is the best way to make sure that IFN-g cytokine spot are from antigen specific T cell alone and not NK cells.

[Esther Schoutrop]

Hej Jane,

Ok in case you want to investigate cytokine release by antigen-specific lymphocytes in response to peptide stimulation, the usage of anti-CD3 or anti-CD16 to activate either T cells or NK cells, respectively, is not recommended as they promote polyclonal stimulation. My previous answer and the reference added would be more relevant in the context of co-cultures in the FluoroSpot plate or pre-culturing your cells prior to FluoroSpot depending on the research question of course.

To follow-up on your second question, instead of performing a T cell isolation procedure you can go for NK cell depletion from your PBMCs. To remove NK cells from your PBMCs you could choose to deplete NK cells by performing magnetic separation using CD56 microbeads or the NK cell isolation kit (e.g. from Miltenyi or Stemcell technologies). Flow cytometry based sorting can also be used to remove the NK cell fraction from your PBMCs.

-        When using the CD56 microbeads, your NK cells and a minor fraction of CD56+CD3+ NK T cells will bind to the magnetic column while the non-NK cells (T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes) are collected in a separate tube. This process can be referred to as NK cell or CD56+ depletion.

-        When using an NK cell isolation kit, all the non-NK cells will bind to the magnetic column while the NK cells are collected in a separate tube. Of note, in this procedure your T cells will be magnetically labelled and when targeting CD3 this might induce some basal activation.

Using either magnetic separation or flow cytometry based sorting will provide efficient depletion of NK cells with minimal impact on your remaining PBMC fraction. Of course this method can also be used with regards to your previous question, as you can choose to deplete the T cell fraction from your PBMCs using CD3 microbeads or flow cytometry based sorting. 

Hope this answers your questions!