Journal: Virulence

Article Title: Dendritic cells maturated by co-culturing with HIV-1 latently infected Jurkat T cells or stimulating with AIDS-associated pathogens secrete TNF-α to reactivate HIV-1 from latency

doi: 10.1080/21505594.2017.1356535

Figure Lengend Snippet: HIV-1 reactivation during the co-culture with MDDCs. (A) Scheme for HIV latency reactivation assay. (B, C) MDDCs-triggered HIV-1 reactivation from C11 during co-culture. C11 cells (4 × 105) were co-cultured or not with immature MDDCs or LPS-treated MDDCs (ratio C11:DC, 2:1) for 24 h. C11 cells (CD11c−) were distinguished from MDDCs with CD11c immunostaining (B), and 6 independent repeats were summarized and analyzed (C). (D, E) Separation of C11 from MDDCs abolishes HIV-1 reactivation. A transwell culture plate with a 0.4-μm insert membrane was used or not to separate the C11 from MDDCs (D), and HIV-1 reactivation in C11 cells was detected with flow cytometry and analyzed as above (E). (F,G) HIV-1 reactivation from the resting primary CD4+ T cells. Resting CD4+ T-cells were isolated from cART-treated patients and further treated with LPS or cultured with immature MDDCs or LPS-treated MDDCs or with their related culture medium, respectively, for 3 d. Viral reactivation was detected by semi-quantifying gag mRNA transcripts and normalized with GAPDH mRNA. The relative gag mRNA products were calculated. * P < 0.05, ** P < 0.01 *** P < 0.001, were considered as significant difference in ANOVA analysis.

Article Snippet: To characterize cell phenotype, the following antibodies or isotype-matched IgG were used for flow cytometry (clone numbers and sources were given in parentheses): APC-Alexa Fluor750-CD11c (B-ly6; BD Biosciences PharMingen); phycoerythrin (PE)-CD14 (61D3; eBioscience); PE-CD83 (H1B19; eBioscience); PE-CD86 (IT2.2; eBioscience), APC-cy7-HLA-DR (LN3; eBioscience) and PE-CD40 (KPL-1; eBioscience).

Techniques: Co-Culture Assay, Cell Culture, Immunostaining, Flow Cytometry, Isolation

Journal: Virulence

Article Title: Dendritic cells maturated by co-culturing with HIV-1 latently infected Jurkat T cells or stimulating with AIDS-associated pathogens secrete TNF-α to reactivate HIV-1 from latency

doi: 10.1080/21505594.2017.1356535

Figure Lengend Snippet: Co-cultured C11 cells mature MDDCs and promote TNF-α secretion for reactivating HIV-1. (A) Phenotype characterization of MDDCs. immature MDDCs or LPS-treated MDDCs were co-cultured with or without C11 cells as above, and MDDCs were distinguished with CD11c+ immunostaining and the expressions of CD83, CD86 and HLA-DR were detected with flow cytometry. The percentage of positive cells was labeled. (B) Assay for TNF-α expression. MDDCs were co-cultured with or without C11 cells for 6 h, and C11 cells stimulated with or without TNF-α (20 ng/ml) for 6 h were also prepared. TNF-α expression was detected at mRNA level by qRT-PCR and normalized with β-actin. Data were presented as mean ± standard deviation (SD). (C, D) The addition of anti-TNF-α neutralizing antibodies during C11-MDDCs co-culture abolishes HIV-1 reactivation. 20 μg/ml anti-TNF-α neutralizing antibodies or IgG control were added during C11-MDDCs co-culture for 24 h (C), and viral reactivation was detected with flow cytometry (D). One representative result from at least 3 independent repeats was shown. * P < 0.05 and *** P < 0.001 were considered as significant difference in ANOVA analysis.

Article Snippet: To characterize cell phenotype, the following antibodies or isotype-matched IgG were used for flow cytometry (clone numbers and sources were given in parentheses): APC-Alexa Fluor750-CD11c (B-ly6; BD Biosciences PharMingen); phycoerythrin (PE)-CD14 (61D3; eBioscience); PE-CD83 (H1B19; eBioscience); PE-CD86 (IT2.2; eBioscience), APC-cy7-HLA-DR (LN3; eBioscience) and PE-CD40 (KPL-1; eBioscience).

Techniques: Cell Culture, Immunostaining, Flow Cytometry, Labeling, Expressing, Quantitative RT-PCR, Standard Deviation, Co-Culture Assay

Journal: Virulence

Article Title: Dendritic cells maturated by co-culturing with HIV-1 latently infected Jurkat T cells or stimulating with AIDS-associated pathogens secrete TNF-α to reactivate HIV-1 from latency

doi: 10.1080/21505594.2017.1356535

Figure Lengend Snippet: Co-cultured C11 cells mature iDCs through CD40-CD40L signaling pathway. (A) CD40 expression on MDDCs. Immature MDDCs or LPS-treated MDDCs were co-cultured with or without C11 cells for 24 h, and MDDCs were distinguished with CD11c+ immunostaining and the expression of CD40 was detected with flow cytometry, and the positive percentage was shown. (B) CD40 expression detected at mRNA level. MDDCs were co-cultured with or without C11 cells for 6 h, and C11 cells stimulated with or without TNF-α (20 ng/ml) for 6 h were also prepared. CD40 expression was detected at mRNA level by qRT-PCR and normalized with β-actin. Data were presented as mean ± SD. (C, D, E) CD40 knockdown impairs iMDDCs-purged HIV-1 reactivation from latency. IMDDCs were transfected with specific CD40 siRNAs or off-target control for 48 h, and CD40 expression was detected by Western blotting (upper panel) and flow cytometry (lower panel, MFI for CD40-positive staining was calculated) at protein level (C), and by mRNA quantification at transcription level (D). These transfected MDDCs were further co-cultured with C11 cells for additional 24 h and HIV-1 reactivation was assessed with flow cytometry (E). (F, G, H) CD40L knockdown on C11 diminishes the response to MDDCs stimulation for HIV-1 reactivation. C11 cells were nucleofected with CD40L specific siRNAs or off-target control for 48 h, and CD40L expression was detected by Western blotting at protein level (F) and mRNA quantification at transcription level (G), and these nucleofected C11 cells were co-cultured with immature MDDCs for additional 24 h, and HIV-1 reactivation was assessed with flow cytometry (H). (I, J) The supply with recombinant CD40Ligand protein complements HIV-1 reactivation from C11 cells. Recombinant CD40 ligand protein (1 μg/ml) was supplied during transwell-culture (pore size, 0.4 μm) of C11 cells with iDCs for 24 h, and C11 cells were harvested and GFP expression was detected with flow cytometry (I), and the supernatant was collected for detecting TNF-α with ELISA (J). One representative result from at least 3 independent repeats was shown. ** P < 0.01 and *** P < 0.001 were considered as significant difference in ANOVA analysis.

Article Snippet: To characterize cell phenotype, the following antibodies or isotype-matched IgG were used for flow cytometry (clone numbers and sources were given in parentheses): APC-Alexa Fluor750-CD11c (B-ly6; BD Biosciences PharMingen); phycoerythrin (PE)-CD14 (61D3; eBioscience); PE-CD83 (H1B19; eBioscience); PE-CD86 (IT2.2; eBioscience), APC-cy7-HLA-DR (LN3; eBioscience) and PE-CD40 (KPL-1; eBioscience).

Techniques: Cell Culture, Expressing, Immunostaining, Flow Cytometry, Quantitative RT-PCR, Transfection, Western Blot, Staining, Recombinant, Enzyme-linked Immunosorbent Assay