Detailed step-by-step operating procedures for NK cell and CTL degranulation assays

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1 Supplemental methods Detailed step-by-step operating procedures for NK cell and CTL degranulation assays Materials PBMC isolated from patients, relatives and healthy donors as control K562 cells (ATCC, CCL-243) Complete medium (RPMI 1640 medium supplemented with 10% FBS, 1% Penicillin/Streptomycin and 2mM L-Glutamin) Recombinant interleukin-2 (IL-2, Proleukin, Novartis) Phytohaemagglutinin (PHA) (Sigma-Aldrich) Dynabeads CD3/CD28 T cell expander (Invitrogen Dynal) FACS buffer (PBS supplemented with 2% FBS and 2mMEDTA) Fluorochrome-conjugated monoclonal antibodies: - anti-cd107a-pe (clone H4A3, BD Biosciences) - anti-cd3-percp (clone SK7, BD Biosciences) - anti-cd56-apc (clone NCAM16.2, BD Biosciences) - anti-cd8-fitc (clone SK1, BD Biosciences) PROTOCOL 1 This protocol is performed either on fresh PBMC or on PHA/IL-2 blasts cultured for hours to allow the analysis of the degranulation of resting and activated NK cells and CTL, respectively. Timetable Day 0 (day of blood sample delivery) - Ficoll separation of PBMC - Resting NK cell degranulation assay after K562 stimulation - Resuspend PBMC at /ml in complete medium supplemented with IL-2 (final concentration of 100 IU/ml) and PHA (final concentration of 1.25 μg/ml) and culture in 24 well plate to obtain activated NK and CTL Day +2 Activated NK cell degranulation assay after K562 stimulation CTL degranulation assay after anti-cd3/cd28 stimulation Degranulation assay procedure Specific for NK cell assay: Resuspend the K562 cells at cells/ml in complete medium. In a 96-well V-bottom plate, add 100 µl of K562 cell suspensions or 100 µl of medium per well, as indicated for stimulated or un-stimulated samples, respectively. Specific for CTL assay: Prepare CTL stimulation mix (15 μl dynabeads and 85 μl medium per sample) In a 96-well V-bottom plate, add 100 µl CTL stimulation mix or 100 µl of medium per well, as indicated for stimulated or un-stimulated samples, respectively.

2 Common for NK cell and CTL assay: Adjust the concentration of PBMC or blasts to cells/ml with complete medium. Add 100 µl of PBMC or blasts per well, as indicated. Centrifugate the plate at 30 g for 3 min. Incubate the plate for 3 hours at 37 C, 5% CO 2. For flow cytometry staining, centrifugate the plate at 450 g for 3 min. Flick off the supernatant. Prepare staining mix: Use per sample: 1 µl CD8-FITC 1 µl CD107a-PE 1 µl CD3-PerCP 1 µl CD56-APC 46 µl FACS buffer Resuspend the cells in 50 µl of staining mix. Incubate the cells for 30 min at 4 C protected from light. Centrifuge the cells at 450 g for 3 min, discard the supernatant. Resuspend the cells in 150 µl FACS buffer. Analyze the cells on a flow cytometer. Notes: It may be necessary to titrate fluorochrome-conjugated antibody concentrations for maximal sensitivity of detection. Tubes with single fluorochrome stainings for compensation settings are necessary (at least with cells from the healthy control). To analyze expression of CD107a on NK cells upon co-culture with K562 cells, gate on CD3 - CD56 + lymphocytes. To analyze expression of CD107a on CTL, gate on CD3 + CD8 + lymphocytes. Data analysis: FlowJo data file analysis (NK cells): 1) On a FSC/SSC plot, create a lymphocyte gate; 2) On a lymphocyte plot, gate on CD3 CD56 + NK cells; 3) On a histogram of unstimulated NK cells from a healthy control, set a CD107a + cell gate which renders between 0.5% and 1.0% positive cells. FlowJo data file analysis (CTL): 1) On a FSC/SSC plot, create a lymphocyte gate; 2) On a lymphocyte plot, gate on CD3 + CD8 + CTL; 3) Create a histogram overlay with stimulated and unstimulated CTL and determine the MFI difference of CD107 between the two samples. NK cell data are expressed as ΔCD107a = difference between the % of cells expressing surface CD107a following K562 stimulation and the % of cells expressing surface CD107a after incubation with medium alone. CTL data are expressed as the MFI difference of CD107 between the two samples. For further analysis details on analysis and troubleshooting, see a more detailed methods paper by Bryceson et al. 1

3 PROTOCOL 2 This protocol is performed on PBMC incubated overnight either in medium alone or supplemented with IL-2 to allow the analysis, at the same time, of the degranulation of resting and activated NK cells, respectively. For degranulation of CTL, the same procedure described in protocol 1 is followed using d3-d7 PHA-blasts. Timetable Day 0 (day of blood sample delivery) - Ficoll separation of PBMC - Resuspend PBMC at 2x10 6 /ml either in complete medium alone or supplemented with IL-2 (600 IU/ml) and culture in 96-well U-bottom plate (1-2x10 6 cells in each condition) Day +1 - Resting and activated NK cell degranulation assay after K562 stimulation Procedure Resuspend the K562 cells at cells/ml in complete medium. In a 96-well V-bottom plate, add 100 µl of K562 cell suspensions or 100 µl of medium per well, as indicated for stimulated or un-stimulated samples, respectively. Collect PBMC cultured overnight in medium alone or supplemented with IL-2 and adjust the concentration to cells/ml with complete medium. Add 100 µl of PBMC suspension per well, as indicated. Add 5 μl of anti-cd107a-pe to each well. Centrifugate the plate at 30 g for 3 min. Incubate the plate for 2 hours at 37 C, 5% CO 2. For flow cytometry staining, harvest cells into FACS tubes, wash with FACS buffer. Prepare the staining mix, per sample: 1 μl of anti-cd107a-pe 4 μl of anti-cd3-percp 4 μl of anti-cd56-apc 41 μl of FACS buffer Resuspend the cells in 50 μl of staining mix. Incubate the cells for 30 min at 4 C protected from light. Add FACS buffer and centrifuge the cells at 450 g for 3 min, discard the supernatant. Resuspend the cells in 200 µl FACS buffer containing formaldehyde. Analyze the cells on a flow cytometer. Notes and Data analysis: see PROTOCOL 1

4 Intracellular staining for perforin For each staining, PBMC were surface stained with anti-cd3-percp, anti-cd16-fitc and anti-cd56-apc in FACS buffer. For intracellular staining, cells were fixed and permeabilized with cytofix/cytoperm solution (BD Bioscience) according to the manufacturer s instructions and stained intracellularly with anti-perforin-pe (δg9, IgG 2b ), a mouse IgG 2b -PE isotype control (27-35) as a negative control and anti-cd107a-pe (H4A3, IgG 1 ) as a positive control for the intracellular staining (all BD Bioscience). Data were acquired on a FACS Calibur (Beckton Dickinson) or a Navios (Beckman Coulter) flow cytometer and analyzed using FlowJo or Cellquest software (Treestar and BD Bioscience, respectively). Intracellular staining for SAP and XIAP PBMC were surface stained with anti-cd3-percp and anti-cd56-apc. Intracellular staining was performed as described above using purified anti-xiap IgG1 (BD Bioscience), and purified anti-sap IgG 2a (Clone 1C9, Abnova) in the same staining followed by anti-igg 1 PE and anti IgG 2a FITC (BD Bioscience). Mouse IgG 2a and mouse IgG 1 (BD Bioscience) were used as isotype controls. Resting NK cell cytotoxicity assay To a 96-well round-bottom plate, 150 μl PBMC at cells/ml in complete medium were plated in duplicate. By transferring 50 μl from this first well, five 3-fold dilutions of PBMC were made in complete medium, with a final volume of 100 μl per well. K562 target cells were pelleted and incubated for 1 h at 37 C in 100 μl 51 Cr-solution (Perkin Elmer, cat.no. NEZ030S002MC; 1mCi/ml in saline solution). To account for a loss of activity of the 51 Crsolution, 130 μl were used in the second week after delivery, 160 μl in the third week and 200 μl in the fourth week. Thereafter, cells were washed three times and resuspended in complete medium at cells/ml. To each well, 100 μl of 51 Cr-labelled K562 target cells were added. The same amount of target cells were added to 3 wells filled with 100 μl medium to assess spontaneous release and to 3 wells filled with 100 μl 2N HCl to assess maximum release. The plates were centrifuged for 3 min at 300 g, and thereafter incubated for 4 hours at 37 C. Thereafter, 70 μl of the supernatant was transferred to plastic tube and counted in a γ-counter. Specific lysis was calculated as follows: 100% (value sample value min ) / (value max value min ), where value sample was the value of lysis for each individual sample, value min was the spontaneous release, and value max was the maximal release. Activated T cell cytotoxicity assay PHA blasts were stimulated as described in the methods section of this report, initially with PBMC plated for PHA/IL-2 stimulation, harvested between d3 and d5, and purified by Ficoll density centrifugation. After washing twice, PHA blasts were replated into a 24- well flat-bottom plate in 1 ml of complete medium supplemented with 100 IU/ml IL-2. Between d7 and d9 after setting up the initial culture, the cells were harvested and washed. L1210 or P815 target (both ATCC) cells were pelleted and incubated for 1 h at 37 C in 100 μl 51 Cr-solution as described above. The effector PHA/IL-2 blasts were plated in duplicates into a 96-well V-bottom plate and diluted by 3-fold titration for a final concentration of effector cells in a volume of 100 μl in the first well. Thus, a total of PHA/IL-2 blasts were used for the assay. The cells were added 50 μl anti-cd3 antibody (clone UCHT1; BD Pharmingen) diluted 1:20 in complete medium and incubated at 37 C for 15 minutes. Thereafter, to each well, 50 μl of 51 Cr labelled L1210 or P815 target cells were added at a concentration of cells/ml. The same amount of target cells were added to 3 wells filled with 100 μl medium to assess spontaneous release and to 3 wells filled with 100 μl 2N HCl. After 4 hours of incubation, 70 μl of the supernatant was transferred to plastic tube and counted in a γ-counter. Specific lysis was calculated as above.

5 References 1. Bryceson YT, Fauriat C, Nunes JM, et al. Functional analysis of human NK cells by flow cytometry. Method Mol Biol. 2010;612:

6 Supplemental tables Table S1: Overview of patients analyzed at the 4 centers contibuting to this study. ST = Stockholm, FR = Freiburg, G = Genoa, L = London. Group ST FR G L All FHL3-5, CHS, GS FHL2, XLP Secondary HLH Unclassified Table S2: Comparative analysis of patients with secondary HLH displaying abnormal and normal results for resting and/or activated NK cell degranulation. Parameter Abnormal degranulation Normal degranulation Age (patients <2y) 6/14 20/52 Therapy 7/14 immunosuppression 25/52 immunosuppression (at the time of analysis) 2/14 HLH /52 HLH /14 no treatment 24/52 no treatment Suspected trigger 8/14 infection 36/52 infection 4/14 rheumatic disease 13/52 rheumatic disease 2/14 malignancy 3/52 malignancy

7 Supplemental figure legends Figure S1: Comparison of NK cell degranulation elicited by K562 cells from different centers. (A, B) Degranulation of (A) resting and (B) activated NK cells from two healthy control donors analyzed with protocol 1 and stimulated with K562 cells derived from different centers, as indicated.

8 Fig. S1 A resting NK cells B activated NK cells Δ CD107% Δ CD107% 70 Freiburg 60 Genoa 50 Stockholm Freiburg Genoa Stockholm 0 0 Co1 Co2 Co1 Co2