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

Tumor-resident DCs promoted the proliferation of CCR6+Tregs in situ.(A) CD11c+ DCs, macrophage or B cells were purified from TILs by MACS. 2×105 CCR6+Tregs or CCR6−Tregs were cultured with CD11c+ DCs, macrophage or B cells from 4T1 bearing mice at a 1∶2 ratio respectively. 3 days later, the proliferation were determined by [3H]thymidine incorporation assay. CD11c+ DCs were purified from TILs and DLNs by MACS. Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at indicated ratio was determined (B). (C) Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at a ratio of 1∶2 was determined at indicated time point as above described. (D) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-MHC class II antibody (10 ug/ml) for 3days. (E) The inhibitory activity of CCR6+Tregs or DC-expanded CCR6+Tregs was determined. (F) 4×105 CD11c+ DCs were purified from TILs and intratumorally injected into 4T1 syngeniec bearing mice (n = 8). 10 days later, the proliferation of CCR6+Tregs and CCR6−Tregs were analyzed by BrdU incorporation assay respectively as above described. One representative out of three independent experiments was shown. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105045&req=5

pone-0020282-g003: Tumor-resident DCs promoted the proliferation of CCR6+Tregs in situ.(A) CD11c+ DCs, macrophage or B cells were purified from TILs by MACS. 2×105 CCR6+Tregs or CCR6−Tregs were cultured with CD11c+ DCs, macrophage or B cells from 4T1 bearing mice at a 1∶2 ratio respectively. 3 days later, the proliferation were determined by [3H]thymidine incorporation assay. CD11c+ DCs were purified from TILs and DLNs by MACS. Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at indicated ratio was determined (B). (C) Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at a ratio of 1∶2 was determined at indicated time point as above described. (D) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-MHC class II antibody (10 ug/ml) for 3days. (E) The inhibitory activity of CCR6+Tregs or DC-expanded CCR6+Tregs was determined. (F) 4×105 CD11c+ DCs were purified from TILs and intratumorally injected into 4T1 syngeniec bearing mice (n = 8). 10 days later, the proliferation of CCR6+Tregs and CCR6−Tregs were analyzed by BrdU incorporation assay respectively as above described. One representative out of three independent experiments was shown. * p<0.05.

Mentions: Since the local proliferation was crucial for the in vivo accumulation of CCR6+Tregs. Next we tried to find whether tumor-resident APCs could promote the proliferation of CCR6+ Tregs. Tumor-resident CD11c+ DCs, macrophages or B cells were isolated from tumor mass of 4T1 bearing mice and then cultured with CCR6+Tregs or CCR6−Tregs at 2∶1 ratio in vitro. It was shown that tumor-resident DCs significantly stimulated the proliferation of CCR6+ Tregs in vitro in a time and dose dependent manner (Fig. 3a,b and c, p<0.05), while no such effect was observed for any of the other APCs isolated from tumor mass (Fig. 3a, p>0.05). Moreover, there were moderate effect of tumor-resident DCs on CCR6−Tregs (Fig. 3a, p>0.05). In addition, DCs derived form DLNs had little effect on the proliferation of CCR6+Tregs (Fig. 3b,c), partially explaining why lower proliferation of CCR6+Tregs in DLNs was found than in TILs. Moreover, the DC-expanded CCR6+Tregs sustained their expression of Foxp3 and CCR6 (data not shown) and the suppressive function (Fig. 3e). Moreover, DCs induced the proliferation of CCR6+Treg in MHC-class II-dependent way (Fig. 3d, p<0.05). To further confirm the effect of DCs on the proliferation of CCR6+Tregs, we further intratumoral injected DCs into tumor mass in 4T1 bearing syngeneic mice and found that DCs could also significantly promote the proliferation of CCR6+Tregs in tumor mass (Fig. 3e, p<0.05). Similar to above data, we also find little effect of DCs on the proliferation of CCR6−Tregs in vivo (Fig. 3e, p>0.05). Different from the report that tumor cells could induce the proliferation of Tregs [17], we couldn't observe any effect of 4T1 tumor cells on the proliferation of CCR6+Tregs in vitro (data not shown).


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)

Tumor-resident DCs promoted the proliferation of CCR6+Tregs in situ.(A) CD11c+ DCs, macrophage or B cells were purified from TILs by MACS. 2×105 CCR6+Tregs or CCR6−Tregs were cultured with CD11c+ DCs, macrophage or B cells from 4T1 bearing mice at a 1∶2 ratio respectively. 3 days later, the proliferation were determined by [3H]thymidine incorporation assay. CD11c+ DCs were purified from TILs and DLNs by MACS. Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at indicated ratio was determined (B). (C) Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at a ratio of 1∶2 was determined at indicated time point as above described. (D) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-MHC class II antibody (10 ug/ml) for 3days. (E) The inhibitory activity of CCR6+Tregs or DC-expanded CCR6+Tregs was determined. (F) 4×105 CD11c+ DCs were purified from TILs and intratumorally injected into 4T1 syngeniec bearing mice (n = 8). 10 days later, the proliferation of CCR6+Tregs and CCR6−Tregs were analyzed by BrdU incorporation assay respectively as above described. One representative out of three independent experiments was shown. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020282-g003: Tumor-resident DCs promoted the proliferation of CCR6+Tregs in situ.(A) CD11c+ DCs, macrophage or B cells were purified from TILs by MACS. 2×105 CCR6+Tregs or CCR6−Tregs were cultured with CD11c+ DCs, macrophage or B cells from 4T1 bearing mice at a 1∶2 ratio respectively. 3 days later, the proliferation were determined by [3H]thymidine incorporation assay. CD11c+ DCs were purified from TILs and DLNs by MACS. Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at indicated ratio was determined (B). (C) Proliferation of CCR6+Tregs after culturing with DCs from TILs or DLNs at a ratio of 1∶2 was determined at indicated time point as above described. (D) Proliferation of CCR6+Tregs after culturing with DCs at a ratio of 1∶2 in the presence of anti-MHC class II antibody (10 ug/ml) for 3days. (E) The inhibitory activity of CCR6+Tregs or DC-expanded CCR6+Tregs was determined. (F) 4×105 CD11c+ DCs were purified from TILs and intratumorally injected into 4T1 syngeniec bearing mice (n = 8). 10 days later, the proliferation of CCR6+Tregs and CCR6−Tregs were analyzed by BrdU incorporation assay respectively as above described. One representative out of three independent experiments was shown. * p<0.05.
Mentions: Since the local proliferation was crucial for the in vivo accumulation of CCR6+Tregs. Next we tried to find whether tumor-resident APCs could promote the proliferation of CCR6+ Tregs. Tumor-resident CD11c+ DCs, macrophages or B cells were isolated from tumor mass of 4T1 bearing mice and then cultured with CCR6+Tregs or CCR6−Tregs at 2∶1 ratio in vitro. It was shown that tumor-resident DCs significantly stimulated the proliferation of CCR6+ Tregs in vitro in a time and dose dependent manner (Fig. 3a,b and c, p<0.05), while no such effect was observed for any of the other APCs isolated from tumor mass (Fig. 3a, p>0.05). Moreover, there were moderate effect of tumor-resident DCs on CCR6−Tregs (Fig. 3a, p>0.05). In addition, DCs derived form DLNs had little effect on the proliferation of CCR6+Tregs (Fig. 3b,c), partially explaining why lower proliferation of CCR6+Tregs in DLNs was found than in TILs. Moreover, the DC-expanded CCR6+Tregs sustained their expression of Foxp3 and CCR6 (data not shown) and the suppressive function (Fig. 3e). Moreover, DCs induced the proliferation of CCR6+Treg in MHC-class II-dependent way (Fig. 3d, p<0.05). To further confirm the effect of DCs on the proliferation of CCR6+Tregs, we further intratumoral injected DCs into tumor mass in 4T1 bearing syngeneic mice and found that DCs could also significantly promote the proliferation of CCR6+Tregs in tumor mass (Fig. 3e, p<0.05). Similar to above data, we also find little effect of DCs on the proliferation of CCR6−Tregs in vivo (Fig. 3e, p>0.05). Different from the report that tumor cells could induce the proliferation of Tregs [17], we couldn't observe any effect of 4T1 tumor cells on the proliferation of CCR6+Tregs in vitro (data not shown).

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