Limits...
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.

Show MeSH

Related in: MedlinePlus

DCs triggerred the proliferation of CCR6+Treg in a TGF-β dependent manner.The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. These mice also were simultaneously intratumorally injected 0.1-ml aliquots of Anti-TGF-β antibody (50 ug) (▪) (n = 6) or rat IgG2a isotype control (□) (n = 6) every other day. 8 hrs after last injection of BrdU, The TILs were collected. The proliferation (A) and proportion (B) of CCR6+Tregs in TILs were analyzed by FACS and calculated. (C) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-TGF-β or isotype control antibody (10 ug/ml) for 3 days. (D) CD11c+ DCs were purified from TILs by MACS. 1×105 DCs was cultured and transfected with TGF-β RNAi or Scramble control. 72 hrs later, the supernatant concentration of TGF-β were determined by ELSA assay. (E) 1×105 CCR6+ Tregs were cocultured with DCs transfected with TGF-β RNAi or scramble control at a ratio of 1∶2. 72 hrs later, the proliferation of CCR6+Tregs was determined. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105045&req=5

pone-0020282-g004: DCs triggerred the proliferation of CCR6+Treg in a TGF-β dependent manner.The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. These mice also were simultaneously intratumorally injected 0.1-ml aliquots of Anti-TGF-β antibody (50 ug) (▪) (n = 6) or rat IgG2a isotype control (□) (n = 6) every other day. 8 hrs after last injection of BrdU, The TILs were collected. The proliferation (A) and proportion (B) of CCR6+Tregs in TILs were analyzed by FACS and calculated. (C) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-TGF-β or isotype control antibody (10 ug/ml) for 3 days. (D) CD11c+ DCs were purified from TILs by MACS. 1×105 DCs was cultured and transfected with TGF-β RNAi or Scramble control. 72 hrs later, the supernatant concentration of TGF-β were determined by ELSA assay. (E) 1×105 CCR6+ Tregs were cocultured with DCs transfected with TGF-β RNAi or scramble control at a ratio of 1∶2. 72 hrs later, the proliferation of CCR6+Tregs was determined. * p<0.05.

Mentions: Previous finding demonstrated TGF-β was critical for Treg proliferation in vivo [18], [19]. Hence, anti-TGF-β antibody was administered to mice before analyzing the BrdU+CCR6+Tregs. As shown in fig. 4a, anti-TGF-β antibody treatment significantly reduced the proliferation and frequency of CCR6+Tregs in tumor mass, suggesting TGF-β was critical for the in situ CCR6+Tregs proliferation. Then, the expression level of TGF-β in tumor-resident DCs was analyzed. As shown in fig. 4d, tumor-resident DCs expressed high level of TGF-β. anti-TGF-β treatment also abrogated nearly 65% in vitro proliferation of CCR6+Tregs triggered by DCs (Fig. 4c, p<0.05). To exclude the influence of CCR6+Tregs derived TGF-β on the effects of DCs, we transfected TGF-β RNAi into DCs. As shown in fig. 4d, TGF-β RNAi could significantly reduce the TGF-β secretion of DCs. Importantly, the proliferation of CCR6+Tregs in TGF-β-RNAi transfected DCs group decreased significantly compared with control group (Fig. 4e, p<0.05). These data indicated that tumor-derived CD11c+DCs triggered the proliferation of CCR6+Treg in a TGF-β dependent manner.


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)

DCs triggerred the proliferation of CCR6+Treg in a TGF-β dependent manner.The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. These mice also were simultaneously intratumorally injected 0.1-ml aliquots of Anti-TGF-β antibody (50 ug) (▪) (n = 6) or rat IgG2a isotype control (□) (n = 6) every other day. 8 hrs after last injection of BrdU, The TILs were collected. The proliferation (A) and proportion (B) of CCR6+Tregs in TILs were analyzed by FACS and calculated. (C) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-TGF-β or isotype control antibody (10 ug/ml) for 3 days. (D) CD11c+ DCs were purified from TILs by MACS. 1×105 DCs was cultured and transfected with TGF-β RNAi or Scramble control. 72 hrs later, the supernatant concentration of TGF-β were determined by ELSA assay. (E) 1×105 CCR6+ Tregs were cocultured with DCs transfected with TGF-β RNAi or scramble control at a ratio of 1∶2. 72 hrs later, the proliferation of CCR6+Tregs was determined. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020282-g004: DCs triggerred the proliferation of CCR6+Treg in a TGF-β dependent manner.The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. These mice also were simultaneously intratumorally injected 0.1-ml aliquots of Anti-TGF-β antibody (50 ug) (▪) (n = 6) or rat IgG2a isotype control (□) (n = 6) every other day. 8 hrs after last injection of BrdU, The TILs were collected. The proliferation (A) and proportion (B) of CCR6+Tregs in TILs were analyzed by FACS and calculated. (C) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-TGF-β or isotype control antibody (10 ug/ml) for 3 days. (D) CD11c+ DCs were purified from TILs by MACS. 1×105 DCs was cultured and transfected with TGF-β RNAi or Scramble control. 72 hrs later, the supernatant concentration of TGF-β were determined by ELSA assay. (E) 1×105 CCR6+ Tregs were cocultured with DCs transfected with TGF-β RNAi or scramble control at a ratio of 1∶2. 72 hrs later, the proliferation of CCR6+Tregs was determined. * p<0.05.
Mentions: Previous finding demonstrated TGF-β was critical for Treg proliferation in vivo [18], [19]. Hence, anti-TGF-β antibody was administered to mice before analyzing the BrdU+CCR6+Tregs. As shown in fig. 4a, anti-TGF-β antibody treatment significantly reduced the proliferation and frequency of CCR6+Tregs in tumor mass, suggesting TGF-β was critical for the in situ CCR6+Tregs proliferation. Then, the expression level of TGF-β in tumor-resident DCs was analyzed. As shown in fig. 4d, tumor-resident DCs expressed high level of TGF-β. anti-TGF-β treatment also abrogated nearly 65% in vitro proliferation of CCR6+Tregs triggered by DCs (Fig. 4c, p<0.05). To exclude the influence of CCR6+Tregs derived TGF-β on the effects of DCs, we transfected TGF-β RNAi into DCs. As shown in fig. 4d, TGF-β RNAi could significantly reduce the TGF-β secretion of DCs. Importantly, the proliferation of CCR6+Tregs in TGF-β-RNAi transfected DCs group decreased significantly compared with control group (Fig. 4e, p<0.05). These data indicated that tumor-derived CD11c+DCs triggered the proliferation of CCR6+Treg in a TGF-β dependent manner.

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