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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 promote differentiation of human Treg in culture.(A) Purified CD3+CD4+ T cells were labeled with CFSE and cultured as described in Materials and Methods ± TMV or DC-derived MV (5 µg/mL). On days 3, 5 and 8, the frequency of CD4+CD25+FOXP3+ Treg among proliferating T cells was determined by flow cytometry. The data (means ± SD) represent three independent experiments (*p<0.01). (B) Proliferating CD3+CD4+ T cells (squares) were tested for co-expression of CD25 in a representative co-culture ± TMV. A higher proportion of proliferating CD4+ T cells expressed CD25 in the co-culture with TMV than without TMV. (C) The proliferating CD4+CD25+ T cells in the co-cultures with TMV were evaluated for the frequency of FOXP3+ T cells upon gating on the CD4+CD25high subset (see box). Over 90% of these cells also expressed intracellular FOXP3. Data are representative for one out of 6 cultures tested.
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pone-0011469-g002: TMV promote differentiation of human Treg in culture.(A) Purified CD3+CD4+ T cells were labeled with CFSE and cultured as described in Materials and Methods ± TMV or DC-derived MV (5 µg/mL). On days 3, 5 and 8, the frequency of CD4+CD25+FOXP3+ Treg among proliferating T cells was determined by flow cytometry. The data (means ± SD) represent three independent experiments (*p<0.01). (B) Proliferating CD3+CD4+ T cells (squares) were tested for co-expression of CD25 in a representative co-culture ± TMV. A higher proportion of proliferating CD4+ T cells expressed CD25 in the co-culture with TMV than without TMV. (C) The proliferating CD4+CD25+ T cells in the co-cultures with TMV were evaluated for the frequency of FOXP3+ T cells upon gating on the CD4+CD25high subset (see box). Over 90% of these cells also expressed intracellular FOXP3. Data are representative for one out of 6 cultures tested.

Mentions: TMV (1–60 µg) were co-incubated with purified CD3+CD4+ T cells previously labeled with CFSE and activated with plate-bound OKT3, soluble anti-CD28 Ab and IL-2. The percent of CD4+CD25+FOXP3+ T cells increased upon co-incubation with TMV in a dose dependent manner, and the optimal TMV concentration for Treg induction was 5 µg/1×106 cells (Figure 1E). MV derived from DC did not induce expansion of CD3+CD25+FOXP3+ Treg, as also previously reported [5]. Next, purified CD3+CD4+ T cells were labeled with CFSE, stimulated as described in Methods and cultured in the presence of TMV or DC-derived MV. The frequency of CD4+CD25+FOXP3+ T cells was measured on days 3, 5 and 8, it was increased at all time points relative to the baseline, and it was significantly greater (p<0.05) in co-cultures containing TMV (Figure 2A). Co-staining of proliferating CD4+ T cells for CD25 indicated that in the presence of TMV, over 60% of these cells were CD4+CD25+. In contrast, CD4+ T cells cultured without TMV contained fewer (p<0.05) CD4+CD25+ T cells (Figure 2B). Gating in these cultures on the CD4+CD25high subset indicated that over 90% co-expressed FOXP3 (Figure 2C). These data suggest that TMV but not DC-derived MV promote the generation of CD4+CD25+FOXP3+ T cells in culture.


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 promote differentiation of human Treg in culture.(A) Purified CD3+CD4+ T cells were labeled with CFSE and cultured as described in Materials and Methods ± TMV or DC-derived MV (5 µg/mL). On days 3, 5 and 8, the frequency of CD4+CD25+FOXP3+ Treg among proliferating T cells was determined by flow cytometry. The data (means ± SD) represent three independent experiments (*p<0.01). (B) Proliferating CD3+CD4+ T cells (squares) were tested for co-expression of CD25 in a representative co-culture ± TMV. A higher proportion of proliferating CD4+ T cells expressed CD25 in the co-culture with TMV than without TMV. (C) The proliferating CD4+CD25+ T cells in the co-cultures with TMV were evaluated for the frequency of FOXP3+ T cells upon gating on the CD4+CD25high subset (see box). Over 90% of these cells also expressed intracellular FOXP3. Data are representative for one out of 6 cultures tested.
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Related In: Results  -  Collection

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pone-0011469-g002: TMV promote differentiation of human Treg in culture.(A) Purified CD3+CD4+ T cells were labeled with CFSE and cultured as described in Materials and Methods ± TMV or DC-derived MV (5 µg/mL). On days 3, 5 and 8, the frequency of CD4+CD25+FOXP3+ Treg among proliferating T cells was determined by flow cytometry. The data (means ± SD) represent three independent experiments (*p<0.01). (B) Proliferating CD3+CD4+ T cells (squares) were tested for co-expression of CD25 in a representative co-culture ± TMV. A higher proportion of proliferating CD4+ T cells expressed CD25 in the co-culture with TMV than without TMV. (C) The proliferating CD4+CD25+ T cells in the co-cultures with TMV were evaluated for the frequency of FOXP3+ T cells upon gating on the CD4+CD25high subset (see box). Over 90% of these cells also expressed intracellular FOXP3. Data are representative for one out of 6 cultures tested.
Mentions: TMV (1–60 µg) were co-incubated with purified CD3+CD4+ T cells previously labeled with CFSE and activated with plate-bound OKT3, soluble anti-CD28 Ab and IL-2. The percent of CD4+CD25+FOXP3+ T cells increased upon co-incubation with TMV in a dose dependent manner, and the optimal TMV concentration for Treg induction was 5 µg/1×106 cells (Figure 1E). MV derived from DC did not induce expansion of CD3+CD25+FOXP3+ Treg, as also previously reported [5]. Next, purified CD3+CD4+ T cells were labeled with CFSE, stimulated as described in Methods and cultured in the presence of TMV or DC-derived MV. The frequency of CD4+CD25+FOXP3+ T cells was measured on days 3, 5 and 8, it was increased at all time points relative to the baseline, and it was significantly greater (p<0.05) in co-cultures containing TMV (Figure 2A). Co-staining of proliferating CD4+ T cells for CD25 indicated that in the presence of TMV, over 60% of these cells were CD4+CD25+. In contrast, CD4+ T cells cultured without TMV contained fewer (p<0.05) CD4+CD25+ T cells (Figure 2B). Gating in these cultures on the CD4+CD25high subset indicated that over 90% co-expressed FOXP3 (Figure 2C). These data suggest that TMV but not DC-derived MV promote the generation of CD4+CD25+FOXP3+ T cells in culture.

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