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Tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg).

Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL - PLoS ONE (2010)

Bottom Line: Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells.Neutralizing Abs specific for TGF-beta1 and/or IL-10 significantly inhibited TMV ability to expand Treg.They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4(+)CD25(high)FOXP3(+) Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/principal findings: TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4(+)CD25(neg) T cells into CD4(+)CD25(high)FOXP3(+) Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-beta1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-beta1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/significance: This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.

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Related in: MedlinePlus

TMV increase suppressor activity of Treg.The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4+CD25neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (A) and greater inhibition of RC proliferation (B). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD+ RC was lower (C) as was RC proliferation inhibition (D). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (C) and inhibited RC proliferation (D).
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pone-0011469-g005: TMV increase suppressor activity of Treg.The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4+CD25neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (A) and greater inhibition of RC proliferation (B). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD+ RC was lower (C) as was RC proliferation inhibition (D). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (C) and inhibited RC proliferation (D).

Mentions: The FLOCA was used to test whether TMV enhanced the ability of Treg to mediate suppression. This assay measures not only the inhibition of RC proliferation but also simultaneously discriminates between CFSE-labeled/7AAD+ (dead) and unlabeled/7AADneg (live) cells [24]. CD4+CD25+ T cells used as suppressor cells (S) were pre-incubated with TMV for 24 h and then co-cultured with autologous RC at the 1∶1 and 1∶5 ratios. The percent of dead RC was increased + TMV-treated S (Figure 5A), and concomitantly, proliferative responses of RC were inhibited (p<0.05) compared to co-cultures with untreated S (Figure 5B). We have reported that human Treg can mediate suppression using either the perforin/GrB or the Fas/FasL pathway [24], [27]. When Treg were pre-treated with Concanamycin A, which inhibits perforin activation, or with GrB inhibitor I, TMV no longer up-regulated suppressor functions of these Treg, as illustrated in Figures 5C and D. This suggests that TMV increase the ability of Treg to mediate suppression/death of RC by up-regulating activity of the perforin/GrB pathway in Treg. In contrast, anti-FasL Ab treatment of Treg had no effect on their ability to kill RC or inhibit RC proliferation ± TMV in these assays (Figures 5C and D).


Tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg).

Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL - PLoS ONE (2010)

TMV increase suppressor activity of Treg.The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4+CD25neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (A) and greater inhibition of RC proliferation (B). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD+ RC was lower (C) as was RC proliferation inhibition (D). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (C) and inhibited RC proliferation (D).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908536&req=5

pone-0011469-g005: TMV increase suppressor activity of Treg.The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4+CD25neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (A) and greater inhibition of RC proliferation (B). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD+ RC was lower (C) as was RC proliferation inhibition (D). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (C) and inhibited RC proliferation (D).
Mentions: The FLOCA was used to test whether TMV enhanced the ability of Treg to mediate suppression. This assay measures not only the inhibition of RC proliferation but also simultaneously discriminates between CFSE-labeled/7AAD+ (dead) and unlabeled/7AADneg (live) cells [24]. CD4+CD25+ T cells used as suppressor cells (S) were pre-incubated with TMV for 24 h and then co-cultured with autologous RC at the 1∶1 and 1∶5 ratios. The percent of dead RC was increased + TMV-treated S (Figure 5A), and concomitantly, proliferative responses of RC were inhibited (p<0.05) compared to co-cultures with untreated S (Figure 5B). We have reported that human Treg can mediate suppression using either the perforin/GrB or the Fas/FasL pathway [24], [27]. When Treg were pre-treated with Concanamycin A, which inhibits perforin activation, or with GrB inhibitor I, TMV no longer up-regulated suppressor functions of these Treg, as illustrated in Figures 5C and D. This suggests that TMV increase the ability of Treg to mediate suppression/death of RC by up-regulating activity of the perforin/GrB pathway in Treg. In contrast, anti-FasL Ab treatment of Treg had no effect on their ability to kill RC or inhibit RC proliferation ± TMV in these assays (Figures 5C and D).

Bottom Line: Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells.Neutralizing Abs specific for TGF-beta1 and/or IL-10 significantly inhibited TMV ability to expand Treg.They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4(+)CD25(high)FOXP3(+) Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/principal findings: TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4(+)CD25(neg) T cells into CD4(+)CD25(high)FOXP3(+) Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-beta1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-beta1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/significance: This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.

Show MeSH
Related in: MedlinePlus