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Results: 6

1.
Figure 5

Figure 5. From: HIV envelope-mediated, CCR5/?4?7-dependent killing of CD4-negative ?? T cells which are lost during progression to AIDS.

gp120 V2 and V3 loops mediate binding to Vγ2Vδ2 cells. (A-B) Fluorescein-conjugated HIV gp120/YU2 (A) or CN54 gp120 (B) was pre-incubated with specific antibodies against (20 μg/mL) for 30 minutes at room temperature, and then the mixtures were added to Vγ2Vδ2 T cells for staining (A) or cell killing assays (B). Inhibition of binding or cell death was calculated in relation to gp120 killing or binding is relative to gp120 in the absence of added antibodies. Data are representative of 3 independent experiments (error bars, SD).

Haishan Li, et al. Blood. 2011 November 24;118(22):5824-5831.
2.
Figure 6

Figure 6. From: HIV envelope-mediated, CCR5/?4?7-dependent killing of CD4-negative ?? T cells which are lost during progression to AIDS.

gp120-induced Vγ2Vδ2 T cell death depends on p38-caspase pathway. (A) Vγ2Vδ2 T cells were untreated or pretreated with specific caspase inhibitors (2μM) for 1 hour at 37°C before incubated with CN54 gp120 (10 μg/mL) for 24 hours. (B) Vγ2Vδ2 T cells were incubated with or without Bal or CN54 gp120 protein for 10 hours. Cells were then collected for Western blot assay with specific antibodies. (C) Vγ2Vδ2 T cells were untreated or pretreated with specific inhibitors for 1 hour at 37°C before incubating with CN54 gp120 for 24 hours. (D) Vγ2Vδ2 T cells were untreated or pretreated with SB203580 or ZVAD for 1 hour at 37°C before incubated with CN54 gp120 for 10 hours. Cells were collected for Western blot assay with specific antibodies. Inhibition of cell death was calculated in relation to gp120-induced cell death with untreated Vγ2Vδ2 T cells. Data are representative of 3 independent experiments (error bars, SD).

Haishan Li, et al. Blood. 2011 November 24;118(22):5824-5831.
3.
Figure 2

Figure 2. From: HIV envelope-mediated, CCR5/?4?7-dependent killing of CD4-negative ?? T cells which are lost during progression to AIDS.

Very high levels of α4β7 and CCR5 on Vγ2Vδ2 T cells. Vγ2Vδ2 T cells were stained with specific antibodies to CD4 (A), DC-SIGN (B), Mannose receptor (C), α4 (D), β7 (E), or CCR5 (G red lines), or isotype controls (blue lines), and analyzed with flow cytometry. (F) Expression of β7 on Vγ2Vδ2 T cells (blue line), CD4 T cells (red line), and NK cells (green line) was analyzed with flow cytometry. (H) Vγ2Vδ2 T cells and 3 standard HeLa cell lines various in the level of cell-surface CCR5 (JC10, JC57 and JC53) were stained with CCR5-PE and isotype control, and then analyzed with flow cytometry. Data are representative of at least 3 independent experiments.

Haishan Li, et al. Blood. 2011 November 24;118(22):5824-5831.
4.
Figure 4

Figure 4. From: HIV envelope-mediated, CCR5/?4?7-dependent killing of CD4-negative ?? T cells which are lost during progression to AIDS.

α4β7 and CCR5 form complexes on Vγ2Vδ2 T cells. (A) Flow cytometry assay for expression of CCR5 and β7 on Vγ2Vδ2 T cells. (B) Vγ2Vδ2 T cells were stained with β7 mAb (green) and CCR5 mAb (red), and viewed under a confocal microscope. Yellow in the merged panel represents the colocalization of CCR5 and β7. (C) Vγ2Vδ2 T cells were treated or not with the crosslinking reagent DTSSP followed by coprecipitation with protein G magnetic beads and the rat IgG2a+DTSSP (lane 1), β7 mAb FIB27 (lane 2), β7 mAb FIB27 + DTSSP (lane 3). Cell lysates were run as a positive control (lane 4). CCR5 and β7 were detected by Western blot with specific antibodies. Data are representative of 3 independent experiments.

Haishan Li, et al. Blood. 2011 November 24;118(22):5824-5831.
5.
Figure 3

Figure 3. From: HIV envelope-mediated, CCR5/?4?7-dependent killing of CD4-negative ?? T cells which are lost during progression to AIDS.

HIV envelope-induced killing of Vγ2Vδ2 T cells depends on α4β7 and CCR5. (A-B) Vγ2Vδ2 T cells were stained with fluoresceine conjugated HIV gp120 (green) and Vδ2 mAb (red) and viewed under a confocal microscope (A) or analyzed with flow cytometry (B). (C) Vγ2Vδ2 T cells were stained with the fluorescein-conjugated HIV gp120 in the absence or presence of soluble CD4, and then analyzed with flow cytometry. (D) Vγ2Vδ2 T cells were incubated with Maraviroc (1μM), MAdCAM1 (20 μg/mL), or both for 1 hour before staining with HIV gp120. (E) Vγ2Vδ2 T cells were incubated with specific antibodies against CCR5, β7 or α4 (20 μg/mL) for 30 minutes before staining with HIV gp120. (F) Cell death was evaluated after Vγ2Vδ2 T cells were incubated with soluble HIV gp120 protein (CN54, 10 μg/mL) in the absence or presence of blocking reagents for α4β7 and CCR5. (G-J) Vγ2Vδ2 T cells were cultured in the absence or presence of retinoic acid. The expression of β7 (G) and CCR5 (H), the binding of gp120 (I), and gp120-induced cell death (J) were examined as previously described. Data are representative of 3 independent experiments (error bars, SD).

Haishan Li, et al. Blood. 2011 November 24;118(22):5824-5831.
6.
Figure 1

Figure 1. From: HIV envelope-mediated, CCR5/?4?7-dependent killing of CD4-negative ?? T cells which are lost during progression to AIDS.

HIV R5-tropic envelope glycoprotein induces Vγ2Vδ2 T cell death. (A) Vγ2Vδ2 T cells were expanded from healthy donor PBMC after stimulating with isopentenyl pyrophosphate (IPP, 15μM) plus IL-2 (100U/mL) for 14 days, and then purified by negative selection to ≥ 97% purity. (B-D) Cell death was evaluated after Vγ2Vδ2 T cells were incubated with soluble HIV gp120 proteins (BaL, CN54 or IIIB; 10 μg/mL) (A), HeLa cells or HeLa cells expressing HIV ADA-envelope (B), 293 cells transfected with empty vector or 293 cells expressing R5-tropic envelopes (BaL, SF162, JRFL, ADA, or YU2) (C) for 24 hours as described in “Methods.” Data are representative of 3 independent experiments (error bars, SD). The statistical significance compared with control was analyzed (*P < .05, **P < .005, ***P < .0001; Student t test). (E) Vγ2Vδ2 T cells were treated with HeLa cells or HeLa cells expressing HIV ADA-envelope for 24 hours and then stimulated with the TCR ligand IPP (15μM) for 4 hours. Cells were stained with annexin V and 7AAD. Data are representative of 3 independent experiments.

Haishan Li, et al. Blood. 2011 November 24;118(22):5824-5831.

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