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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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HIV-1 transfer correlates directly with ps20 expression levels in primary CD4+ T cell clones. (A) Mean percentage of Gag+ dye-labelled ps20low, ps20inter and ps20high primary target CD4+ clones 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. Mean relative copy number of ps20 mRNA of each clone is given along the x-axis. (B) Correlation coefficient comparing the relative expression of ps20 in Clones 1,3,4,6,7,8 with their corresponding level of HIV-1 transfer 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. (C) Clone 7 (ps20high) was used as the target population and seeded at 1 × 105 cells in the presence or the absence of 5 μg/ml of T-20 or 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 40% 2044-infected donor cells at T:D ratio of 1:0.2. The percentage of Gag+ target cells 48 hours post co-culture is shown. All data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using an unpaired t-test (Figure A), a two-tailed non-parametric Spearman's r correlation (Figure B) or paired t-test (Figure C). *P ≤0.05; **P ≤0.01; ***P ≤0.001; ****P ≤0.001.
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Figure 3: HIV-1 transfer correlates directly with ps20 expression levels in primary CD4+ T cell clones. (A) Mean percentage of Gag+ dye-labelled ps20low, ps20inter and ps20high primary target CD4+ clones 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. Mean relative copy number of ps20 mRNA of each clone is given along the x-axis. (B) Correlation coefficient comparing the relative expression of ps20 in Clones 1,3,4,6,7,8 with their corresponding level of HIV-1 transfer 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. (C) Clone 7 (ps20high) was used as the target population and seeded at 1 × 105 cells in the presence or the absence of 5 μg/ml of T-20 or 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 40% 2044-infected donor cells at T:D ratio of 1:0.2. The percentage of Gag+ target cells 48 hours post co-culture is shown. All data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using an unpaired t-test (Figure A), a two-tailed non-parametric Spearman's r correlation (Figure B) or paired t-test (Figure C). *P ≤0.05; **P ≤0.01; ***P ≤0.001; ****P ≤0.001.

Mentions: A panel of six CD4+ T cell clones from multiple donors (Additional file 1 figure S1D) were examined. Clones 1 (ps20low) and 6 (ps20inter) were gut-derived isogenic clones. Clones 3 (ps20inter), 7 (ps20high), 4 (ps20inter) and 8 (ps20high) were all blood-derived, with clones 3 and 7 being isogenic (Figure 3A). Cells were co-cultured for 4 hours with Jwt-ps20inter donor cells that were 60% productively infected with the X4-HIV-1 strain, 2044 and in each case, ps20high clones had a higher frequency of Gag+ cells as compared to the ps20inter or ps20low counterparts. Differences between these clone pairs were as follows: 1.6-fold between clone 2 and clone 6, 16-fold between clone 3 and clone 7 and 5-fold between clone 4 and clone 8 (Figure 3A). Furthermore, comparison of all the ps20 low and intermediate clones (C1, C3, C4, C6) versus the ps20 high clones (C7, C8) highlighted statistically higher virus infection of the ps20 high clones (Mann-Whitney p = 0.0009) (Figure 3A). Indeed, a significant positive correlation was noted between HIV-1 transfer and ps20 mRNA expression in these clones (Two-tailed non-parametric Spearman's correlation, p < 0.0001, Figure 3B).


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)

HIV-1 transfer correlates directly with ps20 expression levels in primary CD4+ T cell clones. (A) Mean percentage of Gag+ dye-labelled ps20low, ps20inter and ps20high primary target CD4+ clones 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. Mean relative copy number of ps20 mRNA of each clone is given along the x-axis. (B) Correlation coefficient comparing the relative expression of ps20 in Clones 1,3,4,6,7,8 with their corresponding level of HIV-1 transfer 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. (C) Clone 7 (ps20high) was used as the target population and seeded at 1 × 105 cells in the presence or the absence of 5 μg/ml of T-20 or 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 40% 2044-infected donor cells at T:D ratio of 1:0.2. The percentage of Gag+ target cells 48 hours post co-culture is shown. All data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using an unpaired t-test (Figure A), a two-tailed non-parametric Spearman's r correlation (Figure B) or paired t-test (Figure C). *P ≤0.05; **P ≤0.01; ***P ≤0.001; ****P ≤0.001.
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Figure 3: HIV-1 transfer correlates directly with ps20 expression levels in primary CD4+ T cell clones. (A) Mean percentage of Gag+ dye-labelled ps20low, ps20inter and ps20high primary target CD4+ clones 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. Mean relative copy number of ps20 mRNA of each clone is given along the x-axis. (B) Correlation coefficient comparing the relative expression of ps20 in Clones 1,3,4,6,7,8 with their corresponding level of HIV-1 transfer 4 hours post co-culture with 40% Jwt ps20inter 2044-infected donor cells at a T:D ratio of 1:0.2. (C) Clone 7 (ps20high) was used as the target population and seeded at 1 × 105 cells in the presence or the absence of 5 μg/ml of T-20 or 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 40% 2044-infected donor cells at T:D ratio of 1:0.2. The percentage of Gag+ target cells 48 hours post co-culture is shown. All data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using an unpaired t-test (Figure A), a two-tailed non-parametric Spearman's r correlation (Figure B) or paired t-test (Figure C). *P ≤0.05; **P ≤0.01; ***P ≤0.001; ****P ≤0.001.
Mentions: A panel of six CD4+ T cell clones from multiple donors (Additional file 1 figure S1D) were examined. Clones 1 (ps20low) and 6 (ps20inter) were gut-derived isogenic clones. Clones 3 (ps20inter), 7 (ps20high), 4 (ps20inter) and 8 (ps20high) were all blood-derived, with clones 3 and 7 being isogenic (Figure 3A). Cells were co-cultured for 4 hours with Jwt-ps20inter donor cells that were 60% productively infected with the X4-HIV-1 strain, 2044 and in each case, ps20high clones had a higher frequency of Gag+ cells as compared to the ps20inter or ps20low counterparts. Differences between these clone pairs were as follows: 1.6-fold between clone 2 and clone 6, 16-fold between clone 3 and clone 7 and 5-fold between clone 4 and clone 8 (Figure 3A). Furthermore, comparison of all the ps20 low and intermediate clones (C1, C3, C4, C6) versus the ps20 high clones (C7, C8) highlighted statistically higher virus infection of the ps20 high clones (Mann-Whitney p = 0.0009) (Figure 3A). Indeed, a significant positive correlation was noted between HIV-1 transfer and ps20 mRNA expression in these clones (Two-tailed non-parametric Spearman's correlation, p < 0.0001, Figure 3B).

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