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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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Related in: MedlinePlus

CCR6+Foxp3+ regulatory T cells were enriched in tumor mass during tumor progress.(A) Tumor infiltrating cells (TILs) from 4T1 bearing mice on day 10 or day 28 were stained by anti-CD4, anti-CD25, anti-Foxp3 and anti-CCR6 antibodies and analyzed by Flow cytometry. The gating strategy and expression of Foxp3 were shown. Dot plots showing examples of CCR6/Foxp3 expression in CD4+ CD25neg, CD4+ CD25low, and CD4+ CD25high gated populations of TILs from 4T1 bearing mice are reported. (B) FACS analysis of the phenotypes of CCR6− regulatory T cells and CCR6+ regulatory T cells. Dot lines here represent the isotype control. (C) Suppressive capacity of the sorted CCR6− regulatory T cells and CCR6+ regulatory T cells against CD4+ CD25−Teff cells, determined by [3H]-thymidine incorporation respectively. (D) As gated on fig. 1a, the percentage of CCR6− Foxp3+ regulatory T cells and CCR6+ Foxp3+ regulatory T cells in CD4+ T cells from peripheral blood mononuclear cells (PBMCs), draining lymph nodes(DLNs) and TILs were analyzed at day 10 or day 28 (termed as early or late stage) (n = 12) by FACS and calculated. *p<0.05.
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pone-0020282-g001: CCR6+Foxp3+ regulatory T cells were enriched in tumor mass during tumor progress.(A) Tumor infiltrating cells (TILs) from 4T1 bearing mice on day 10 or day 28 were stained by anti-CD4, anti-CD25, anti-Foxp3 and anti-CCR6 antibodies and analyzed by Flow cytometry. The gating strategy and expression of Foxp3 were shown. Dot plots showing examples of CCR6/Foxp3 expression in CD4+ CD25neg, CD4+ CD25low, and CD4+ CD25high gated populations of TILs from 4T1 bearing mice are reported. (B) FACS analysis of the phenotypes of CCR6− regulatory T cells and CCR6+ regulatory T cells. Dot lines here represent the isotype control. (C) Suppressive capacity of the sorted CCR6− regulatory T cells and CCR6+ regulatory T cells against CD4+ CD25−Teff cells, determined by [3H]-thymidine incorporation respectively. (D) As gated on fig. 1a, the percentage of CCR6− Foxp3+ regulatory T cells and CCR6+ Foxp3+ regulatory T cells in CD4+ T cells from peripheral blood mononuclear cells (PBMCs), draining lymph nodes(DLNs) and TILs were analyzed at day 10 or day 28 (termed as early or late stage) (n = 12) by FACS and calculated. *p<0.05.

Mentions: Our previous data found that CCR6+Foxp3+ regulatory T cells were dominantly enriched in tumor mass and closely related to poor prognosis of breast cancer patients [14], to further investigate the mechanism of the enrichment of this tumor-resident Treg subset, the frequency of CCR6+ Tregs in TILs during tumor progression was observed. As shown in fig. 1a, the expression of CCR6 and Foxp3 were evaluated inside the CD4+ CD25neg, CD4+ CD25low and CD4+ CD25high gated populations of TILs from 4T1 bearing mice. We found that CCR6 was preferentially expressed on CD4+ CD25high Foxp3+ regulatory T cells and defined a distinct subset of CCR6+CD4+ CD25high Foxp3+ regulatory T cells in 4T1 bearing mice (Fig. 1a). Next, we analyzed the phenotype and inhibitory function of CCR6+Tregs. The expression level of Foxp3, GITR, CTLA-4, CCR4 and CCR8 on CCR6+ Tregs were similar to those on CCR6− Tregs indicating both CCR6+ and CCR6− Tregs displayed regulatory phenotype (Fig. 1b). And the inhibitory effect of CCR6+Tregs on responder CD4+ CD25−T cells was comparable to that of CCR6− Tregs (Fig. 1c, p>0.05). Of note, CCR6+ Tregs expressed low level of CD62L and high level of CD44, displaying an effector/memory phenotype (Fig. 1b). We further analyzed the frequency of CCR6+Tregs and CCR6−Tregs at day 10 or 28 (defined as early and late stage respectively) and found that the CCR6−Treg frequency in TILs (6.1%) at late stage of tumor was equal to that (5.9%) at early stage of tumor; while the frequency of CCR6+Tregs in TILs (12.5%) at late stage of tumor was significantly higher than that (5.7%) at early stage of tumor (Fig. 1d, p<0.05). Moreover, CCR6+Treg frequency at late stage of tumor in TILs was higher than that in DLNs; In contrast, frequency of CCR6− Tregs in TILs at late stage was similar to that in DLNs (Fig. 1d, p>0.05). In addition, there wasn't any change in the frequency of CCR6+Tregs or CCR6−Tregs in PBMCs during tumor progression. These data showed that CCR6+ Tregs, but not their CCR6− counterpart, accumulated in tumor mass during tumor progression.


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)

CCR6+Foxp3+ regulatory T cells were enriched in tumor mass during tumor progress.(A) Tumor infiltrating cells (TILs) from 4T1 bearing mice on day 10 or day 28 were stained by anti-CD4, anti-CD25, anti-Foxp3 and anti-CCR6 antibodies and analyzed by Flow cytometry. The gating strategy and expression of Foxp3 were shown. Dot plots showing examples of CCR6/Foxp3 expression in CD4+ CD25neg, CD4+ CD25low, and CD4+ CD25high gated populations of TILs from 4T1 bearing mice are reported. (B) FACS analysis of the phenotypes of CCR6− regulatory T cells and CCR6+ regulatory T cells. Dot lines here represent the isotype control. (C) Suppressive capacity of the sorted CCR6− regulatory T cells and CCR6+ regulatory T cells against CD4+ CD25−Teff cells, determined by [3H]-thymidine incorporation respectively. (D) As gated on fig. 1a, the percentage of CCR6− Foxp3+ regulatory T cells and CCR6+ Foxp3+ regulatory T cells in CD4+ T cells from peripheral blood mononuclear cells (PBMCs), draining lymph nodes(DLNs) and TILs were analyzed at day 10 or day 28 (termed as early or late stage) (n = 12) by FACS and calculated. *p<0.05.
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Related In: Results  -  Collection

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pone-0020282-g001: CCR6+Foxp3+ regulatory T cells were enriched in tumor mass during tumor progress.(A) Tumor infiltrating cells (TILs) from 4T1 bearing mice on day 10 or day 28 were stained by anti-CD4, anti-CD25, anti-Foxp3 and anti-CCR6 antibodies and analyzed by Flow cytometry. The gating strategy and expression of Foxp3 were shown. Dot plots showing examples of CCR6/Foxp3 expression in CD4+ CD25neg, CD4+ CD25low, and CD4+ CD25high gated populations of TILs from 4T1 bearing mice are reported. (B) FACS analysis of the phenotypes of CCR6− regulatory T cells and CCR6+ regulatory T cells. Dot lines here represent the isotype control. (C) Suppressive capacity of the sorted CCR6− regulatory T cells and CCR6+ regulatory T cells against CD4+ CD25−Teff cells, determined by [3H]-thymidine incorporation respectively. (D) As gated on fig. 1a, the percentage of CCR6− Foxp3+ regulatory T cells and CCR6+ Foxp3+ regulatory T cells in CD4+ T cells from peripheral blood mononuclear cells (PBMCs), draining lymph nodes(DLNs) and TILs were analyzed at day 10 or day 28 (termed as early or late stage) (n = 12) by FACS and calculated. *p<0.05.
Mentions: Our previous data found that CCR6+Foxp3+ regulatory T cells were dominantly enriched in tumor mass and closely related to poor prognosis of breast cancer patients [14], to further investigate the mechanism of the enrichment of this tumor-resident Treg subset, the frequency of CCR6+ Tregs in TILs during tumor progression was observed. As shown in fig. 1a, the expression of CCR6 and Foxp3 were evaluated inside the CD4+ CD25neg, CD4+ CD25low and CD4+ CD25high gated populations of TILs from 4T1 bearing mice. We found that CCR6 was preferentially expressed on CD4+ CD25high Foxp3+ regulatory T cells and defined a distinct subset of CCR6+CD4+ CD25high Foxp3+ regulatory T cells in 4T1 bearing mice (Fig. 1a). Next, we analyzed the phenotype and inhibitory function of CCR6+Tregs. The expression level of Foxp3, GITR, CTLA-4, CCR4 and CCR8 on CCR6+ Tregs were similar to those on CCR6− Tregs indicating both CCR6+ and CCR6− Tregs displayed regulatory phenotype (Fig. 1b). And the inhibitory effect of CCR6+Tregs on responder CD4+ CD25−T cells was comparable to that of CCR6− Tregs (Fig. 1c, p>0.05). Of note, CCR6+ Tregs expressed low level of CD62L and high level of CD44, displaying an effector/memory phenotype (Fig. 1b). We further analyzed the frequency of CCR6+Tregs and CCR6−Tregs at day 10 or 28 (defined as early and late stage respectively) and found that the CCR6−Treg frequency in TILs (6.1%) at late stage of tumor was equal to that (5.9%) at early stage of tumor; while the frequency of CCR6+Tregs in TILs (12.5%) at late stage of tumor was significantly higher than that (5.7%) at early stage of tumor (Fig. 1d, p<0.05). Moreover, CCR6+Treg frequency at late stage of tumor in TILs was higher than that in DLNs; In contrast, frequency of CCR6− Tregs in TILs at late stage was similar to that in DLNs (Fig. 1d, p>0.05). In addition, there wasn't any change in the frequency of CCR6+Tregs or CCR6−Tregs in PBMCs during tumor progression. These data showed that CCR6+ Tregs, but not their CCR6− counterpart, accumulated in tumor mass during tumor progression.

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