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Plasmid pNL4-3 BaL+ containing the backbone of NL4-3 and the Env of BaL (referred to as BaL), TZMbl cell line and antibody Act-1 were from NIH AIDS Research & Reference Reagent Program, Division of AIDS, NIH (Germantown, MD, USA). QT6, a fibrosarcoma cell line derived from Japanese quail, was from American Tissue Culture Collection (ATCC, Cambridge, MA, USA). Integrin expressing plasmids including human pα4, pαE, pβ7 and pβ1, mouse pα4 and pβ7, rat pα4 and pβ7 were amplified from cDNA subtracted from PBMCs of corresponding species using primers listed in Table 1 (Table 1) and inserted into the expression vector pcDNA3.1 (+), respectively. Antibody 2B4 and mouse anti-human IgG were from R & D Systems (Minneapolis, MN, USA). HP2/1 was from AbD Serotec (Oxford, UK). RPA-T4, anti-CD3-PE, anti-CD4-FITC, anti-CD8-FITC, anti-CCR5-PE, anti-human β7-PE, anti-mouse β1-PE, anti-rat β7-PE, anti-mouse IgG-APC and matched isotype control antibodies were from BD Pharmingen (San Jose, CA, USA). Leu3A, anti-human β7-APC and control IgG-APC were from Biolegend (San Diego, CA, USA). Reagents for ELISA assay were from Beckman Coulter (Brea, CA, USA), with a limit of sensitivity of 30 pg/mL.
Name Sequence (5' to 3') Usage α4 forward GAACTAGCTAGCGCATGGCTTGGGAAGCGAGa PCR amplification of human α4 from cDNA α4 reverse GCTCCTGCCTCGAGTCAATTTGAAAGAAGTCCTTAATC β7 forward GAAACACGAATTCTTGGGATCTCGGGCATGGTGG PCR amplification of human β7 from cDNA β7 reverse CCTATTCTAGAGGGTAAGTGTCCCTCCCTCCTTCAGA αE forward GAACTAGCTAGCGCTCCAGCAAGGATGTGGCTCTTC PCR amplification of human αE from cDNA αE reverse TTATGCCTCGAGTCTCCCAGTGGATAGCAGGTCC β1 forward AACGGAATTCAAGATGAATTTACAACCAATTTTCTG PCR amplification of human β1 from cDNA β1 reverse TAGCTCGTCTAGAAGTACTCATTTTCCCTCATACTTC m. α4 forward GACCTAGCTAGCTGTTGAATGTTCTCCACCAAGAGCG PCR amplification of mouse α4 from cDNA m. α4 reverse TATGCCTCGAGGTCTTCAGTCATCATTGCTTTTGCT m. β7 forward AATATGAATTCTGCTCCTCCTCAAGCACCTGCCATG PCR amplification of mouse β7 from cDNA m. β7 reverse CACCTGGTCTAGAACTGTCCTCCAAGACAAGAATCCTAAGTC r. α4forward GACCTAGCTAGCTGTTGAATGTTCCCCACCAAGAGTG PCR amplification of rat α4from cDNA r. α4reverse TTACCTCGAGAGTCTTCAGTCATCATTGCTTTTGCTGT r. β7 forward ACCTAGCTAGCGCCATGGTGGATTCATCAACTGTTC PCR amplification of rat β7 from cDNA r. β7 reverse TTACCTCGAGCTAAGTCAGTCAGCCTCCTGGGTCAG iα4 #1 forward TGCTCCGTGTTATCAAGATTATTTCAAGAGAATAATCTTGATAACACGGAGCTTTTTTG shRNA targeting α4 iα4 #1 reverse TCGACAAAAAAGCTCCGTGTTATCAAGATTATTCTCTTGAAATAATCTTGATAACACGGAGCA iα4 #2 forward TCGGGAGCAGTAATGAATGCAATTCAAGAGATTGCATTCATTACTGCTCCCGTTTTTTG shRNA targeting α4 iα4 #2 reverse TCGACAAAAAACGGGAGCAGTAATGAATGCAATCTCTTGAATTGCATTCATTACTGCTCCCGA iβ1 forward TGCCTTGCATTACTGCTGATATTTCAAGAGAATATCAGCAGTAATGCAAGGCTTTTTTC shRNA targeting β1 iβ1 reverse TCGAGAAAAAAGCCTTGCATTACTGCTGATATTTCAAGAGAATATCAGCAGTAATGCAAGGCA iCCR5 forward TGAGCATGACTGACATCTACTTCAAGAGAGTAGATGTCAGTCATGCTCTTTTTTG shRNA targeting CCR5 iCCR5 reverse TCGACAAAAAAGAGCATGACTGACATCTACTCTCTTGAAGTAGATGTCAGTCATGCTCA scrambled forward TCCTAAGGTTAAGTCGCCCTTTCAAGAGAAGGGCGACTTAACCTTAGGTTTTTTG non-targeting shRNA scrambled reverse TCGACAAAAAACCTAAGGTTAAGTCGCCCTTCTCTTGAAAGGGCGACTTAACCTTAGGA The underlined positions are ezymatic restriction sites, sense or anti-sense sequences of the oligos as implicated. Table 1. Primers and shRNA oligos
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The complete coding sequences of α4 and β7 were amplified from constructed pα4 and pβ7 and subcloned into the lentiviral vectors pLJM1 (Addgene, Cambridge, MA, USA, with a substitution of CMV-EGFP into CMV-2aRFP) and pLenti6.3/V5-DEST (Invitrogen, Grand Island, NY, USA, with an insertion of a MCS-IRES2-EGFP sequence after the CMV promoter), respectively. The constructed pLJM1-CMV-α4-2a-RFP or pLenti6.3-CMV-β7-IRES2-EGFP/V5-DEST vector was co-transfected with psPAX2 and pMG2.G (Addgene, Cambridge, MA, USA) into 293T cells to produce lentiviruses which were subsequently used to infect CHO cells at optimal multiplicity of infection. Cells with high and stable α4β7 expression were selected by culture in the presence of antibiotics puromycin and blasticidine, and further purified by limited dilution (Wurm F M, 2004).
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All human blood samples were collected under protocols approved by the Local Research Ethics Committee. PBMCs were isolated from single buffy coats, and stimulated with 20 U/mL interluekin-2 (R & D Systems, Minneapolis, MN, USA)and 1 μg/mL phytohaemagglutinin (Sigma-Aldrich, St. Louis, MO, USA) for 3 days. For α4β7 activation, 10 nmol/L retinoic acid (RA, SigmaAldrich, St. Louis, MO, USA) was added to the medium at the beginning of the 3-day culture, followed by an additional 4-day culture in the presence of IL-2 and RA. CD4+ T cells were prepared from PBMCs by negative selection and cultured as PBMCs (Miltenyi, Bergisch Gladbach, NRW, Germany). CD8+ T cells with high or low/negative α4β7 expression were sorted from activated PBMCs by gating on the CD8+ lymphocytes with corresponding β7 expression.
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For cell transfectants and CHO-α4β7 cell lines, cells were detached by trypsin and recovered at 37 ℃ for 2 hours. For CD8+ T cells, after sorting by flow cytometry, cells were cultured at 37 ℃ for 2 days prior to binding assay. For each condition, 1 × 106 cell-line cells or 5 × 105 primary cells were incubated with 100 ng p24 of virus at 37 ℃ for 2 hours (for transfectants) or 1 hour (for primary cells) with end-to-end rotation. Inhibitors were present as indicated and pre-incubated with the cells for 1 hour (for transfectants) or 30 min (for primary cells). The incubation medium was supplemented DMEM with the addition of 1 mmol/L MnCl2. After incubation, the cells were washed 3 times using HEPES buffer supplemented with 1 mmol/L MnCl2 and 100 μmol/L CaCl2 to remove unbound virus, lysed using 1% Triton X-100, then subjected to p24 antigen quantification.
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Short-hairpin RNA interference was carried out as described previously (Qin X F, et al., 2003). In brief, shRNA oligo sequences targeting human integrin α4were downloaded from the RNAi Consortium(http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/ProjTRC.shtml), analyzed for suitability and potential efficacy according to general guidelines for RNAi design (Birmingham A, et al., 2007; Qin X F, et al., 2003; Tiscornia G, et al., 2003), synthesized, annealed and inserted into the lentiviral vector pLentiLox3.7 (referred to as pLL3.7) (Addgene, Cambridge, MA, USA). Scrambled shRNA and shRNA sequence targeting CCR5 were from Addgene or synthesized as described (Qin X F, et al., 2003) (Table 1), and subsequently inserted into the lentiviral vector pLL3.7. The constructed pLL3.7-shRNA or empty vector were co-transfected with psPAX2 and pMG2.G into 293T cells to produce lentivirus which were then titrated and used to transduce RA-treated CD4+ T cells. 4–6 days post-transduction, the positively transduced cells (GFP+) were analyzed for gene expression by FCM and sorted for downstream virus infection.
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Pseudotyped reporter viruses were prepared as described (Hu Q, et al., 2005). In brief, 293T cells were co-transfected with plasmids expressing HIV-1 Env or VSV-G and pNL4–3.Luc. R E using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. Infectious HIV-1 was produced by transfecting plasmid BaL into 293T cells using Lipofectamine 2000 according to the manufacturer's instructions. PBMC-originated viruses were produced by infection of PBMCs with 293T-derived viruses. All viral stocks were titrated by p24 ELISA.
For virus infection assays, 2 × 105 PBMCs or CD4+ T cells were infected with 2 ng p24 of virus followed by extensive washes to remove unbound virus. Cells were cultured in supplemented RPMI-1640 for 12 days. Supernatants were collected every 3 days post-infection, lysed and then stored at -80 ℃ until detection for p24 antigen.
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For expression assays, 5 × 106 cells were used for each condition. In brief, cells were harvested, counted and washed once using PBS with 3% FBS. For integrin transfectants and α4β7-expressing cell lines, cells were washed using HEPES buffer supplemented with 1 mmol/L MnCl2 and 100 μmol/L CaCl2. Cells were stained with indicated antibody for 30 minutes on ice, followed by staining with secondary antibody for another 30 minutes in some cases. The stained cells were washed twice, fixed with 500 μL of 1% paraformaldehyde in PBS and analyzed on a FACSAria Ⅲ (BD, San Jose, CA, USA) cytometer.
For cell sorting, 5 × 107 cells were used for each sample. Cells were stained as described above except that cells were kept in ice-cold washing buffer and immediately sorted after staining.
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Data are presented as mean ± SD. The difference of mean value was analyzed by the two-tailed student's t test.p < 0.05 was considered statistically significant.