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Blocking the recruitment of naive CD4 + T cells reverses immunosuppression in breast cancer

View Article: PubMed Central - PubMed

ABSTRACT

The origin of tumor-infiltrating Tregs, critical mediators of tumor immunosuppression, is unclear. Here, we show that tumor-infiltrating naive CD4+ T cells and Tregs in human breast cancer have overlapping TCR repertoires, while hardly overlap with circulating Tregs, suggesting that intratumoral Tregs mainly develop from naive T cells in situ rather than from recruited Tregs. Furthermore, the abundance of naive CD4+ T cells and Tregs is closely correlated, both indicating poor prognosis for breast cancer patients. Naive CD4+ T cells adhere to tumor slices in proportion to the abundance of CCL18-producing macrophages. Moreover, adoptively transferred human naive CD4+ T cells infiltrate human breast cancer orthotopic xenografts in a CCL18-dependent manner. In human breast cancer xenografts in humanized mice, blocking the recruitment of naive CD4+ T cells into tumor by knocking down the expression of PITPNM3, a CCL18 receptor, significantly reduces intratumoral Tregs and inhibits tumor progression. These findings suggest that breast tumor-infiltrating Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Inhibiting naive CD4+ T cell recruitment into tumors by interfering with PITPNM3 recognition of CCL18 may be an attractive strategy for anticancer immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Naive CD4+ T cells are converted to functional Tregs by tumor-infiltrating DCs and tumor conditioned medium (CM). (A-C) Naive CD4+ T cells from peripheral blood of patients with invasive breast carcinoma were co-cultured with or without autologous pDCs isolated from tumor (TI) or peripheral blood (PB) for 9 days in the presence or absence of 30% CM from autologous tumor slices or adjacent normal tissue slices. (A, B) Non-adherent cells from co-cultures were stained for CD3, CD4, CD25 and intracellular Foxp3, and analyzed by flow cytometry. Representative plots of gated CD3+CD4+ cells (A) and quantification of percentage of Foxp3+CD25+ cells among CD3+CD4+ cells (B) are shown (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 by Student's t-test). (C) Expression of Treg-associated genes, assessed by qRT-PCR normalized to GAPDH, in sorted CD4+ T cells, relative to expression in cultures without DCs or CM (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 compared with naive CD4+ T cells cultured alone by Student's t-test). (D-G) Effect of naive CD4+ T cell-derived Tregs, obtained by co-culture with TI pDCs and tumor CM as above, on function of autologous tumor-specific CD8+ T cells. Tumor-specific CD8+ T cells were generated for each subject by stimulating autologous PB CD8+ T cells with autologous tumor lysate-pulsed autologous DCs. Tregs were recovered from co-cultures by magnetic sorting. (D) CFSE-labeled CD8+ T cells were incubated with tumor lysate-pulsed DCs in the presence of induced Tregs at the indicated ratios and proliferation was assessed by flow cytometry. Numbers denote the percentage of cells undergoing at least one cellular division (mean ± SEM, n = 12, **P < 0.01, ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (E-G) Tumor-specific CD8+ T cells were incubated with autologous primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and stained for CD3, CD8, intracellular perforin (E) or granzyme B (F) and gated CD3+CD8+ cells were analyzed by flow cytometry. Numbers indicate the percentage of gated cells stained for perforin or granzyme B (mean ± SEM, n = 7; ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (G) Tumor-specific CD8+ T cells were incubated with autologous dioctadecyloxacarbocyanine (DIOC18)-labeled primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and the death of tumor cells was assessed by propidium iodide (PI) uptake by flow cytometry. The numbers shown indicate the percentage of PI+ tumor cells (mean ± SEM, n = 4; ***P < 0.001 compared with CD8+ T cells cultured without Tregs).
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fig3: Naive CD4+ T cells are converted to functional Tregs by tumor-infiltrating DCs and tumor conditioned medium (CM). (A-C) Naive CD4+ T cells from peripheral blood of patients with invasive breast carcinoma were co-cultured with or without autologous pDCs isolated from tumor (TI) or peripheral blood (PB) for 9 days in the presence or absence of 30% CM from autologous tumor slices or adjacent normal tissue slices. (A, B) Non-adherent cells from co-cultures were stained for CD3, CD4, CD25 and intracellular Foxp3, and analyzed by flow cytometry. Representative plots of gated CD3+CD4+ cells (A) and quantification of percentage of Foxp3+CD25+ cells among CD3+CD4+ cells (B) are shown (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 by Student's t-test). (C) Expression of Treg-associated genes, assessed by qRT-PCR normalized to GAPDH, in sorted CD4+ T cells, relative to expression in cultures without DCs or CM (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 compared with naive CD4+ T cells cultured alone by Student's t-test). (D-G) Effect of naive CD4+ T cell-derived Tregs, obtained by co-culture with TI pDCs and tumor CM as above, on function of autologous tumor-specific CD8+ T cells. Tumor-specific CD8+ T cells were generated for each subject by stimulating autologous PB CD8+ T cells with autologous tumor lysate-pulsed autologous DCs. Tregs were recovered from co-cultures by magnetic sorting. (D) CFSE-labeled CD8+ T cells were incubated with tumor lysate-pulsed DCs in the presence of induced Tregs at the indicated ratios and proliferation was assessed by flow cytometry. Numbers denote the percentage of cells undergoing at least one cellular division (mean ± SEM, n = 12, **P < 0.01, ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (E-G) Tumor-specific CD8+ T cells were incubated with autologous primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and stained for CD3, CD8, intracellular perforin (E) or granzyme B (F) and gated CD3+CD8+ cells were analyzed by flow cytometry. Numbers indicate the percentage of gated cells stained for perforin or granzyme B (mean ± SEM, n = 7; ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (G) Tumor-specific CD8+ T cells were incubated with autologous dioctadecyloxacarbocyanine (DIOC18)-labeled primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and the death of tumor cells was assessed by propidium iodide (PI) uptake by flow cytometry. The numbers shown indicate the percentage of PI+ tumor cells (mean ± SEM, n = 4; ***P < 0.001 compared with CD8+ T cells cultured without Tregs).

Mentions: Our data so far suggest that naive CD4+ T cells may differentiate into Tregs within breast tumors. Dendritic cells (DCs) have been shown to induce naive T cell conversion into Tregs in the presence of cancer cell-secreted immunomodulators19,20. To determine whether naive CD4+ T cells can develop into Tregs in the tumor microenvironment13, we co-cultured naive CD4+ T cells, isolated from the PB of breast cancer patients, with autologous plasmacytoid DCs (pDCs), isolated from the blood or breast cancer tissues in normal culture medium or medium that was supplemented with 30% conditioned medium (CM) from autologous cancer tissue slices (Figure 3A and 3B). TI pDCs, but not PB pDCs, converted 7.5% ± 1.7% of naive CD4+ T cells to CD25+Foxp3+ Tregs in unsupplemented medium. Tumor CM on its own without DCs did not induce Treg differentiation of naive CD4+ T cells, but PB pDCs caused Treg differentiation when tumor CM was added. However, the greatest effect, 23.2% ± 3.4% Tregs, was seen when both TI pDCs and tumor CM were added. The co-cultures that increased the numbers of Tregs, defined by CD25 and Foxp3 expression, also increased the mRNA expression of Treg-associated genes, TGFB1, CTLA4, IL10 and GITR by quantitative reverse transcription (qRT)-PCR (Figure 3C). Increases in expression of these genes were in proportion to the generation of CD25+Foxp3+ cells in the cultures, being greatest for naive cells cultured in the presence of both TI pDCs and CM. We observed similar results when myeloid DCs were substituted for pDCs (Supplementary information, Figure S5).


Blocking the recruitment of naive CD4 + T cells reverses immunosuppression in breast cancer
Naive CD4+ T cells are converted to functional Tregs by tumor-infiltrating DCs and tumor conditioned medium (CM). (A-C) Naive CD4+ T cells from peripheral blood of patients with invasive breast carcinoma were co-cultured with or without autologous pDCs isolated from tumor (TI) or peripheral blood (PB) for 9 days in the presence or absence of 30% CM from autologous tumor slices or adjacent normal tissue slices. (A, B) Non-adherent cells from co-cultures were stained for CD3, CD4, CD25 and intracellular Foxp3, and analyzed by flow cytometry. Representative plots of gated CD3+CD4+ cells (A) and quantification of percentage of Foxp3+CD25+ cells among CD3+CD4+ cells (B) are shown (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 by Student's t-test). (C) Expression of Treg-associated genes, assessed by qRT-PCR normalized to GAPDH, in sorted CD4+ T cells, relative to expression in cultures without DCs or CM (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 compared with naive CD4+ T cells cultured alone by Student's t-test). (D-G) Effect of naive CD4+ T cell-derived Tregs, obtained by co-culture with TI pDCs and tumor CM as above, on function of autologous tumor-specific CD8+ T cells. Tumor-specific CD8+ T cells were generated for each subject by stimulating autologous PB CD8+ T cells with autologous tumor lysate-pulsed autologous DCs. Tregs were recovered from co-cultures by magnetic sorting. (D) CFSE-labeled CD8+ T cells were incubated with tumor lysate-pulsed DCs in the presence of induced Tregs at the indicated ratios and proliferation was assessed by flow cytometry. Numbers denote the percentage of cells undergoing at least one cellular division (mean ± SEM, n = 12, **P < 0.01, ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (E-G) Tumor-specific CD8+ T cells were incubated with autologous primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and stained for CD3, CD8, intracellular perforin (E) or granzyme B (F) and gated CD3+CD8+ cells were analyzed by flow cytometry. Numbers indicate the percentage of gated cells stained for perforin or granzyme B (mean ± SEM, n = 7; ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (G) Tumor-specific CD8+ T cells were incubated with autologous dioctadecyloxacarbocyanine (DIOC18)-labeled primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and the death of tumor cells was assessed by propidium iodide (PI) uptake by flow cytometry. The numbers shown indicate the percentage of PI+ tumor cells (mean ± SEM, n = 4; ***P < 0.001 compared with CD8+ T cells cultured without Tregs).
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fig3: Naive CD4+ T cells are converted to functional Tregs by tumor-infiltrating DCs and tumor conditioned medium (CM). (A-C) Naive CD4+ T cells from peripheral blood of patients with invasive breast carcinoma were co-cultured with or without autologous pDCs isolated from tumor (TI) or peripheral blood (PB) for 9 days in the presence or absence of 30% CM from autologous tumor slices or adjacent normal tissue slices. (A, B) Non-adherent cells from co-cultures were stained for CD3, CD4, CD25 and intracellular Foxp3, and analyzed by flow cytometry. Representative plots of gated CD3+CD4+ cells (A) and quantification of percentage of Foxp3+CD25+ cells among CD3+CD4+ cells (B) are shown (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 by Student's t-test). (C) Expression of Treg-associated genes, assessed by qRT-PCR normalized to GAPDH, in sorted CD4+ T cells, relative to expression in cultures without DCs or CM (mean ± SEM, n = 19; *P < 0.05, **P < 0.01,***P < 0.001 compared with naive CD4+ T cells cultured alone by Student's t-test). (D-G) Effect of naive CD4+ T cell-derived Tregs, obtained by co-culture with TI pDCs and tumor CM as above, on function of autologous tumor-specific CD8+ T cells. Tumor-specific CD8+ T cells were generated for each subject by stimulating autologous PB CD8+ T cells with autologous tumor lysate-pulsed autologous DCs. Tregs were recovered from co-cultures by magnetic sorting. (D) CFSE-labeled CD8+ T cells were incubated with tumor lysate-pulsed DCs in the presence of induced Tregs at the indicated ratios and proliferation was assessed by flow cytometry. Numbers denote the percentage of cells undergoing at least one cellular division (mean ± SEM, n = 12, **P < 0.01, ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (E-G) Tumor-specific CD8+ T cells were incubated with autologous primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and stained for CD3, CD8, intracellular perforin (E) or granzyme B (F) and gated CD3+CD8+ cells were analyzed by flow cytometry. Numbers indicate the percentage of gated cells stained for perforin or granzyme B (mean ± SEM, n = 7; ***P < 0.001 compared with CD8+ T cells cultured without Tregs). (G) Tumor-specific CD8+ T cells were incubated with autologous dioctadecyloxacarbocyanine (DIOC18)-labeled primary breast cancer cells for 18 h in the presence or absence of Tregs (CD8:Treg 2:1) and the death of tumor cells was assessed by propidium iodide (PI) uptake by flow cytometry. The numbers shown indicate the percentage of PI+ tumor cells (mean ± SEM, n = 4; ***P < 0.001 compared with CD8+ T cells cultured without Tregs).
Mentions: Our data so far suggest that naive CD4+ T cells may differentiate into Tregs within breast tumors. Dendritic cells (DCs) have been shown to induce naive T cell conversion into Tregs in the presence of cancer cell-secreted immunomodulators19,20. To determine whether naive CD4+ T cells can develop into Tregs in the tumor microenvironment13, we co-cultured naive CD4+ T cells, isolated from the PB of breast cancer patients, with autologous plasmacytoid DCs (pDCs), isolated from the blood or breast cancer tissues in normal culture medium or medium that was supplemented with 30% conditioned medium (CM) from autologous cancer tissue slices (Figure 3A and 3B). TI pDCs, but not PB pDCs, converted 7.5% ± 1.7% of naive CD4+ T cells to CD25+Foxp3+ Tregs in unsupplemented medium. Tumor CM on its own without DCs did not induce Treg differentiation of naive CD4+ T cells, but PB pDCs caused Treg differentiation when tumor CM was added. However, the greatest effect, 23.2% ± 3.4% Tregs, was seen when both TI pDCs and tumor CM were added. The co-cultures that increased the numbers of Tregs, defined by CD25 and Foxp3 expression, also increased the mRNA expression of Treg-associated genes, TGFB1, CTLA4, IL10 and GITR by quantitative reverse transcription (qRT)-PCR (Figure 3C). Increases in expression of these genes were in proportion to the generation of CD25+Foxp3+ cells in the cultures, being greatest for naive cells cultured in the presence of both TI pDCs and CM. We observed similar results when myeloid DCs were substituted for pDCs (Supplementary information, Figure S5).

View Article: PubMed Central - PubMed

ABSTRACT

The origin of tumor-infiltrating Tregs, critical mediators of tumor immunosuppression, is unclear. Here, we show that tumor-infiltrating naive CD4+ T cells and Tregs in human breast cancer have overlapping TCR repertoires, while hardly overlap with circulating Tregs, suggesting that intratumoral Tregs mainly develop from naive T cells in situ rather than from recruited Tregs. Furthermore, the abundance of naive CD4+ T cells and Tregs is closely correlated, both indicating poor prognosis for breast cancer patients. Naive CD4+ T cells adhere to tumor slices in proportion to the abundance of CCL18-producing macrophages. Moreover, adoptively transferred human naive CD4+ T cells infiltrate human breast cancer orthotopic xenografts in a CCL18-dependent manner. In human breast cancer xenografts in humanized mice, blocking the recruitment of naive CD4+ T cells into tumor by knocking down the expression of PITPNM3, a CCL18 receptor, significantly reduces intratumoral Tregs and inhibits tumor progression. These findings suggest that breast tumor-infiltrating Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Inhibiting naive CD4+ T cell recruitment into tumors by interfering with PITPNM3 recognition of CCL18 may be an attractive strategy for anticancer immunotherapy.

No MeSH data available.


Related in: MedlinePlus