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Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy.

Tran TA, de Goër de Herve MG, Hendel-Chavez H, Dembele B, Le Névot E, Abbed K, Pallier C, Goujard C, Gasnault J, Delfraissy JF, Balazuc AM, Taoufik Y - PLoS ONE (2008)

Bottom Line: Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes.The half-life of the Treg reservoir was estimated at 20 months.Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.

View Article: PubMed Central - PubMed

Affiliation: INSERM U802, Université Paris 11, Le Kremlin Bicêtre, France.

ABSTRACT

Background: In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4+CD25+ regulatory T cells (Tregs) are a CD4+ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART.

Methodology/principal findings: We found evidence of infection of resting Tregs (HLADR(-)CD69(-)CD25(hi)FoxP3+CD4+ T cells) purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir.

Conclusions: Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.

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Related in: MedlinePlus

Definition of the resting Treg and non-Treg working cell populations.Fig. 1A: FoxP3 expression was examined by flow cytometry following cell permeabilization of PBMCs isolated from HIV-infected patients on long-term HAART (see Methods). FoxP3, CD25 and CD127 expression was analyzed in the HLADR− CD4+ small lymphocyte gate. In Fig. 1B, mRNA was extracted from resting Tregs (CD25hiHLADR− CD69−CD4+ small size T cells) and non-Tregs (CD25−HLADR−CD4+ small size T cells). Quantitative RT-PCR was then used to quantify FoxP3 and GAPDH cDNA. Results are expressed as relative units: FoxP3/GAPDH cDNA ratios (atograms/femtograms) and correspond to the mean±SEM of values obtained in 3 donors. Fig. 1C: Sorted resting non-Tregs (CD25−HLADR−CD4+small size T cells) were activated with plate-bound anti-CD3 mAb in the absence or presence of resting Tregs (CD25hiHLADR− CD4+ small size T cells) (>99% FoxP3+) at ratios ranging from 1/10 to 1/1. Cell proliferation was measured. Results (mean±SEM, n = 2) were expressed as the percentage inhibition of proliferation compared to control cultures (without resting Tregs). Fig. 1D: 100 000 sorted CD25−HLADR−CD4+ T cells (black columns) or CD25+HLADR−CD4+T cells (white columns) (obtained as described in Methods) were cultured with plate-bound anti-CD3 mAb±soluble anti-CD28 mAb or with PHA+IL-2. Controls were untreated cells. In Fig. 1E, 100 000 freshly purified CD25−HLADR−CD4+ (black columns) or CD25+HLADR−CD4+ (white columns) T cells (see methods) were also co-cultured with 20 000 mature monocyte-derived DCs loaded in the immature state with a mix of recall antigens (Cytomegalovirus CMV, Purified Protein Derivative PPD, Tetanus toxoid TT and HIV p24). Controls were co-cultures of non-antigen-loaded mature DCs and T cells. Proliferation was evaluated on day 3 for anti-CD3±anti-CD28 activation and on days 3 and 7 for co-culture with mature DCs by measuring thymidine incorporation. Results are expressed as the difference between activated wells and control wells. The results are means±SEM for 4 patients.
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pone-0003305-g001: Definition of the resting Treg and non-Treg working cell populations.Fig. 1A: FoxP3 expression was examined by flow cytometry following cell permeabilization of PBMCs isolated from HIV-infected patients on long-term HAART (see Methods). FoxP3, CD25 and CD127 expression was analyzed in the HLADR− CD4+ small lymphocyte gate. In Fig. 1B, mRNA was extracted from resting Tregs (CD25hiHLADR− CD69−CD4+ small size T cells) and non-Tregs (CD25−HLADR−CD4+ small size T cells). Quantitative RT-PCR was then used to quantify FoxP3 and GAPDH cDNA. Results are expressed as relative units: FoxP3/GAPDH cDNA ratios (atograms/femtograms) and correspond to the mean±SEM of values obtained in 3 donors. Fig. 1C: Sorted resting non-Tregs (CD25−HLADR−CD4+small size T cells) were activated with plate-bound anti-CD3 mAb in the absence or presence of resting Tregs (CD25hiHLADR− CD4+ small size T cells) (>99% FoxP3+) at ratios ranging from 1/10 to 1/1. Cell proliferation was measured. Results (mean±SEM, n = 2) were expressed as the percentage inhibition of proliferation compared to control cultures (without resting Tregs). Fig. 1D: 100 000 sorted CD25−HLADR−CD4+ T cells (black columns) or CD25+HLADR−CD4+T cells (white columns) (obtained as described in Methods) were cultured with plate-bound anti-CD3 mAb±soluble anti-CD28 mAb or with PHA+IL-2. Controls were untreated cells. In Fig. 1E, 100 000 freshly purified CD25−HLADR−CD4+ (black columns) or CD25+HLADR−CD4+ (white columns) T cells (see methods) were also co-cultured with 20 000 mature monocyte-derived DCs loaded in the immature state with a mix of recall antigens (Cytomegalovirus CMV, Purified Protein Derivative PPD, Tetanus toxoid TT and HIV p24). Controls were co-cultures of non-antigen-loaded mature DCs and T cells. Proliferation was evaluated on day 3 for anti-CD3±anti-CD28 activation and on days 3 and 7 for co-culture with mature DCs by measuring thymidine incorporation. Results are expressed as the difference between activated wells and control wells. The results are means±SEM for 4 patients.

Mentions: Highly purified CD25hiHLADR−CD4+ small size T cells were obtained by cell sorting. For each patient tested, the CD25hi sorting gate in HLADR−CD4+ small size lymphocytes was pre-defined on the basis of intracellular Foxp3 expression (>99% Foxp3+) (see Fig. 1A and methods). Expression of FoxP3 was confirmed by RT-PCR analysis (Fig. 1B) Expression of the activation markers CD30, intracellular CD40 ligand and CD69 in selected cells ranged from less than 0.1% to 0.6%. Cells were mainly CD127low (Fig. 1A) as previously reported [24], [25] and suppressed conventional CD4 T-cell proliferation following polyclonal activation with anti-CD3 (Fig. 1C). Tregs also showed hyporesponsiveness following polyclonal activation or specific activation with recall antigens presented by mature dendritic cells (Fig. 1D and 1E). In subsequent virological studies, in addition to the markers described above, quiescent Tregs cells were also sorted on the basis of negative CD69 expression.


Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy.

Tran TA, de Goër de Herve MG, Hendel-Chavez H, Dembele B, Le Névot E, Abbed K, Pallier C, Goujard C, Gasnault J, Delfraissy JF, Balazuc AM, Taoufik Y - PLoS ONE (2008)

Definition of the resting Treg and non-Treg working cell populations.Fig. 1A: FoxP3 expression was examined by flow cytometry following cell permeabilization of PBMCs isolated from HIV-infected patients on long-term HAART (see Methods). FoxP3, CD25 and CD127 expression was analyzed in the HLADR− CD4+ small lymphocyte gate. In Fig. 1B, mRNA was extracted from resting Tregs (CD25hiHLADR− CD69−CD4+ small size T cells) and non-Tregs (CD25−HLADR−CD4+ small size T cells). Quantitative RT-PCR was then used to quantify FoxP3 and GAPDH cDNA. Results are expressed as relative units: FoxP3/GAPDH cDNA ratios (atograms/femtograms) and correspond to the mean±SEM of values obtained in 3 donors. Fig. 1C: Sorted resting non-Tregs (CD25−HLADR−CD4+small size T cells) were activated with plate-bound anti-CD3 mAb in the absence or presence of resting Tregs (CD25hiHLADR− CD4+ small size T cells) (>99% FoxP3+) at ratios ranging from 1/10 to 1/1. Cell proliferation was measured. Results (mean±SEM, n = 2) were expressed as the percentage inhibition of proliferation compared to control cultures (without resting Tregs). Fig. 1D: 100 000 sorted CD25−HLADR−CD4+ T cells (black columns) or CD25+HLADR−CD4+T cells (white columns) (obtained as described in Methods) were cultured with plate-bound anti-CD3 mAb±soluble anti-CD28 mAb or with PHA+IL-2. Controls were untreated cells. In Fig. 1E, 100 000 freshly purified CD25−HLADR−CD4+ (black columns) or CD25+HLADR−CD4+ (white columns) T cells (see methods) were also co-cultured with 20 000 mature monocyte-derived DCs loaded in the immature state with a mix of recall antigens (Cytomegalovirus CMV, Purified Protein Derivative PPD, Tetanus toxoid TT and HIV p24). Controls were co-cultures of non-antigen-loaded mature DCs and T cells. Proliferation was evaluated on day 3 for anti-CD3±anti-CD28 activation and on days 3 and 7 for co-culture with mature DCs by measuring thymidine incorporation. Results are expressed as the difference between activated wells and control wells. The results are means±SEM for 4 patients.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2551739&req=5

pone-0003305-g001: Definition of the resting Treg and non-Treg working cell populations.Fig. 1A: FoxP3 expression was examined by flow cytometry following cell permeabilization of PBMCs isolated from HIV-infected patients on long-term HAART (see Methods). FoxP3, CD25 and CD127 expression was analyzed in the HLADR− CD4+ small lymphocyte gate. In Fig. 1B, mRNA was extracted from resting Tregs (CD25hiHLADR− CD69−CD4+ small size T cells) and non-Tregs (CD25−HLADR−CD4+ small size T cells). Quantitative RT-PCR was then used to quantify FoxP3 and GAPDH cDNA. Results are expressed as relative units: FoxP3/GAPDH cDNA ratios (atograms/femtograms) and correspond to the mean±SEM of values obtained in 3 donors. Fig. 1C: Sorted resting non-Tregs (CD25−HLADR−CD4+small size T cells) were activated with plate-bound anti-CD3 mAb in the absence or presence of resting Tregs (CD25hiHLADR− CD4+ small size T cells) (>99% FoxP3+) at ratios ranging from 1/10 to 1/1. Cell proliferation was measured. Results (mean±SEM, n = 2) were expressed as the percentage inhibition of proliferation compared to control cultures (without resting Tregs). Fig. 1D: 100 000 sorted CD25−HLADR−CD4+ T cells (black columns) or CD25+HLADR−CD4+T cells (white columns) (obtained as described in Methods) were cultured with plate-bound anti-CD3 mAb±soluble anti-CD28 mAb or with PHA+IL-2. Controls were untreated cells. In Fig. 1E, 100 000 freshly purified CD25−HLADR−CD4+ (black columns) or CD25+HLADR−CD4+ (white columns) T cells (see methods) were also co-cultured with 20 000 mature monocyte-derived DCs loaded in the immature state with a mix of recall antigens (Cytomegalovirus CMV, Purified Protein Derivative PPD, Tetanus toxoid TT and HIV p24). Controls were co-cultures of non-antigen-loaded mature DCs and T cells. Proliferation was evaluated on day 3 for anti-CD3±anti-CD28 activation and on days 3 and 7 for co-culture with mature DCs by measuring thymidine incorporation. Results are expressed as the difference between activated wells and control wells. The results are means±SEM for 4 patients.
Mentions: Highly purified CD25hiHLADR−CD4+ small size T cells were obtained by cell sorting. For each patient tested, the CD25hi sorting gate in HLADR−CD4+ small size lymphocytes was pre-defined on the basis of intracellular Foxp3 expression (>99% Foxp3+) (see Fig. 1A and methods). Expression of FoxP3 was confirmed by RT-PCR analysis (Fig. 1B) Expression of the activation markers CD30, intracellular CD40 ligand and CD69 in selected cells ranged from less than 0.1% to 0.6%. Cells were mainly CD127low (Fig. 1A) as previously reported [24], [25] and suppressed conventional CD4 T-cell proliferation following polyclonal activation with anti-CD3 (Fig. 1C). Tregs also showed hyporesponsiveness following polyclonal activation or specific activation with recall antigens presented by mature dendritic cells (Fig. 1D and 1E). In subsequent virological studies, in addition to the markers described above, quiescent Tregs cells were also sorted on the basis of negative CD69 expression.

Bottom Line: Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes.The half-life of the Treg reservoir was estimated at 20 months.Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.

View Article: PubMed Central - PubMed

Affiliation: INSERM U802, Université Paris 11, Le Kremlin Bicêtre, France.

ABSTRACT

Background: In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4+CD25+ regulatory T cells (Tregs) are a CD4+ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART.

Methodology/principal findings: We found evidence of infection of resting Tregs (HLADR(-)CD69(-)CD25(hi)FoxP3+CD4+ T cells) purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir.

Conclusions: Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.

Show MeSH
Related in: MedlinePlus