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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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In situ expansion of CCR6+Tregs contributed to their prior accumulation in tumor mass.(A) The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. Then TILs was collected and analyzed. The gating strategy was shown. The proliferation and cell cycle of CCR6+Tregs and CCR6−Tregs were analyzed by FACS. (B) CCR6+ Tregs and CCR6−Tregs were purified by FACS from TILs and labeled with CFSE respectivley. Then, 1×106 CFSE-labeled cells were injected into tumor mass in 4T1-bearing mice. 10 days later, the proliferation of CFSE labeled CCR6+Tregs or CCR6−Tregs was analyzed by FACS. (C). 4T1 bearing Balb/c mice were injected with or without 200 ug vinblastine sulfate through tail vein as reported previously [15]. 15 hours later, CCR6+Tregs were sorted by FACS from TILs and the proliferation were analyzed by FACS and calculated. And the percentage of CCR6+Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated (D). (E) The percentage of CD4+CD25high Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated. One representative data of three independent experiments was shown.* p<0.05.
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pone-0020282-g002: In situ expansion of CCR6+Tregs contributed to their prior accumulation in tumor mass.(A) The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. Then TILs was collected and analyzed. The gating strategy was shown. The proliferation and cell cycle of CCR6+Tregs and CCR6−Tregs were analyzed by FACS. (B) CCR6+ Tregs and CCR6−Tregs were purified by FACS from TILs and labeled with CFSE respectivley. Then, 1×106 CFSE-labeled cells were injected into tumor mass in 4T1-bearing mice. 10 days later, the proliferation of CFSE labeled CCR6+Tregs or CCR6−Tregs was analyzed by FACS. (C). 4T1 bearing Balb/c mice were injected with or without 200 ug vinblastine sulfate through tail vein as reported previously [15]. 15 hours later, CCR6+Tregs were sorted by FACS from TILs and the proliferation were analyzed by FACS and calculated. And the percentage of CCR6+Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated (D). (E) The percentage of CD4+CD25high Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated. One representative data of three independent experiments was shown.* p<0.05.

Mentions: We next tried to elucidate whether selective enrichment of CCR6+ Tregs in the tumor mass was related to their preferable recruitment or their prior in site proliferation. And it was found that CCR6+Tregs expressed equal level of CCR4 and CCR8 as that of CCR6−Tregs (Fig. 1b) and displayed simlar response to CCL17 and CCL22 to CCR6−Tregs did (Fig S1). In addition, the concentration of CCL17, CCL20 and CCL22 in supernatant also were determined (Fig S2). However, cultured supernatant of 4T1 tumor cells attracted similar counts of CCR6+ and CCR6−Tregs (Fig S3). It is seemingly recuitment of CCR6+Tregs didn't be mostly responsible for their dominate enrichment in tumor sites. Previous finding showed that Tregs could proliferate in tumor mass. Then, we presumed that the proliferation of CCR6+Tregs may differ from that of CCR6−Tregs, which led to the dominant enrichment of CCR6+Tregs. It was found that 52.6% CCR6+Tregs were BrdU-positive, significantly higher than 13.4% BrdU-positive- CCR6−Tregs (Fig. 2a, p<0.05) in tumor mass. Moreover, the percentage of S phage of CCR6+Tregs was also higher than that of CCR6−Tregs (p<0.05). And the proliferation of tumor infiltrating CCR6+Tregs was higher than that in DLNs and PBMCs (data not shown). To further confirm this observation, CCR6+ or CCR6−Tregs were sorted from 4T1 bearing mice and labeled with CFSE, then injected into tumor mass in 4T1 bearing syngeneic mice respectively. 10 days later, the proliferation of CCR6+Treg cells or CCR6−Treg cells was analyzed. It was found that the percentage of CFSE+ CCR6+Treg cells (41.64%) were significantly higher than that of CFSE+ CCR6−Treg cells (14.21%) (Fig. 2b, p<0.05) indicating that CCR6+Tregs could more powerfully proliferate than CCR6−Tregs in tumor mass.


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)

In situ expansion of CCR6+Tregs contributed to their prior accumulation in tumor mass.(A) The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. Then TILs was collected and analyzed. The gating strategy was shown. The proliferation and cell cycle of CCR6+Tregs and CCR6−Tregs were analyzed by FACS. (B) CCR6+ Tregs and CCR6−Tregs were purified by FACS from TILs and labeled with CFSE respectivley. Then, 1×106 CFSE-labeled cells were injected into tumor mass in 4T1-bearing mice. 10 days later, the proliferation of CFSE labeled CCR6+Tregs or CCR6−Tregs was analyzed by FACS. (C). 4T1 bearing Balb/c mice were injected with or without 200 ug vinblastine sulfate through tail vein as reported previously [15]. 15 hours later, CCR6+Tregs were sorted by FACS from TILs and the proliferation were analyzed by FACS and calculated. And the percentage of CCR6+Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated (D). (E) The percentage of CD4+CD25high Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated. One representative data 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-g002: In situ expansion of CCR6+Tregs contributed to their prior accumulation in tumor mass.(A) The 4T1-bearing mice were treated with 2 mg BrdU i.p. every other day up to a cumulative dose of 8 mg BrdU. Then TILs was collected and analyzed. The gating strategy was shown. The proliferation and cell cycle of CCR6+Tregs and CCR6−Tregs were analyzed by FACS. (B) CCR6+ Tregs and CCR6−Tregs were purified by FACS from TILs and labeled with CFSE respectivley. Then, 1×106 CFSE-labeled cells were injected into tumor mass in 4T1-bearing mice. 10 days later, the proliferation of CFSE labeled CCR6+Tregs or CCR6−Tregs was analyzed by FACS. (C). 4T1 bearing Balb/c mice were injected with or without 200 ug vinblastine sulfate through tail vein as reported previously [15]. 15 hours later, CCR6+Tregs were sorted by FACS from TILs and the proliferation were analyzed by FACS and calculated. And the percentage of CCR6+Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated (D). (E) The percentage of CD4+CD25high Tregs in PBMCs, DLNs and TILs also were analyzed by FACS and calculated. One representative data of three independent experiments was shown.* p<0.05.
Mentions: We next tried to elucidate whether selective enrichment of CCR6+ Tregs in the tumor mass was related to their preferable recruitment or their prior in site proliferation. And it was found that CCR6+Tregs expressed equal level of CCR4 and CCR8 as that of CCR6−Tregs (Fig. 1b) and displayed simlar response to CCL17 and CCL22 to CCR6−Tregs did (Fig S1). In addition, the concentration of CCL17, CCL20 and CCL22 in supernatant also were determined (Fig S2). However, cultured supernatant of 4T1 tumor cells attracted similar counts of CCR6+ and CCR6−Tregs (Fig S3). It is seemingly recuitment of CCR6+Tregs didn't be mostly responsible for their dominate enrichment in tumor sites. Previous finding showed that Tregs could proliferate in tumor mass. Then, we presumed that the proliferation of CCR6+Tregs may differ from that of CCR6−Tregs, which led to the dominant enrichment of CCR6+Tregs. It was found that 52.6% CCR6+Tregs were BrdU-positive, significantly higher than 13.4% BrdU-positive- CCR6−Tregs (Fig. 2a, p<0.05) in tumor mass. Moreover, the percentage of S phage of CCR6+Tregs was also higher than that of CCR6−Tregs (p<0.05). And the proliferation of tumor infiltrating CCR6+Tregs was higher than that in DLNs and PBMCs (data not shown). To further confirm this observation, CCR6+ or CCR6−Tregs were sorted from 4T1 bearing mice and labeled with CFSE, then injected into tumor mass in 4T1 bearing syngeneic mice respectively. 10 days later, the proliferation of CCR6+Treg cells or CCR6−Treg cells was analyzed. It was found that the percentage of CFSE+ CCR6+Treg cells (41.64%) were significantly higher than that of CFSE+ CCR6−Treg cells (14.21%) (Fig. 2b, p<0.05) indicating that CCR6+Tregs could more powerfully proliferate than CCR6−Tregs in tumor mass.

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