A Rapid Ex Vivo Clinical Diagnostic Assay for Fas Receptor-Induced T Lymphocyte Apoptosis

Journal of Clinical Immunology

Online Resource Supplementary Data

A rapid ex vivo clinical diagnostic assay for Fas receptor-induced T lymphocyte apoptosis

Bernice Lo, Madhu Ramaswamy, Joie Davis, Susan Price, V. Koneti Rao, Richard M. Siegel, Michael J. Lenardo

Corresponding author:

Michael J. Lenardo

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Email:

Supplementary Figure 1

The FasT Kill assay results corroborate with the findings of the traditional Fas apoptosis assay and can identify apoptosis defects in ALPS-U patients. (a-d, upper panels) In each experiment, PBMCs from two normal controls (NC 1, 2) and a prospective ALPS-U patient were tested for Fas-mediated apoptosis using the FasT Kill assay. Cells were incubated for 4-8 hours with varying doses of crosslinked APO-1-3, then stained and analyzed by flow cytometry. (a-d, lower panels) In each experiment, previously activated T cells cycling in IL-2 from two healthy donors and a potential ALPS-U patient were assessed using the traditional Fas apoptosis assay. Cells were incubated with increasing doses of APO-1-3 crosslinked with Protein A for 18-24 hours then stained with propidium iodide and analyzed by flow cytometry. (a-b) Defective apoptosis in the ALPS-U patients is evident with the traditional and FasT Kill assay. (c-d) The FasT Kill assay validated the findings of the conventional apoptosis assay, showing normal apoptosis induction in patients with an ALPS-like phenotype. All data are shown as mean + SD of triplicate wells.

Supplementary Methods

Traditional Fas apoptosis assay. PBMCs (1x106 cells/mL) were activated with 1mg/mL anti-CD3 (BD Biosciences) and 1 mg/mL anti-CD28 (BD Biosciences) for 3 days. The activated cells were washed 2-3 times with PBS then cultured for at least another 7 days in complete RPMI supplemented with 100 units/mL recombinant human IL-2. The activated cycling T cells were plated in triplicate at 1x105 cells/well in a 96-well round bottom plate and treated with varying concentrations of agonistic Fas antibody plus Protein A at one-tenth the concentration of anti-Fas for crosslinking of the antibody. Cells were incubated for 18-24 hours at 37oC 5% CO2 then stained with 5 mg/mL propidium iodide (PI). Using flow cytometry, cells were acquired on constant time and cell death was assessed by PI incorporation. The percentage of cell loss was calculated using the following formula: [1-(number of live cells after treatment/number of untreated live cells)] x 100.