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In situ prior proliferation of CD4+ CCR6+ regulatory T cells facilitated by TGF-β secreting DCs is crucial for their enrichment and suppression in tumor immunity.

Xu L, Xu W, Wen Z, Xiong S - PLoS ONE (2011)

Bottom Line: The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated.We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ.Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-β dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunobiology and Department of Immunology, Shanghai Medical College of Fudan University, Shanghai, China.

ABSTRACT

Background: CD4(+)CD25(+) regulatory T cells (Tregs), a heterogeneous population, were enrichment in tumor mass and played an important role in modulating anti-tumor immunity. Recently, we reported a Treg subset, CCR6(+) Tregs but not CCR6(-)Tregs, were enriched in tumor mass and closely related to poor prognosis of breast cancer patients. However, the underlying mechanism remains elusive. Here, we carefully evaluate the enrichment of CCR6(+)Tregs in tumor mass during progression of breast cancer and explore its possible mechanism.

Methodology/principal findings: The frequency of CCR6(+)Tregs in tumor infiltrating lymphocytes (TILs ) was analyzed at early stage and at late stage of tumor in a murine breast cancer model by FACS respectively. The expansion of CCR6(+)Tregs and their CCR6(-) counterpart in tumor mass were determined by BrdU incorporation assay. The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated. The role of local expansion of CCR6(+)Tregs in their enrichment and suppression in vivo also was evaluated in adoptive cell transfer assay. We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ. Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-β dependent manner. Adoptive transfer of CCR6(+)Tregs was found to potently inhibit the function of CD8(+)T cells in vivo, which was dependent on their proliferation and subsequently enrichment in tumor mass.

Conclusions/significance: Our finding suggested that CCR6(+) Tregs, a distinct subset of Tregs, exert its predominant suppressive role in tumor immunity through prior in situ expansion, which might ultimately provide helpful thoughts for the designing of Treg-based immunotherapy for tumor in the future.

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The proliferation of CCR6+Tregs in situ was crucial for their suppressive function in vivo.Balb/c mice were immunized with inactivated 4T1 tumor cells 3 times, with 1-week-interval. Then CD8+ T cells were sorted from splenocytes and re-stimulated with inactivated 4T1 tumor cells for 24 h in vitro. CCR6+Tregs and CCR6−Tregs were purified from TILs and treated with or with vinblastine (50 ng/ml) for 12 hrs. Then CD8+T cells were transferred with CCR6+Treg or CCR6−Tregs pretreated with or without vinblastine at a ratio of 2∶1 into syngeneic 4T1-bearing nude mice (n = 8). 10 days later, the proportion of CCR6+Tregs or CCR6−Tregs in TILs of 4T1 bearing nude mice was analyzed by FACS and calculated (A). CD8+T cells in tumor mass also were obtained. The percentage of IFN-γ secreting CD8+ T cells (B), as well as the expression of CD107a and Granzyme B on CD8+T cells (C), were analyzed by FACS. Symbols were representative of three independent experiments. Medians were indicated. (D) And the proliferation of CD8+T cells were also obtained by BrdU incorporation assay and calculated. (E) 21 day later, the tumor size in each group also was obtained.* p<0.05.
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pone-0020282-g005: The proliferation of CCR6+Tregs in situ was crucial for their suppressive function in vivo.Balb/c mice were immunized with inactivated 4T1 tumor cells 3 times, with 1-week-interval. Then CD8+ T cells were sorted from splenocytes and re-stimulated with inactivated 4T1 tumor cells for 24 h in vitro. CCR6+Tregs and CCR6−Tregs were purified from TILs and treated with or with vinblastine (50 ng/ml) for 12 hrs. Then CD8+T cells were transferred with CCR6+Treg or CCR6−Tregs pretreated with or without vinblastine at a ratio of 2∶1 into syngeneic 4T1-bearing nude mice (n = 8). 10 days later, the proportion of CCR6+Tregs or CCR6−Tregs in TILs of 4T1 bearing nude mice was analyzed by FACS and calculated (A). CD8+T cells in tumor mass also were obtained. The percentage of IFN-γ secreting CD8+ T cells (B), as well as the expression of CD107a and Granzyme B on CD8+T cells (C), were analyzed by FACS. Symbols were representative of three independent experiments. Medians were indicated. (D) And the proliferation of CD8+T cells were also obtained by BrdU incorporation assay and calculated. (E) 21 day later, the tumor size in each group also was obtained.* p<0.05.

Mentions: Our recent work indicated that adoptive transfer of CCR6+Tregs more effectively suppressed the anti-tumor CD8+T cells than their CCR6− counterparts did [20]. Then, whether in situ prior expansion of CCR6+Tregs might be responsible for their enrichment and subsequently suppressive effects in vivo was investigated. As shown in fig. 5a,b, CCR6+ or CCR6−Tregs were sorted from TILs and pretreated with or without vinblastine. Then cells were transferred into 4T1 bearing syngeneic nude mice with 4T1 specific CD8+T cells at a ratio of 1∶2 respectively. As shown in fig. 5a, vinblastine pre-treatment obviously decreased CCR6+Treg frequency in tumor mass from 5.43% to 2.13% (p<0.05). Correspondingly, IFN-γ production of CD8+T cells in CCR6+ Tregs co-transferred mice was 2.89%, while it increased dramatically to 6.11% when the co-transferred CCR6+Tregs were pre-treated with vinblastine (Fig. 5b, p<0.05). Similar results were obtained on the proliferation, as well as expression of CD107a and Granzyme B, of CD8+T cells (Fig. 5c,d, p<0.05). Finally, the size of tumor mass in CCR6+Tregs co-transferred mice was significantly elevated compared with that in CD8+T cells transferred group and CCR6−Tregs co-transferred group, while it decreased obviously in vinblastine pre-treated CCR6+ Tregs co-transferred group(Fig. 5e, p<0.05).


In situ prior proliferation of CD4+ CCR6+ regulatory T cells facilitated by TGF-β secreting DCs is crucial for their enrichment and suppression in tumor immunity.

Xu L, Xu W, Wen Z, Xiong S - PLoS ONE (2011)

The proliferation of CCR6+Tregs in situ was crucial for their suppressive function in vivo.Balb/c mice were immunized with inactivated 4T1 tumor cells 3 times, with 1-week-interval. Then CD8+ T cells were sorted from splenocytes and re-stimulated with inactivated 4T1 tumor cells for 24 h in vitro. CCR6+Tregs and CCR6−Tregs were purified from TILs and treated with or with vinblastine (50 ng/ml) for 12 hrs. Then CD8+T cells were transferred with CCR6+Treg or CCR6−Tregs pretreated with or without vinblastine at a ratio of 2∶1 into syngeneic 4T1-bearing nude mice (n = 8). 10 days later, the proportion of CCR6+Tregs or CCR6−Tregs in TILs of 4T1 bearing nude mice was analyzed by FACS and calculated (A). CD8+T cells in tumor mass also were obtained. The percentage of IFN-γ secreting CD8+ T cells (B), as well as the expression of CD107a and Granzyme B on CD8+T cells (C), were analyzed by FACS. Symbols were representative of three independent experiments. Medians were indicated. (D) And the proliferation of CD8+T cells were also obtained by BrdU incorporation assay and calculated. (E) 21 day later, the tumor size in each group also was obtained.* p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105045&req=5

pone-0020282-g005: The proliferation of CCR6+Tregs in situ was crucial for their suppressive function in vivo.Balb/c mice were immunized with inactivated 4T1 tumor cells 3 times, with 1-week-interval. Then CD8+ T cells were sorted from splenocytes and re-stimulated with inactivated 4T1 tumor cells for 24 h in vitro. CCR6+Tregs and CCR6−Tregs were purified from TILs and treated with or with vinblastine (50 ng/ml) for 12 hrs. Then CD8+T cells were transferred with CCR6+Treg or CCR6−Tregs pretreated with or without vinblastine at a ratio of 2∶1 into syngeneic 4T1-bearing nude mice (n = 8). 10 days later, the proportion of CCR6+Tregs or CCR6−Tregs in TILs of 4T1 bearing nude mice was analyzed by FACS and calculated (A). CD8+T cells in tumor mass also were obtained. The percentage of IFN-γ secreting CD8+ T cells (B), as well as the expression of CD107a and Granzyme B on CD8+T cells (C), were analyzed by FACS. Symbols were representative of three independent experiments. Medians were indicated. (D) And the proliferation of CD8+T cells were also obtained by BrdU incorporation assay and calculated. (E) 21 day later, the tumor size in each group also was obtained.* p<0.05.
Mentions: Our recent work indicated that adoptive transfer of CCR6+Tregs more effectively suppressed the anti-tumor CD8+T cells than their CCR6− counterparts did [20]. Then, whether in situ prior expansion of CCR6+Tregs might be responsible for their enrichment and subsequently suppressive effects in vivo was investigated. As shown in fig. 5a,b, CCR6+ or CCR6−Tregs were sorted from TILs and pretreated with or without vinblastine. Then cells were transferred into 4T1 bearing syngeneic nude mice with 4T1 specific CD8+T cells at a ratio of 1∶2 respectively. As shown in fig. 5a, vinblastine pre-treatment obviously decreased CCR6+Treg frequency in tumor mass from 5.43% to 2.13% (p<0.05). Correspondingly, IFN-γ production of CD8+T cells in CCR6+ Tregs co-transferred mice was 2.89%, while it increased dramatically to 6.11% when the co-transferred CCR6+Tregs were pre-treated with vinblastine (Fig. 5b, p<0.05). Similar results were obtained on the proliferation, as well as expression of CD107a and Granzyme B, of CD8+T cells (Fig. 5c,d, p<0.05). Finally, the size of tumor mass in CCR6+Tregs co-transferred mice was significantly elevated compared with that in CD8+T cells transferred group and CCR6−Tregs co-transferred group, while it decreased obviously in vinblastine pre-treated CCR6+ Tregs co-transferred group(Fig. 5e, p<0.05).

Bottom Line: The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated.We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ.Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-β dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunobiology and Department of Immunology, Shanghai Medical College of Fudan University, Shanghai, China.

ABSTRACT

Background: CD4(+)CD25(+) regulatory T cells (Tregs), a heterogeneous population, were enrichment in tumor mass and played an important role in modulating anti-tumor immunity. Recently, we reported a Treg subset, CCR6(+) Tregs but not CCR6(-)Tregs, were enriched in tumor mass and closely related to poor prognosis of breast cancer patients. However, the underlying mechanism remains elusive. Here, we carefully evaluate the enrichment of CCR6(+)Tregs in tumor mass during progression of breast cancer and explore its possible mechanism.

Methodology/principal findings: The frequency of CCR6(+)Tregs in tumor infiltrating lymphocytes (TILs ) was analyzed at early stage and at late stage of tumor in a murine breast cancer model by FACS respectively. The expansion of CCR6(+)Tregs and their CCR6(-) counterpart in tumor mass were determined by BrdU incorporation assay. The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated. The role of local expansion of CCR6(+)Tregs in their enrichment and suppression in vivo also was evaluated in adoptive cell transfer assay. We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ. Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-β dependent manner. Adoptive transfer of CCR6(+)Tregs was found to potently inhibit the function of CD8(+)T cells in vivo, which was dependent on their proliferation and subsequently enrichment in tumor mass.

Conclusions/significance: Our finding suggested that CCR6(+) Tregs, a distinct subset of Tregs, exert its predominant suppressive role in tumor immunity through prior in situ expansion, which might ultimately provide helpful thoughts for the designing of Treg-based immunotherapy for tumor in the future.

Show MeSH
Related in: MedlinePlus