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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.

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ps20high CD4+ T cells form a higher frequency of conjugates, multiple conjugates and VS with HIV-1 infected donor cells. 1 × 105 DDAO vital dye labelled ps20inter (Figure 5A) and ps20high (Figure 5B) target cells were co-cultured with 60% 1 × 105 2044-infected donor cells for 1 hour on Poly-L-lysine coated glass cover slips. Cells were then fixed and stained with a PE anti-Gag (red) and a FITC anti-CD4 (green) Abs. The frequency of target cells that were involved in a single conjugate was defined as the percentage of dye-labelled target cells apposed to an HIV-1-infected donor cell. Representative high power fields captured at 63× magnification are shown, which depict single conjugates for between (A) J-ps20inter or (B) J-ps20high cells and HIV-infected donor cells. Left panels depict conjugates with no HIV-Gag CD4 polarization to conjugate interface. Right panels depict conjugates with HIV-Gag and CD4 polarization (yellow) to conjugate interfaces. (C) Picture shows a representative high power field of dye labelled targets (white arrows) involved in multiple conjugates, defined as a target cell apposed to two or more HIV-1-infected donor cells. (D) Picture shows a representative high power field of a polysynapse, defined as a cell with two or more virological synapses (yellow) at conjugate interfaces. (E) Picture shows a representative high power field of remote contacts (RC) (filopodial bridges or nanotubes) that connect uninfected target cells to HIV-infected targets. (F) Mean frequency of J-ps20inter vs. J-ps20high cells involved in single conjugate, multiple conjugates, or which contain virological synapses, polysynapses or are in contact through remote contacts are shown. A total of 600 random target cells were assessed across quadruplicate experiments. (G) Qualitative analysis of the junction diameter of a conjugates was measured using LEICA TCS SP2 software, where the diameter was measured across the medial section of a conjugate. Graph depicts the mean conjugate interface diameter (μM) between J-ps20inter vs. J-ps20high cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across quadruplicate experiments. Asterisk denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05).
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Figure 5: ps20high CD4+ T cells form a higher frequency of conjugates, multiple conjugates and VS with HIV-1 infected donor cells. 1 × 105 DDAO vital dye labelled ps20inter (Figure 5A) and ps20high (Figure 5B) target cells were co-cultured with 60% 1 × 105 2044-infected donor cells for 1 hour on Poly-L-lysine coated glass cover slips. Cells were then fixed and stained with a PE anti-Gag (red) and a FITC anti-CD4 (green) Abs. The frequency of target cells that were involved in a single conjugate was defined as the percentage of dye-labelled target cells apposed to an HIV-1-infected donor cell. Representative high power fields captured at 63× magnification are shown, which depict single conjugates for between (A) J-ps20inter or (B) J-ps20high cells and HIV-infected donor cells. Left panels depict conjugates with no HIV-Gag CD4 polarization to conjugate interface. Right panels depict conjugates with HIV-Gag and CD4 polarization (yellow) to conjugate interfaces. (C) Picture shows a representative high power field of dye labelled targets (white arrows) involved in multiple conjugates, defined as a target cell apposed to two or more HIV-1-infected donor cells. (D) Picture shows a representative high power field of a polysynapse, defined as a cell with two or more virological synapses (yellow) at conjugate interfaces. (E) Picture shows a representative high power field of remote contacts (RC) (filopodial bridges or nanotubes) that connect uninfected target cells to HIV-infected targets. (F) Mean frequency of J-ps20inter vs. J-ps20high cells involved in single conjugate, multiple conjugates, or which contain virological synapses, polysynapses or are in contact through remote contacts are shown. A total of 600 random target cells were assessed across quadruplicate experiments. (G) Qualitative analysis of the junction diameter of a conjugates was measured using LEICA TCS SP2 software, where the diameter was measured across the medial section of a conjugate. Graph depicts the mean conjugate interface diameter (μM) between J-ps20inter vs. J-ps20high cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across quadruplicate experiments. Asterisk denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05).

Mentions: The quality and quantity of cell-cell conjugates formed in the presence of ps20 were next assessed. To avoid inherent differences in primary clonal populations, these studies were performed using the Jurkat model system. First the number of conjugates formed was assessed. Conjugates were defined as a target cell closely apposed to an infected donor cell, and multiple conjugates (MCs) as a target cell closely apposed to two or more infected donor cells (Figures 5A, B, C). We observed a significant 2.3-fold and 5.4-fold higher frequency of conjugate and MC formation and a 2.75-fold higher frequency of Gag and CD4 polarization to conjugate interfaces (VS formation - see Figure 5D) in J-ps20high vs. J-ps20inter populations respectively (Figure 5F). However, the proportion of conjugates containing VS was similar, 14.6% vs. 17.3% in J-ps20inter vs. J-ps20high conjugates, in keeping with the notion that the number of VS formed is determined by the number of conjugates. Previously, it has been shown postulated that the formation of multiple virological synapses (termed polysynapses-PS) in conjugates of uninfected targets and HIV-infected donors is an efficient mode of virus dissemination [12]. We, therefore, enumerated conjugates (target or donor) containing two or more synapses simultaneously (Figure 5D). A marked 28-fold higher frequency of PS in co-cultures of J-ps20high vs. J-ps20inter cells was noted (Figure 5F). However, the frequency of remote contacts (filopodial bridges and nanotubes) formed between uninfected target and infected donor cells did not differ between J-ps20high vs. J-ps20inter cells (Figure 5F). Interestingly, ps20high cells were observed to be more closely apposed to infected donor cells compared to ps20inter cells (Figure 5A vs. 5B). To quantify this observation, the medial diameter of conjugate interfaces was measured and found to be significantly larger in conjugates with ps20high targets. J-ps20high vs. J-ps20inter conjugates had a mean diameter of 7.46 uM (± 0.41) vs. 4.25 uM (± 0.23) respectively (Figure 5G). Together, these data highlight ps20 to impact the fundamental biologic process of cell-cell conjugation.


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)

ps20high CD4+ T cells form a higher frequency of conjugates, multiple conjugates and VS with HIV-1 infected donor cells. 1 × 105 DDAO vital dye labelled ps20inter (Figure 5A) and ps20high (Figure 5B) target cells were co-cultured with 60% 1 × 105 2044-infected donor cells for 1 hour on Poly-L-lysine coated glass cover slips. Cells were then fixed and stained with a PE anti-Gag (red) and a FITC anti-CD4 (green) Abs. The frequency of target cells that were involved in a single conjugate was defined as the percentage of dye-labelled target cells apposed to an HIV-1-infected donor cell. Representative high power fields captured at 63× magnification are shown, which depict single conjugates for between (A) J-ps20inter or (B) J-ps20high cells and HIV-infected donor cells. Left panels depict conjugates with no HIV-Gag CD4 polarization to conjugate interface. Right panels depict conjugates with HIV-Gag and CD4 polarization (yellow) to conjugate interfaces. (C) Picture shows a representative high power field of dye labelled targets (white arrows) involved in multiple conjugates, defined as a target cell apposed to two or more HIV-1-infected donor cells. (D) Picture shows a representative high power field of a polysynapse, defined as a cell with two or more virological synapses (yellow) at conjugate interfaces. (E) Picture shows a representative high power field of remote contacts (RC) (filopodial bridges or nanotubes) that connect uninfected target cells to HIV-infected targets. (F) Mean frequency of J-ps20inter vs. J-ps20high cells involved in single conjugate, multiple conjugates, or which contain virological synapses, polysynapses or are in contact through remote contacts are shown. A total of 600 random target cells were assessed across quadruplicate experiments. (G) Qualitative analysis of the junction diameter of a conjugates was measured using LEICA TCS SP2 software, where the diameter was measured across the medial section of a conjugate. Graph depicts the mean conjugate interface diameter (μM) between J-ps20inter vs. J-ps20high cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across quadruplicate experiments. Asterisk denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05).
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Figure 5: ps20high CD4+ T cells form a higher frequency of conjugates, multiple conjugates and VS with HIV-1 infected donor cells. 1 × 105 DDAO vital dye labelled ps20inter (Figure 5A) and ps20high (Figure 5B) target cells were co-cultured with 60% 1 × 105 2044-infected donor cells for 1 hour on Poly-L-lysine coated glass cover slips. Cells were then fixed and stained with a PE anti-Gag (red) and a FITC anti-CD4 (green) Abs. The frequency of target cells that were involved in a single conjugate was defined as the percentage of dye-labelled target cells apposed to an HIV-1-infected donor cell. Representative high power fields captured at 63× magnification are shown, which depict single conjugates for between (A) J-ps20inter or (B) J-ps20high cells and HIV-infected donor cells. Left panels depict conjugates with no HIV-Gag CD4 polarization to conjugate interface. Right panels depict conjugates with HIV-Gag and CD4 polarization (yellow) to conjugate interfaces. (C) Picture shows a representative high power field of dye labelled targets (white arrows) involved in multiple conjugates, defined as a target cell apposed to two or more HIV-1-infected donor cells. (D) Picture shows a representative high power field of a polysynapse, defined as a cell with two or more virological synapses (yellow) at conjugate interfaces. (E) Picture shows a representative high power field of remote contacts (RC) (filopodial bridges or nanotubes) that connect uninfected target cells to HIV-infected targets. (F) Mean frequency of J-ps20inter vs. J-ps20high cells involved in single conjugate, multiple conjugates, or which contain virological synapses, polysynapses or are in contact through remote contacts are shown. A total of 600 random target cells were assessed across quadruplicate experiments. (G) Qualitative analysis of the junction diameter of a conjugates was measured using LEICA TCS SP2 software, where the diameter was measured across the medial section of a conjugate. Graph depicts the mean conjugate interface diameter (μM) between J-ps20inter vs. J-ps20high cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across quadruplicate experiments. Asterisk denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05).
Mentions: The quality and quantity of cell-cell conjugates formed in the presence of ps20 were next assessed. To avoid inherent differences in primary clonal populations, these studies were performed using the Jurkat model system. First the number of conjugates formed was assessed. Conjugates were defined as a target cell closely apposed to an infected donor cell, and multiple conjugates (MCs) as a target cell closely apposed to two or more infected donor cells (Figures 5A, B, C). We observed a significant 2.3-fold and 5.4-fold higher frequency of conjugate and MC formation and a 2.75-fold higher frequency of Gag and CD4 polarization to conjugate interfaces (VS formation - see Figure 5D) in J-ps20high vs. J-ps20inter populations respectively (Figure 5F). However, the proportion of conjugates containing VS was similar, 14.6% vs. 17.3% in J-ps20inter vs. J-ps20high conjugates, in keeping with the notion that the number of VS formed is determined by the number of conjugates. Previously, it has been shown postulated that the formation of multiple virological synapses (termed polysynapses-PS) in conjugates of uninfected targets and HIV-infected donors is an efficient mode of virus dissemination [12]. We, therefore, enumerated conjugates (target or donor) containing two or more synapses simultaneously (Figure 5D). A marked 28-fold higher frequency of PS in co-cultures of J-ps20high vs. J-ps20inter cells was noted (Figure 5F). However, the frequency of remote contacts (filopodial bridges and nanotubes) formed between uninfected target and infected donor cells did not differ between J-ps20high vs. J-ps20inter cells (Figure 5F). Interestingly, ps20high cells were observed to be more closely apposed to infected donor cells compared to ps20inter cells (Figure 5A vs. 5B). To quantify this observation, the medial diameter of conjugate interfaces was measured and found to be significantly larger in conjugates with ps20high targets. J-ps20high vs. J-ps20inter conjugates had a mean diameter of 7.46 uM (± 0.41) vs. 4.25 uM (± 0.23) respectively (Figure 5G). Together, these data highlight ps20 to impact the fundamental biologic process of cell-cell conjugation.

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