Limits...
WFDC1 expression identifies memory CD4 T-lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions.

Alvarez RA, Thorborn G, Reading JL, Reddy SK, Vyakarnam A - Retrovirology (2011)

Bottom Line: Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer.Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, King's College London, UK.

ABSTRACT

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes.

Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.

Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

Show MeSH

Related in: MedlinePlus

Jurkat CD4+ T cells stably transduced to express full-length human ps20 are rendered more susceptible to T-T HIV-1 transfer. (A) Representative dot plots of dye labelled target cells co-cultured with uninfected (transfer control) or infected donor cells at 4 and 24 hours post co-culture. (B) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with 36% NL4-3 Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (C) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 24 hours post co-culture with 36% NL4-3 infected Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (D) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with YU2 infected Jwt-ps20inter donor cells at T:D ratio of 1:0.2. Data represent mean of three replicate assays. (E) Target cells co-cultured with 40% YU2 infected donor cells were sorted for dye-positive single cells based on both FSC height vs. width followed by SSC height vs. width, on a BD FACS Aria II cell sorter. DNA extracted from these sorted singlet cells was subject to qDNA PCR for HIV-1 LTR. The level of HIV-1 LTR in J-ps20inter vs. J-ps20high target cells is shown relative to β-actin expression and normalized against DNA isolated from 8E5 cells. Asterisks denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05; **P ≤0.01; ***P ≤0.001)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3108927&req=5

Figure 1: Jurkat CD4+ T cells stably transduced to express full-length human ps20 are rendered more susceptible to T-T HIV-1 transfer. (A) Representative dot plots of dye labelled target cells co-cultured with uninfected (transfer control) or infected donor cells at 4 and 24 hours post co-culture. (B) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with 36% NL4-3 Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (C) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 24 hours post co-culture with 36% NL4-3 infected Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (D) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with YU2 infected Jwt-ps20inter donor cells at T:D ratio of 1:0.2. Data represent mean of three replicate assays. (E) Target cells co-cultured with 40% YU2 infected donor cells were sorted for dye-positive single cells based on both FSC height vs. width followed by SSC height vs. width, on a BD FACS Aria II cell sorter. DNA extracted from these sorted singlet cells was subject to qDNA PCR for HIV-1 LTR. The level of HIV-1 LTR in J-ps20inter vs. J-ps20high target cells is shown relative to β-actin expression and normalized against DNA isolated from 8E5 cells. Asterisks denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05; **P ≤0.01; ***P ≤0.001)

Mentions: We next probed the role of ps20 in cell-to-cell HIV transfer using a flow cytometry assay [10,12,15] (see Figure 1A). HIV-infected WT Jurkat cells (Jwt-ps20inter) served as infected donor cells. J-ps20high and empty vector transduced J-ps20inter target cells were co-cultured with donor cells that were 40% Gag+ following infection with NL4-3 virus at 1:1 or 1:0.2 target:donor (T:D) cell ratios and the percentage of Gag+ target cells enumerated at 4 (Figure 1B) and 24 hours (Figure 1C) post co-culture. At both time points and ratios tested, a higher proportion of Gag+ cells were detected in J-ps20high cells. However, a significant 2-fold difference between the J-ps20high vs. J-ps20inter population was only observed at the lower T:D ratio of 1:0.2, similar to our previous study that highlighted ps20-dependency of HIV-1 to be most marked at low virus challenge doses [23].


WFDC1 expression identifies memory CD4 T-lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions.

Alvarez RA, Thorborn G, Reading JL, Reddy SK, Vyakarnam A - Retrovirology (2011)

Jurkat CD4+ T cells stably transduced to express full-length human ps20 are rendered more susceptible to T-T HIV-1 transfer. (A) Representative dot plots of dye labelled target cells co-cultured with uninfected (transfer control) or infected donor cells at 4 and 24 hours post co-culture. (B) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with 36% NL4-3 Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (C) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 24 hours post co-culture with 36% NL4-3 infected Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (D) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with YU2 infected Jwt-ps20inter donor cells at T:D ratio of 1:0.2. Data represent mean of three replicate assays. (E) Target cells co-cultured with 40% YU2 infected donor cells were sorted for dye-positive single cells based on both FSC height vs. width followed by SSC height vs. width, on a BD FACS Aria II cell sorter. DNA extracted from these sorted singlet cells was subject to qDNA PCR for HIV-1 LTR. The level of HIV-1 LTR in J-ps20inter vs. J-ps20high target cells is shown relative to β-actin expression and normalized against DNA isolated from 8E5 cells. Asterisks denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05; **P ≤0.01; ***P ≤0.001)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108927&req=5

Figure 1: Jurkat CD4+ T cells stably transduced to express full-length human ps20 are rendered more susceptible to T-T HIV-1 transfer. (A) Representative dot plots of dye labelled target cells co-cultured with uninfected (transfer control) or infected donor cells at 4 and 24 hours post co-culture. (B) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with 36% NL4-3 Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (C) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 24 hours post co-culture with 36% NL4-3 infected Jwt-ps20inter donor cells at T:D ratio of 1:1 and 1:0.2. Data represent mean of three replicate assays. (D) Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells at 4 hours post co-culture with YU2 infected Jwt-ps20inter donor cells at T:D ratio of 1:0.2. Data represent mean of three replicate assays. (E) Target cells co-cultured with 40% YU2 infected donor cells were sorted for dye-positive single cells based on both FSC height vs. width followed by SSC height vs. width, on a BD FACS Aria II cell sorter. DNA extracted from these sorted singlet cells was subject to qDNA PCR for HIV-1 LTR. The level of HIV-1 LTR in J-ps20inter vs. J-ps20high target cells is shown relative to β-actin expression and normalized against DNA isolated from 8E5 cells. Asterisks denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05; **P ≤0.01; ***P ≤0.001)
Mentions: We next probed the role of ps20 in cell-to-cell HIV transfer using a flow cytometry assay [10,12,15] (see Figure 1A). HIV-infected WT Jurkat cells (Jwt-ps20inter) served as infected donor cells. J-ps20high and empty vector transduced J-ps20inter target cells were co-cultured with donor cells that were 40% Gag+ following infection with NL4-3 virus at 1:1 or 1:0.2 target:donor (T:D) cell ratios and the percentage of Gag+ target cells enumerated at 4 (Figure 1B) and 24 hours (Figure 1C) post co-culture. At both time points and ratios tested, a higher proportion of Gag+ cells were detected in J-ps20high cells. However, a significant 2-fold difference between the J-ps20high vs. J-ps20inter population was only observed at the lower T:D ratio of 1:0.2, similar to our previous study that highlighted ps20-dependency of HIV-1 to be most marked at low virus challenge doses [23].

Bottom Line: Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer.Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, King's College London, UK.

ABSTRACT

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes.

Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.

Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

Show MeSH
Related in: MedlinePlus