Transduction of lentivirus to human primary CD4+ T cells

Transcription

1 Transduction of lentivirus to human primary CD4 + T cells Human primary CD4 T cells were stimulated with anti-cd3/cd28 antibodies (10 µl/2 5 10^6 cells of Dynabeads CD3/CD28 T cell expander, Invitrogen) for 16 hours, followed by IL-2 (10ng/ml) for 5 hours in RPMI + 10% FCS. Lentivirus expressing GFP + c-myb-1, and c-myb-3, or scrambled sequence control shrna were freshly prepared in 293FT cells (Invitrogen). The Lentivirus were added to the stimulated CD4 + T cells (CD4 + T cells 5 10^6/ml:viral supernatant = 1:1.5 vol/vol, Polybrene 8 µg/ml). Five days post-transduction, GFP positive cells were sorted by the FACSAria cell sorter (BD Biosciences). Transduction efficiency ranged from % of live cells (Fig. S1). The GFP positive cells were further sorted using magnetic beads conjugated with anti-cd45ro + antibody (CD45 MicroBeads, Mileni Biotec). Alternatively, GFP + CD4 + T cells were sorted with CD45RA-PE antibody using the FACSAria cell sorter. The cells selected negatively with CD45RO, or positively CD45RA positive cells, were classified as naïve CD4 + T cells. ChIP assay for histone modifications of locus in Jurkat cells and human primary CD4 + T cells. Jurkat cells were infected with Lentivirus expressing GFP + shrna targeted to c-myb, or a scrambled (SCR) shrna sequence control. GFP positive cells were sorted and used to establish stable cell lines expressing c-myb or control shrna. The efficiency of silencing was ~60% in c-myb shrna expressing Jurkat cells. The shrna expressing Jurkat cells were then transfected with c-myb1 and c-myb3 sirna using Lonza s transfection system (Lonza). SCR control shrna expressing Jurkat cells were transfected with control sirna. This method further decreases protein expression to ~10 20% of that found in wild type Jurkat cells (Fig. 4C). At 48 hr post transfection, a cross-link reaction for the ChIP assay was performed. Chromatin captured by each antibody was then amplified by QRT-PCR (Fig. 7A) using the locus primer pairs shown in Table S2. In experiments employing primary human CD4 + T cells, we utilized the shrna lentivirus silencing system described above. The sorted GFP + CD45RA + and GFP + CD45RA cells were stimulated under Th2 promoting conditions for 7 and 4 days respectively, after which time cross-linking reactions were carried out. QRT-PCR analysis was performed with primer sets #1 to #15 as shown in Table S2. Magna ChIP TM G kit (Millipore) was employed for ChIP assays. For each experiment, CD4 + T cells were used.

2 Table S1. Fold-up or -down change induced by silencing in CD4 + CD45RO + cells PCR Array (Human Th1-Th2-Th3 RT² Profiler PCR Array, SABiosciences) was performed with human primary CD4 + CD45RO + cells transduced with c-myb-1 and 3, or scrambled shrna expression vectors. Fold changed was calculated with reference to results obtained with the scrambled shrna. CCL CCL CCL CCR CCR CCR CCR CD CD CD40LG 1.38 CD CD CD CEBPB 1.82 CREBBP 1.20 CSF CTLA CXCR FASLG 1.69 GATA GFI GLMN 1.12 GPR HAVCR ICOS 1.28 IFNG 2.08 IGSF IL IL12B 1.10 IL12RB IL13 IL13RA IL IL IL18R IL1R IL1R IL IL2RA 6.77 IL IL4R 7.26 IL IL IL6R 2.71 IL IL INHA 1.56 INHBA 3.16 IRF IRF JAK JAK LAG LAT 2.20 MAF 1.58 MAP2K MAPK NFATC NFATC NFATC2IP 2.75 PCGF PTPRC 1.04 SFTPD 1.82 SOCS1 SOCS SOCS SPP STAT STAT STAT TBX TFCP TGFB TLR TLR TMED TNF 1.47 TNFRSF TNFRSF TNFRSF TNFSF TYK YY B2M 1.36 HPRT RPL13A 1.67 GAPDH 1.58 ACTB 1.95

3 Table S2. Primer pairs employed for ChIP assays described in Fig. 7 #1 Forward: 5 -TCTGTACCCGACTGGGTTTC-3 Reverse: 5 -CGGGGTATGTGTGTCCTCTT-3 #2 Forward: 5 -TTTAGAGAGCGCTGTGAGCA-3 Reverse: 5 -GTTTCTCCTGAGCCCACTTG-3 #3 Forward: 5 -GCTCCAGTCAAAGGCATCTC-3 Reverse: 5 -TAGGCACGCATGGATCATTA-3 #4 Forward: 5 -TTGGGTTGCAGTTTCCTTGT-3 Reverse: 5 -CGACGCAACTTAAGGAGGTT-3 #5 Forward: 5 -GAGAGAGACGGAGGGAGAGC-3 Reverse: 5 -CTGGGTAGCGAAGAGCAGAG-3 #6 Forward: 5 -CAACCACTTGGGAGGACCTA-3 Reverse: 5 -AGTCCCAGGGCTGACTGTTA-3 #7 Forward: 5 -TTCTGCCGTACCCAGTTTTT-3 Reverse: 5 -AAGCAAAGGTGAGCAAAGGA-3 #8 Forward: 5 -TCACACCCTTCTCCTTGACC-3 Reverse: 5 -GGCGACTTGAAAGCAAAGAC-3 #9 Forward: 5 -CTTCAGGCCTCCTCACTCAC-3 Reverse: 5 -TTGGCCTCTCCAGATACACC-3 #10 Forward: 5 -CTGCTTCATGGATCCCTACC-3 Reverse: 5 -GATGGACGTCTTGGAGAAGG-3 #11 Forward: 5 -TGCCATTCTTCCCATTCTTC-3 Reverse: 5 -TTCCTATCCCAGCTCATTGG-3 #12 Forward: 5 -AGGCTAGCTGGTGGAGTCAA-3 Reverse: 5 -TGCACCCCACTTCTTAATCC-3 #13 Forward: 5 -GAAACGGTTGAAGTCGTGGT-3 Reverse: 5 -AGGTGGCAGTGGAGCTAAGA-3 #14 Forward: 5 -AAGGCAGGGAGTGTGTGAAC-3 Reverse: 5 -CTTCGCTTGGGCTTAATGAG-3 #15 Forward: 5 -GTGGCAACATTCCTTGGTCT-3 Reverse: 5 -TTGGTCACTTCCCATTCACA-3 #16 Forward: 5 -CTGCAGCCAGGAGAGCAG-3 Reverse: 5 -AGTAGAGCCCACAGGCATTG-3 #17 Forward: 5 -GCTGAACAGGGAGGAGACAG-3 Reverse: 5 -GAAAAAGAAGCAGGGTGCTG-3 #18 Forward: 5 -TTCGACTTGCATTTTTGCAG-3 Reverse: 5 -ACAGATGGGGTCCAGATTCA-3 #19 Forward: 5 -CTGGCCACAGTTGTTTGATG-3 Reverse: 5 -GCTCTCTGAAACCCTCAACG-3 #20 Forward: 5 -TGCCTGCCTTGGATATTTTC-3 Reverse: 5 -CTCTGTCCAGGCAGATCACA-3

4 Figure S1. Typical flow analysis of shrna transduced cells The live cells were 46.8 % of all events, and GFP + cells were 41.3% of live cells (19.0% of all cells). β-actin shrna SCR shrna Figure S2. protein suppression by shrna Lentivirus system in the naive T cells under Th2 promoting conditions Normal human peripheral blood CD4 + T cells were transduced with lentivirus constructs expressing GFP + c-myb shrna. Sorted GFP positive CD4 + CD45RO cells were cultured under Th2 promoting conditions for 5 days. Western blotting was carried out with anti antibody. SCR= scrambled shrn.

5 IL-4 APC RBP-J? NF-kB? STAT6 Menin Pol II promoter Naive CD4+ T Developing Th2 Effector Th2 MLL? Menin Histone modification promoter Memory Th2 Figure S3 Model for the regulation of the expression during Th2 cell development. IL-4/STAT6 signaling initiates expression leading to the development of Th2 cells. Subsequently, activates its own promoter by forming an auto-regulatory complex with and Menin that markedly increases expression. During the process of generating memory Th2 cell, the / complex recruits MLL. These events sustain expression, and are permissive of cytokine production as a result of hypothesized changes in histone modifications at the locus (Memory Th2). Transcriptionally active proteins identified to regulate expression are shown clustered on the promoter (thick blue line). The pink filled circles with dashed lines denote the presence of as yet unidentified proteins that likely play a role as co-transcriptional regulators.