Programmed differentiated natural killer cells kill leukemia cells by engaging SLAM family receptors SUPPLEMENTARY MATERIALS Samples

Keywords: CD34, CD3, CD56, CD48, CD226, NKp30, NKp44, NKp46, CD94, NKG2C, NKG2D, CD11a, CD62L, CD25, CD122, CD244, CXCR3, CCR6, IFN-γ, TNF-α, perforin, granzyme B, and CD107

Decidual samples were obtained from normal donors with elective pregnancy terminations at the Maternity and Child Care Hospital, Hefei. Human PB samples were obtained at Hefei Blood Bank. BM samples were obtained from leukemia patients at the time of diagnosis and prior to therapy at Anhui Provincial Hospital. 

Cell lines

A549 (human lung carcinoma), H460 (large cell lung cancer), SGC7901 (human gastric adenocarcinoma), HeLa (cervical cancer), HO8910 (human ovarian carcinoma), HCT116 (epithelial colon colorectal carcinoma), K562 (erythroleukemia), HL60 (acute promyelocytic leukemia), Raji (Burkitt lymphoma), Jurkat (acute T cell leukemia) and THP-1 (acute monocytic leukemia) cells were purchased from the Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China). Karpas-299 (T-cell lymphoma) cells were purchased from DSMZ (Germany). IM9 (multiple myeloma), U937 (histiocytic lymphoma) and NK92 (NK cell line) cells were purchased from the American Type Culture Collection (USA). YT (NK-like cell line) cells were obtained from the NCI (USA). NKL (NK cell line) cells were kindly provided by Professor BQ Jin (Fourth Military Medical University, Xi 'an, PR China). NKG (NK cell line) cells were established in our lab (Department of Immunology, University of Science and Technology of China, Hefei). 

RNA isolation and quantitative real-time PCR

Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo (dT) 20 primers. Then, quantitative real-time PCR was performed with SYBR Premix Ex Taq (TaKaRa) with primers for the relevant target transcripts. The mRNA level of β-actin was used as a normalization control. The data were analyzed with the 2 -ΔΔCt method. 
The gene-specific primers are shown in Supplementary Table 2. 

CFSE-based cytotoxicity assay

Briefly, target cells were labeled with 5 μM CFSE (Sigma) for 15 min at 37°C. After being labeled, the cells were resuspended in RPMI 1640 medium (Hyclone). Effector cells and target cells were added to 96-well plates at different effector-target ratios for 6 hours at 37°C and 5% CO 2 . For the detection of dead cells, 7-AAD (BD Pharmingen) was added, and the samples were directly analyzed by flow cytometry. 

Supplementary Table 2: The gene-specific primers


Gene

Sequence (5'-3') Gene Sequence (5'-3') 
CEBPA-F TATAGGCTGGGCTTCCCCTT IFNG-F TGTCGCCAGCAGCTAAAACA CEBPA-R AGCTTTCTGGTGTGACTCGG IFNG-R TGCAGGCAGGACAACCATTA ZBTB16-F TGGGGTCGAGCTTCCTGATA GZMA-F ATCTGTGCTGGGGCTTTGAT ZBTB16-R GGCCATGTCAGTGCCAGTAT GZMA-R TGCTTTTTCCGTCAGCTGTAA PBX1-F AACTCGGCTGGTTCTTCCAG GZMB-F CCCTGGGAAAACACTCACACA PBX1-R TCCCCATTGAGTGACTGCAC GZMB-R CACAACTCAATGGTACTGTCGT ERG-F TTCACTTGGTCGGAATGGGG PRF1-F CGCCTACCTCAGGCTTATCTC ERG-R CTGACGGCTTTAGTTGCCCT PRF1-R CCTCGACAGTCAGGCAGTC ZNF69-F GCTGTAGTCACAGGAGGTGT CSF2-F AATGTTTGACCTCCAGGAGCC ZNF69-R GAGACTCCTGAAGTTTCTCCTGG CSF2-R CCCTTGGTCCCTCCAAGATG SOX4-F CAGCAAACCAACAATGCCGA IL32-F CGTGTGACACTGAGGACACT SOX4-R GATCTGCGACCACACCATGA IL32-R CGACATCACCTGTCCACGTC BHLHE40-F GAAGCTGCATCTCAAAGCCG OSM-F AGTGGGGACATGGACTGGAA BHLHE40-R GGGTACATCCCTGGTAGGTCT OSM-R TACGTATATAGGGGTCCAGGAGTC MAF-F AGCAAGTCGACCACCTCAAG FASL-F CCTTGGTAGGATTGGGCCTG MAF-R CTGGAATCGCGTGTCAGACT FASL-R TCTGGCTGGTAGACTCTCGG KLF2-F CACACAGGTGAGAAGCCCTACC LTB-F GGAGCCACTTCTCTGGTGAC KLF2-R TCGTCAAGGAGGATCGTGGT LTB-R TCTGAAACCCAGTCCTCCCT EGR2-F GGCTTTTCTGACACTCCAGGTA NTBA-F TTTGGCCCAGGACAACTGAGG EGR2-R TCCAACGACCTCTTCTCTCCA NTBA-R TGCAGGTGTAGTCCTGTTCAC MYBL-F AAAGCGAATTCCATCACAGCC CRACC-F CTGGTCGGTTCCGTTGG MYBL-R TGGGATGGTCTGCAAAGAGG CRACC-R ACTTTAGGCTTTGACAGGTGC HOPX-F GAGGAGACCCAGGGTAGTGAT CD48-F ACTGCTGCCTCTGTCACTCC HOPX-R TCTGTGACGGATCTGCACTC CD48-R TCTTGCTCATTCCCAGTCTTT ETS1-F CGATCTGGAGCTTTTCCCC CD244-F GGGAGAATGGCTCTTTGCCT ETS1-R TGGAGTTAATAGTGGGACATCTGC CD244-R CGGGGTTTCTCAACTTTATCAAATA ID2-F TGAAAGCCTTCAGTCCCGTG SAP-F AAGTACACAAGGTACTACAGGGAT ID2-R AGGGAATTCAGAAGCCTGCAA SAP-R AAGGGACACCAGCCAACTTC TBX21-F CACGTCCACAAACATCCTGT EAT-2-F GACAGCGAGTCGATACCAGG TBX21-R GATCATCACCAAGCAGGGAC EAT-2-R CCTTCTGCAGTCTGTATCCTGT TOX-F AACCGGTGGACTGGAATAAC SHP-1-F TTCGGATCCAGAACTCAGG TOX-R TGGAGAACTGCCTTGACTGT SHP-1-R TGCAAACTCTCAAACTCCTC TOX2-F CACCTGGACTATTACCACGGC SHP-2-F GGAGCTGTCACCCACATCAA TOX2-R TTGTAGGTCTGGCTGGTTGAC SHP-2-R CCGTCATTGCTCTCCCCTTT EOMES-F CTGGCTTCCGTGCCCACGTC SHIP-1-F AAGTGGCCACAACTCTCCTG EOMES-R CATGCGCCTGCCCTGTTTCG SHIP-1-R CGCAATTCCGATACAGCACG ACTIN-F TTGCCGACAGGATGCAGAA ACTIN-R GCCGATCCACACGGAGTACTT F, Forward. R, Reverse.



  




  


Supplementary Table 1: Clinical characteristics of leukemia patients

See Supplementary File 1
Programmed differentiated natural killer cells kill leukemia cells by engaging SLAM family receptors SUPPLEMENTARY MATERIALS Samples