**4.3 NK cell cytotoxicity and degranulation assay evaluation**

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition characterized by persistent macrophage/lymphocyte activation and a multisystemic hyperinflammatory syndrome. The clinical diagnosis of HLH is challenging and requires a composite of several findings. According to the updated criteria of the Histiocyte Society [51], the diagnosis is currently established by fulfilling 5 of 8 clinical and laboratory criteria, or the finding of a genetic defect consistent with HLH. Defects associated with HLH are generally associated with abnormal NK cell function. The characterization of these patients requires genetic and functional studies, among the latter are the study of cytotoxic capacity and degranulation.

In *cytotoxicity assays*, we measure the functional capacity of natural killer cells. In this assay, PBMCs or purified NK cell are co-incubated at different ratios with a target HLA-null cell lines (usually K562) known to be sensitive to NK cell-mediated cytotoxicity [52]. After an incubation period, dead target cells are identified by nucleic acid staining, which specifically penetrates the dead cells and is measured by flow cytometry [53]. Chromium-51 (51Cr) release assays are also commonly used for accurate and precise quantification of cytotoxicity. The main disadvantages of this method are the use of radioactivity, which can be hazardous to health, and are impractical and costly due to the short half-life of 51Cr and to the need for radiological safety training and authorization requirements [54].

Cytotoxicity assay protocol:


Other lineage-specific function tests include evaluation of *NK cell degranulation* by evaluation of CD107a (LAMP1) surface expression, which is normally expressed in cytoplasmic granules, following stimulation with a target cell line (K562) or PMA plus ionomycin. This assay complements the classical NK cell cytotoxicity assay and is particularly useful in screening for the diagnosis of familial HLH. Specifically, the lack of surface expression of CD107a after incubation with K562 target cells is consistent with syntaxin-11 (STX11), uncoordinated mammals 13.4 (UNC13D), syntaxin-2 binding protein (STXBP2), or Rab27A protein defects [55].

NK cell degranulation assay protocol:


**45**

**Figure 9.**

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

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

helper 17 lymphocytes (Th17) as those producing IL-17a and IL-17f [1].

IRAK4 suspicion in defects of the innate immunity [3].

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

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

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

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

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

*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).*

*DOI: http://dx.doi.org/10.5772/intechopen.89743*

for a definitive diagnosis [56].

analysis and interpretation (**Figure 9**).

2.Add 1 μL of GolgiPlug.

3.Incubate for 18 h at 37°C.

Intracellular cytokine staining protocol:

*Innovations in Cell Research and Therapy*

through flow cytometry.

NK cell degranulation assay protocol:

1.Count and adjust patient PBMCs at 106

2.Count and adjust K562 cells at 2 × 105

5.Add 100 μL of IL-2 to other three wells.

7.Resuspend cell button and transfer it to FACS tubes.

11.Add 5 μL of anti-CD107a into all FACS tubes.

12.Incubate all FACS tubes during 3 h at 37°C.

14.Incubate for 20 min at RT and in the dark.

13.Add the appropriate mix of surface antibodies of interest.

16.Discard the PBS after 5 min of centrifugation at 1500 rpm.

17.Add 100 μL of PBS and the sample is ready to be acquired by flow

3.Dispense 100 μL of PBMCs at 106

6.Incubate at 37°C over night.

10.Add 100 μL of K26 (106

15.Wash with 2 mL of PBS.

cytometry.

IL-2 tube.

and in the dark.

7. 5 μL of propidium iodide (PI) are added at each tube to identificate death cells

8.PBMcs mixed with K562 at different dilutions are incubated during 4 h at 37°C

Other lineage-specific function tests include evaluation of *NK cell degranulation* by evaluation of CD107a (LAMP1) surface expression, which is normally expressed in cytoplasmic granules, following stimulation with a target cell line (K562) or PMA plus ionomycin. This assay complements the classical NK cell cytotoxicity assay and is particularly useful in screening for the diagnosis of familial HLH. Specifically, the lack of surface expression of CD107a after incubation with K562 target cells is consistent with syntaxin-11 (STX11), uncoordinated mammals 13.4 (UNC13D),

cells/mL concentration.

cells/mL concentration.

cells/mL in a 96-well plate.

cells/mL) into a tube unstimulated and into a

9.Samples are ready to be acquired by flow cytometry.

syntaxin-2 binding protein (STXBP2), or Rab27A protein defects [55].

4.Leave three wells unstimulated by adding 100 μL of RPMI in each.

8.Add 100 μL of RPMI into a tube unstimulated and into a IL-2 tube.

9.Add 100 μL of PHA into a tube unstimulated and into a IL-2 tube.

**44**
