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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

Quiescent Tregs are sensitive to specific CD8+ T cell cytotoxicity.Fig. 4A: CD25+ cell-depleted PBMCs were activated with PMA-inonomycin then extensively washed. Untreated HLADR−CD25+CD4+ T cells were then added at a physiological ratio. Cells were assayed by ELISPOT for granzyme B. Results correspond to the mean±SEM of results obtained in 2 patients. In Fig. 4B, HIV-specific CTL were co-cultured with CD25+HLADR−CD4+ and CD25−HLADR−CD4+ T cells loaded with HIV peptides. Controls were co-cultures of CD4+ T-cell subsets without peptides. Apoptosis was analyzed following annexin V staining on CD25hiHLADR−CD4+ and CD25−HLADR−CD4+ T cells. Results are expressed as the difference in the percentage of annexin V-positive cells in the presence and absence of HIV peptides. Results correspond to the mean±SEM of results obtained for 7 patients. Statistical comparison involved the Wilcoxon Signed Rank Test.
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pone-0003305-g004: Quiescent Tregs are sensitive to specific CD8+ T cell cytotoxicity.Fig. 4A: CD25+ cell-depleted PBMCs were activated with PMA-inonomycin then extensively washed. Untreated HLADR−CD25+CD4+ T cells were then added at a physiological ratio. Cells were assayed by ELISPOT for granzyme B. Results correspond to the mean±SEM of results obtained in 2 patients. In Fig. 4B, HIV-specific CTL were co-cultured with CD25+HLADR−CD4+ and CD25−HLADR−CD4+ T cells loaded with HIV peptides. Controls were co-cultures of CD4+ T-cell subsets without peptides. Apoptosis was analyzed following annexin V staining on CD25hiHLADR−CD4+ and CD25−HLADR−CD4+ T cells. Results are expressed as the difference in the percentage of annexin V-positive cells in the presence and absence of HIV peptides. Results correspond to the mean±SEM of results obtained for 7 patients. Statistical comparison involved the Wilcoxon Signed Rank Test.

Mentions: Approaches based on cell activation have been proposed to diminish or eliminate the HIV lymphocyte reservoir in patients on prolonged HAART. However, in addition to the hyporesponsiveness of Tregs, which may limit cell activation and virus expression, activated Tregs exert suppressive effects on both CD4+ and CD8+ T cells [20], [21], [30], [31]. Granzyme B is directly involved in CTL-mediated killing, and we found that CD25+CD4+ T-cell depletion strongly increased granzyme B release by CD8+ T cells following nonspecific activation with PMA+ionomycin (Fig. 3A). Depletion of CD25+CD4+ T cells also significantly increased granzyme B secretion in response to 15-mer overlapping HIV peptide pools corresponding to the RT, P24, and Nef gene products (Fig. 3B). These peptides could have activated specific Tregs, which then exerted their suppressive effects. Alternatively, this suppressive effect may be related to the existence, among untreated peripheral blood mononuclear cells (PBMCs), of already activated Tregs that could be continuously stimulated through interaction with self peptides. This latter possibility may explain the high proportion of HLADR+ cells among Tregs shown by ex vivo flow cytometry analysis (the median percentage of HLADR+ cells among CD25hiFoxP3+ CD4+ T cells was 51%, quartiles [46–59%], n = 15 patients on HAART for at least 2 years) (not shown). Activated Tregs might therefore create an environment in which CD8+ T cells are unable to fully exert their cytotoxic functions. In contrast, resting Tregs (HLADR− Tregs without exogenous activation) have no significant inhibitory effects on granzyme B secretion by activated CD8+ T cells (Fig. 4A). Histone deacetylase inhibitors could lead to virus expression without full cell activation [29] (see above). This could be an approach of potential interest for reducing the resting Treg reservoir. We next examined whether resting Tregs but expressing HIV peptides were susceptible to CD8+ T-cell cytolysis. Owing to the low frequency of infected cells and specific CD8+ T cells, we used the following strategy. CD4+-depleted PBMCs were first screened for reactivity to several pools of overlapping peptides corresponding to the Gag, Pol, and Nef sequences (see Methods). The most reactive overlapping peptide pool was then used to amplify specific CTL for testing against peptide-loaded autologous resting Tregs and resting non-Tregs (see methods). As shown in Fig. 4B, similar levels of apoptosis were found in the Treg and non-Treg subsets. This suggested that resting Tregs expressing HIV peptides may be as susceptible as non-Tregs to CD8+ T-cell cytotoxicity.


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)

Quiescent Tregs are sensitive to specific CD8+ T cell cytotoxicity.Fig. 4A: CD25+ cell-depleted PBMCs were activated with PMA-inonomycin then extensively washed. Untreated HLADR−CD25+CD4+ T cells were then added at a physiological ratio. Cells were assayed by ELISPOT for granzyme B. Results correspond to the mean±SEM of results obtained in 2 patients. In Fig. 4B, HIV-specific CTL were co-cultured with CD25+HLADR−CD4+ and CD25−HLADR−CD4+ T cells loaded with HIV peptides. Controls were co-cultures of CD4+ T-cell subsets without peptides. Apoptosis was analyzed following annexin V staining on CD25hiHLADR−CD4+ and CD25−HLADR−CD4+ T cells. Results are expressed as the difference in the percentage of annexin V-positive cells in the presence and absence of HIV peptides. Results correspond to the mean±SEM of results obtained for 7 patients. Statistical comparison involved the Wilcoxon Signed Rank Test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2551739&req=5

pone-0003305-g004: Quiescent Tregs are sensitive to specific CD8+ T cell cytotoxicity.Fig. 4A: CD25+ cell-depleted PBMCs were activated with PMA-inonomycin then extensively washed. Untreated HLADR−CD25+CD4+ T cells were then added at a physiological ratio. Cells were assayed by ELISPOT for granzyme B. Results correspond to the mean±SEM of results obtained in 2 patients. In Fig. 4B, HIV-specific CTL were co-cultured with CD25+HLADR−CD4+ and CD25−HLADR−CD4+ T cells loaded with HIV peptides. Controls were co-cultures of CD4+ T-cell subsets without peptides. Apoptosis was analyzed following annexin V staining on CD25hiHLADR−CD4+ and CD25−HLADR−CD4+ T cells. Results are expressed as the difference in the percentage of annexin V-positive cells in the presence and absence of HIV peptides. Results correspond to the mean±SEM of results obtained for 7 patients. Statistical comparison involved the Wilcoxon Signed Rank Test.
Mentions: Approaches based on cell activation have been proposed to diminish or eliminate the HIV lymphocyte reservoir in patients on prolonged HAART. However, in addition to the hyporesponsiveness of Tregs, which may limit cell activation and virus expression, activated Tregs exert suppressive effects on both CD4+ and CD8+ T cells [20], [21], [30], [31]. Granzyme B is directly involved in CTL-mediated killing, and we found that CD25+CD4+ T-cell depletion strongly increased granzyme B release by CD8+ T cells following nonspecific activation with PMA+ionomycin (Fig. 3A). Depletion of CD25+CD4+ T cells also significantly increased granzyme B secretion in response to 15-mer overlapping HIV peptide pools corresponding to the RT, P24, and Nef gene products (Fig. 3B). These peptides could have activated specific Tregs, which then exerted their suppressive effects. Alternatively, this suppressive effect may be related to the existence, among untreated peripheral blood mononuclear cells (PBMCs), of already activated Tregs that could be continuously stimulated through interaction with self peptides. This latter possibility may explain the high proportion of HLADR+ cells among Tregs shown by ex vivo flow cytometry analysis (the median percentage of HLADR+ cells among CD25hiFoxP3+ CD4+ T cells was 51%, quartiles [46–59%], n = 15 patients on HAART for at least 2 years) (not shown). Activated Tregs might therefore create an environment in which CD8+ T cells are unable to fully exert their cytotoxic functions. In contrast, resting Tregs (HLADR− Tregs without exogenous activation) have no significant inhibitory effects on granzyme B secretion by activated CD8+ T cells (Fig. 4A). Histone deacetylase inhibitors could lead to virus expression without full cell activation [29] (see above). This could be an approach of potential interest for reducing the resting Treg reservoir. We next examined whether resting Tregs but expressing HIV peptides were susceptible to CD8+ T-cell cytolysis. Owing to the low frequency of infected cells and specific CD8+ T cells, we used the following strategy. CD4+-depleted PBMCs were first screened for reactivity to several pools of overlapping peptides corresponding to the Gag, Pol, and Nef sequences (see Methods). The most reactive overlapping peptide pool was then used to amplify specific CTL for testing against peptide-loaded autologous resting Tregs and resting non-Tregs (see methods). As shown in Fig. 4B, similar levels of apoptosis were found in the Treg and non-Treg subsets. This suggested that resting Tregs expressing HIV peptides may be as susceptible as non-Tregs to CD8+ T-cell cytotoxicity.

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