**4.4 Detection of intracellular IFN-γ expression in activated T lymphocytes**

The use of intracellular cytokine staining technique has become extremely useful in recent years to elucidate defects in intracellular signaling as causes of different IDPs. One of the applications of this technique is the characterization of the different subpopulations of T helper (Th) lymphocytes. The strategy of characterization of these subpopulations is to identify T helper 1 lymphocytes (Th1) as cells producing INFγ, T helper 2 lymphocytes (Th2) as those producing IL-4 and IL-13 and T helper 17 lymphocytes (Th17) as those producing IL-17a and IL-17f [1].

In patients with STAT3 or DOCK8 deficiency, a decrease in IL-17 producing T cells (Th17) has been demonstrated. Th17 cells play a role in autoimmunity and defense of extracellular pathogens (fungi, bacteria, and parasites). Therefore, the absence of IL-17 production after stimulation of PBMCs in these patients allows identifying a functional defect. However, mutational genetic analysis is necessary for a definitive diagnosis [56].

Moreover, this technique can be used to determine the production of other cytokines such as INFγ in front MSMD suspicion [57, 58] or the production of TNF-α in IRAK4 suspicion in defects of the innate immunity [3].

In summary, the intracellular cytokine staining technique consists of the stimulation of heparinized whole blood with PMA and ionomycin or other stimuli. After the appropriate incubation time for each cytokine, intracellular and superficial staining is performed. There are three aspects to consider: (A) the results will be compared with the cytokine production of a healthy control; (B) the incubation time with the stimulus can be modified depending on the cytokine we want to study. In case we want to study more than one, we will have to find an ideal incubation time for each of them, that is, leave time for all of them to be synthesized, without degrading any of them; (C) it is a key to know the cell subpopulation producing the cytokine of interest, as we can add surface markers that will facilitate analysis and interpretation (**Figure 9**).

Intracellular cytokine staining protocol:

1.Stimulate 500 μL of heparin whole blood with PMA and ionomycin.


### **Figure 9.**

*Intracellular production of IFNg and IL2 cytokines. In the dotplots, cytokine production is observed after 18 h stimulation in a healthy donor (left) and a patient with an immune deficit (right).*


(This protocol may change slightly depending on the commercial kit used).
