This protocol describes detection of STAT5 tyrosine phosphorylation after cytokine stimulation by whole blood flowcytometry (minimum 2 colour facs, but additional information may be gained with additional flurochromes) in order to screen γc/JAK3 signal transduction pathway in T-B+NK- SCIDs.

0.5 ml EDTA-blood from patient and healthy control (does not need to be age or sex matched)

CellFix (BD Biosciences)

Phospho Perm III (BD Biosciences)

STAT Wash (PBS with 1%FCS)

Phospho Lyse/Fix

Deionised water (BD Biosciences)

IL-2 10⁶U/ml (Chiron proleukin) (other manufacturers eg R&D also work)

37°C water bath, heat block or incubator

ice bath or 4°C refrigerator

Centrifuge, microfuge or immunofuge

*Minimum 2 colour flow cytometer (FACSScan), but more information can be gained from a 4 or 6 colour machine as for example FACSCalibur with 488nm-laser and 635nm-diode-laser or CANTO II (BD Biosciences)

* Equipment of other manufacturer’s may be used

2 hours

Preparation of cells

Whole blood is used.

Step 1: Dilute Phosho Lyse/Fix 1:5 with deionised water (e.g. 2 mls Lyse/Fix + 8 mls water) and place at 37°C.

Step 2: Label FACs Tubes (may use microcentrifuge tubes and transfer to FAC tubes later): control unstim, control stim, patient (initials or other identifier like lab number) unstim, and patient stim. and add 100ul of blood to each tube.

Step 3: Rapidly add 10 ul of IL-2 to each of the “stim.” tubes and place at 37°C for 10 minutes.

Step 4: After 10 minutes, immediately add 2 mls diluted Phospho Lyse/Fix to each tube, mix and place at 37°C for 10 minutes.

Step 5: After the lyse/fix incubation, samples are peletted (centrifuged for 5 min at 300xg, 5 minutes 2000 rpm in a microfuge or 45 seconds on high in an immunofuge) and the supernatant removed.

Step 6: Wash 1 time with STAT wash (add 1ml of STAT wash to cell pellet, resuspend, spin and decant supernatant).

Step 7: Add 1 ml of Phosho Perm buffer III to each tube, cover and place on ice or at 4°C for 30 minutes.

Step 8: Pellet cells, wash 1 x with STAT wash.

Step 9: Add 5ul of STAT5 ptyr antibody and choice of surface markers to each tube. Incubate 30 minutes at room temperature in the dark (in a cabinet or covered with foil).

Step 10: Wash 2 times with STAT wash.

Step 11: Add 250 ul Cell Fix and acquire cells within 24 hours of staining. If not acquired immediately, cover and refrigerate.

Commentary: To diagnose defects in the γc/JAK3 pathway the minimum that is required is to compare STAT5 tyrosine phosphorylation pre and post IL-2 stimulation in lymphocytes (defined by FSC vs SSC gating). Other γc cytokines can be used (e.g. IL-7, IL-15). The addition of cell surface markers enables examination of STAT5 tyrosine phosphorylation in individual cell populations (Note: as Perm III is methanol based, many antibodies bind poorly in it. The antibodies listed will provide discrete positive/negative populations but are often less bright than when used for routine surface staining).

Cells in gate R1 define the lymphocyte population.

Suggested Acquisition setting: Events saved: 10000 within the lymphocyte gate with a time limit of 5 min. It is recommended to record all cells, in order to re-gate lymphocytes if required.

Start by gating cells in the lymphocyte population based on FSC vs SSC in the control unstimulated tube. Create a histogram and with STAT5 ptyr (FL1) on the x-axis. Only show lymphocytes (R1). Depending on software overlay the histogram with the lymphocytes from the stimulated tube. Draw a marker from the bottom of the unstimulated histogram peak (often there is a tail as some cells in the unstimulated sample will be activated by cytokines and hormones [e.g. prolactin] present in the blood) to the right side of the histogram. Obtain the statistics (% and MFI) for the unstimulated and stimulated sample. If overlaying histogram is not possible (e.g. with Diva software), create a 2^nd histogram. Repeat with the patient files.

If cell surface markers were included (e.g. CD4, CD8, CD20), start with the unstimulated sample, gate around lymphocytes (R1), then using R1, create a dot plot: surface marker vs SSC (e.g. CD4 vs SSC) and gate on the positive population (e.g CD4+) (R2). Create a histogram and with STAT5 ptyr (FL1) on the x-axis. Only show positive population (R2). Depending on software, overlay the histogram with the positive population from the stimulated tube. If overlaying histogram is not possible (e.g. with Diva software), create a 2^nd histogram. Repeat with the patient files. Repeat as required for each surface marker used.

The interpretation of the results requires the awareness of the medical history of the patient. The reported normal ranges are only reference values for paediatric and adults Patients with maternal-foetal engraftment often have a small cell population showing STAT5 tyrosine phosphorylation while the majority of cells show absent STAT5 ptyr. If surface markers are used, this positive population is usually found in the CD8 T cells. Unexpected results need to be confirmed.

Report percentage change in STAT5 tyrosine phosphorylation and the change in MFI.

Beside standard methods of quality control, a healthy control should be run with STAT5 ptyr staining of peripheral blood to ensure cytokine activity and detect staining errors. It is essential that the control sample is collected at the same time as the patient sample and they are transported together to control for delays and temperature changes in transport. Over time graphing of percent change and change in MFI from all control samples enables rapid detection of technical problems such as deteriorating antibodies and drift in the flow cytometer among others.

Lack of tyrosine phosphorylation in the control sample after cytokine stimulation is usually caused by degraded cytokine. Store diluted cytokines in aliquots at -70°C and avoid repeated freeze/thawing. Poor red cell lysis occurs when the Lyse/Fix is colder than 37°C or the cells have not been incubated for a full 10 minutes at 37°C. Weak/absent staining of cell surface markers results from using antibody clones that are not stable in methanol.

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