Kaluza 2

Cells were harvested and dissociated using TrypLE™ Select Enzyme, fixed with 1% PFA for 20 min, permeabilized with 90% methanol for 10 min, and stained using 1:40 mouse monoclonal IgG1 TNNT2 and 1:200 mouse monoclonal IgG2b MLC2A (MYL7) for 1 h at RT. Mouse IgG1 and mouse IgG2b isotype controls were used. Secondary staining was performed with 1:1000 Alexa Fluor 488 goat anti-mouse IgG1 and 1:1000 Alexa Fluor 647 goat anti-mouse IgG2b for 30 min at RT. Cells were analyzed using a Navios (Beckman Coulter) flow cytometer. Data were analyzed using Kaluza 2.2 (Beckman Coulter) software.

For isolation of retrovirally transduced cells carrying the marker gene CD2, APC-labeled anti-murine CD2 (clone RM2-5; Biolegend) was used. Healthy hematopoietic stem cells were isolated based on expression of CD34 using PE-labeled anti-CD34 (8G12; BD) and acute myeloid leukemic cells were isolated based on expression of CD33 using BV421-labeled anti-CD33 (WM53; BD). Cells were washed twice with phosphate-buffered saline containing 2% FCS and incubated with fluorochrome-conjugated monoclonal antibodies for 30 min at room temperature. Data acquisition was performed on a fluorescence-activated cell sorter Canto II and a fluorescence-activated cell sorter BD FACS Aria (BD Biosciences). Forward scatter/side scatter was used for gating on viable cells. FSC-H/-A was used for doublet exclusion. Data were analyzed with Kaluza 2.1. (Beckman Coulter).

Single-cell suspensions from tumors were achieved by enzyme digestion and mechanical dissociation using a commercial kit (Miltenyi Biotec) (22 (link)). Briefly, tumors were removed from mice when indicated and minced into small pieces of 2–4 mm in diameter. Tumor pieces were then digested by enzymes provided in the kit for 40 min after dissociation for 1 min. Following digestion, tissues underwent another round of dissociation. Single cell suspensions were achieved by filtering samples through 70-μm strainers and centrifuged (300× g for 10 min at 4°C).
For cell death assessments, a Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide (PI) (Thermo Fisher Scientific) was used for flow cytometry analyses according to the manufacturer's guides.
Tumor cells were then incubated with Fixable Viability Dye eFluro 780 (1:1000 in PBS, eBioscience/Thermo Fisher Scientific) for 30 min at 4°C. Subsequently, cells were blocked with FC blocker (1:100, eBiosience) for 10 min at room temperature. Samples then were incubated with antibodies with designated titrations (listed in Table 1). Cells were applied to an Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed by software Kaluza 2.1 (Beckman Coulter, Brea, CA, USA).

Lymphocytes and monocytes were gated using side scatter (SSC) vs CD45 dot plots after exclusion of diploids and debris. Normal T cells are gated on the expression of CD3, T cells are defined as CD45+CD3+ events, and can be divided into CD4+ T cells and CD8+ T cells by CD4 and CD8. NK cells were gated on the expression of CD56 and the absence of CD3, and NK cells were defined as CD45+CD3-CD56+ events. For each group of cases (AITL, PTCL-NOS, ENKTL-N, ALCL, and T-CUS), the expression pattern of each marker of the clonal T cells studied were assessed. Using normal T cells and NK cells as internal controls, the fluorescence intensities of various intracellular and extracellular markers of clonal T cells were compared using a scatter plot. Data were analyzed and interpreted using Kaluza2.1 (Beckman Coulter, Brea, CA, USA) and Flowjo10.6.2 (BD Biosciences, San Jose, CA, USA).

Platelet count and parameters were determined using an automated hematology analyzer (Sysmex, Kobe, Japan). Platelet reactivity was determined in citrated whole blood (3.2% sodium citrate, Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometry based assay as previously described between 1 and 3 h after blood collection^{52 (link)}. Platelets were ex vivo stimulated with ADP (1.2 and 125 μM; Sigma-Aldrich, Zwijndrecht, The Netherlands) and CRP-XL (a kind gift from Prof. Farndale, Cambridge, UK) for 20 min at room temperature. Platelets were stained using anti-CD61 (Beckman Coulter, Brea. CA, USA), anti-P-selectin (Biolegend, San Diego, CA, USA) and anti-fibrinogen (DAKO, Santa Clara, CA) antibodies and fixated in 0.2% paraformaldehyde. Platelets were identified based on Size (FSC), granularity (SSC) and their expression of CD61. Degranulation was determined as the membrane expression of α-granule protein P-selectin and platelet aggregation was quantified as the amount of fibrinogen binding to the activated integrin αIIbβ3. Platelet reactivity was measured on a FC500 flow cytometer (Beckman Coulter, Brea, USA). Data were extracted using Kaluza 2.1 (Beckman Coulter), normalized against quality controls to ensure measurement stability and are expressed as median fluorescence intensity (MFI). A gating strategy is provided in Supplemental Fig. S1.