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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

CD4-aptamer-siRNA targeting PITPNM3 reduces TI Tregs and inhibits tumor progression in humanized mice with circulating human Tregs. Humanized mice, implanted with MDA-MB-231 tumors and concurrently injected intravenously with autologous Tregs, were intraperitoneally injected daily for 14 days after tumors became palpable with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Tregs were administered every 10 days after the initial injection and mice were sacrificed 30 days after tumor cell inoculation. (A) Experimental schematic. (B, C) Peripheral blood cells of humanized mice were stained for human CD3, CD4 and Foxp3, and analyzed by flow cytometry. A representative flow plot (B) and the percentage (mean ± SEM) of PB CD4+ cells that are CFSE+ Tregs in six mice per group (C) are shown. (D, E) Isolated cells from xenografts were stained for human CD3, CD4 and Foxp3. The percentage (mean ± SEM) of six mice per group (D) and representative flow plot (E) of FoxP3+ Tregs are shown. Most Tregs were CFSE− (i.e., did not come from infused Tregs) and the number of TI Tregs was reduced by knocking down PITPNM3 in CD4+ T cells (***P < 0.001 compared to the PBS group by Student's t-test). (F) Tumor size (mean ± SEM, n = 6 per group; ***P < 0.001 by two-way ANOVA with Bonferroni multiple comparison tests). (G) Lung metastases assessed by qRT-PCR analysis of human HPRT mRNA relative to mouse 18S rRNA in the lungs. Data are shown as mean ± SEM (n = 6 per group; **P < 0.01 by Student's t-test). NS, not statistically significant by Student's t-test.
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fig7: CD4-aptamer-siRNA targeting PITPNM3 reduces TI Tregs and inhibits tumor progression in humanized mice with circulating human Tregs. Humanized mice, implanted with MDA-MB-231 tumors and concurrently injected intravenously with autologous Tregs, were intraperitoneally injected daily for 14 days after tumors became palpable with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Tregs were administered every 10 days after the initial injection and mice were sacrificed 30 days after tumor cell inoculation. (A) Experimental schematic. (B, C) Peripheral blood cells of humanized mice were stained for human CD3, CD4 and Foxp3, and analyzed by flow cytometry. A representative flow plot (B) and the percentage (mean ± SEM) of PB CD4+ cells that are CFSE+ Tregs in six mice per group (C) are shown. (D, E) Isolated cells from xenografts were stained for human CD3, CD4 and Foxp3. The percentage (mean ± SEM) of six mice per group (D) and representative flow plot (E) of FoxP3+ Tregs are shown. Most Tregs were CFSE− (i.e., did not come from infused Tregs) and the number of TI Tregs was reduced by knocking down PITPNM3 in CD4+ T cells (***P < 0.001 compared to the PBS group by Student's t-test). (F) Tumor size (mean ± SEM, n = 6 per group; ***P < 0.001 by two-way ANOVA with Bonferroni multiple comparison tests). (G) Lung metastases assessed by qRT-PCR analysis of human HPRT mRNA relative to mouse 18S rRNA in the lungs. Data are shown as mean ± SEM (n = 6 per group; **P < 0.01 by Student's t-test). NS, not statistically significant by Student's t-test.

Mentions: Because human PB Tregs were rare in the tumor-bearing humanized mice, to better mimic human breast cancer and determine whether PB Tregs infiltrate the tumors, we adoptively transferred autologous Tregs, expanded ex vivo from CB as previously described28,29, 6 weeks after the transplant of hematopoietic stem cells (HSCs) at the time of concomitant tumor implantation and every 10 days thereafter in the humanized tumor model described above. The transferred Tregs were labeled with CFSE (Figure 7A). After transfer, Foxp3+ Tregs accounted for about 4% of human PB CD4+ T cells (Figure 7B and 7C). Most of the PB Tregs were CFSE labeled, indicating that they were mainly adoptively transferred. When the tumors became palpable, the mice were injected intraperitoneally with PBS, CD4-AsiC-con or CD4-AsiC-PI. AsiC treatment did not affect the number of PB Tregs (Figure 7C). In the control mice given PBS or CD4-AsiC-con, Tregs accounted for about 30% of TI CD4+ T cells and most of these were CFSE− (Figure 7D and 7E). The proportion of TI Tregs in the adoptively transferred mice was similar to that in the humanized mice that did not receive transferred Tregs (compare Figure 6F, Figure 7D-7E). These data, together with the paucity of CFSE-stained Tregs in the tumors from adoptively transferred mice, strongly suggest that TI Tregs were mainly derived from recruited naive CD4+ T cells, rather than from circulating Tregs. In fact, in the adoptively transferred tumor-bearing mice, knockdown of PITPNM3 by CD4-AsiC-PI in CD4+ T cells markedly reduced CFSE− TI Tregs, but had no effect on the small number of TI CFSE+ Tregs (Figure 7D and 7E). As before, CD4-AsiC-PI treatment dramatically reduced tumor growth and lung metastasis in the adoptively transferred tumor-bearing mice (Figure 7F and 7G). Together, these results suggest that Tregs are generated from naive CD4+ T cells responding via PITPNM3 to CCL18 produced in the tumor. They also suggest that interfering with PIPTNM3 or CCL18 could be used therapeutically to enhance anti-tumor immunity in breast cancer.


Blocking the recruitment of naive CD4 + T cells reverses immunosuppression in breast cancer
CD4-aptamer-siRNA targeting PITPNM3 reduces TI Tregs and inhibits tumor progression in humanized mice with circulating human Tregs. Humanized mice, implanted with MDA-MB-231 tumors and concurrently injected intravenously with autologous Tregs, were intraperitoneally injected daily for 14 days after tumors became palpable with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Tregs were administered every 10 days after the initial injection and mice were sacrificed 30 days after tumor cell inoculation. (A) Experimental schematic. (B, C) Peripheral blood cells of humanized mice were stained for human CD3, CD4 and Foxp3, and analyzed by flow cytometry. A representative flow plot (B) and the percentage (mean ± SEM) of PB CD4+ cells that are CFSE+ Tregs in six mice per group (C) are shown. (D, E) Isolated cells from xenografts were stained for human CD3, CD4 and Foxp3. The percentage (mean ± SEM) of six mice per group (D) and representative flow plot (E) of FoxP3+ Tregs are shown. Most Tregs were CFSE− (i.e., did not come from infused Tregs) and the number of TI Tregs was reduced by knocking down PITPNM3 in CD4+ T cells (***P < 0.001 compared to the PBS group by Student's t-test). (F) Tumor size (mean ± SEM, n = 6 per group; ***P < 0.001 by two-way ANOVA with Bonferroni multiple comparison tests). (G) Lung metastases assessed by qRT-PCR analysis of human HPRT mRNA relative to mouse 18S rRNA in the lungs. Data are shown as mean ± SEM (n = 6 per group; **P < 0.01 by Student's t-test). NS, not statistically significant by Student's t-test.
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fig7: CD4-aptamer-siRNA targeting PITPNM3 reduces TI Tregs and inhibits tumor progression in humanized mice with circulating human Tregs. Humanized mice, implanted with MDA-MB-231 tumors and concurrently injected intravenously with autologous Tregs, were intraperitoneally injected daily for 14 days after tumors became palpable with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Tregs were administered every 10 days after the initial injection and mice were sacrificed 30 days after tumor cell inoculation. (A) Experimental schematic. (B, C) Peripheral blood cells of humanized mice were stained for human CD3, CD4 and Foxp3, and analyzed by flow cytometry. A representative flow plot (B) and the percentage (mean ± SEM) of PB CD4+ cells that are CFSE+ Tregs in six mice per group (C) are shown. (D, E) Isolated cells from xenografts were stained for human CD3, CD4 and Foxp3. The percentage (mean ± SEM) of six mice per group (D) and representative flow plot (E) of FoxP3+ Tregs are shown. Most Tregs were CFSE− (i.e., did not come from infused Tregs) and the number of TI Tregs was reduced by knocking down PITPNM3 in CD4+ T cells (***P < 0.001 compared to the PBS group by Student's t-test). (F) Tumor size (mean ± SEM, n = 6 per group; ***P < 0.001 by two-way ANOVA with Bonferroni multiple comparison tests). (G) Lung metastases assessed by qRT-PCR analysis of human HPRT mRNA relative to mouse 18S rRNA in the lungs. Data are shown as mean ± SEM (n = 6 per group; **P < 0.01 by Student's t-test). NS, not statistically significant by Student's t-test.
Mentions: Because human PB Tregs were rare in the tumor-bearing humanized mice, to better mimic human breast cancer and determine whether PB Tregs infiltrate the tumors, we adoptively transferred autologous Tregs, expanded ex vivo from CB as previously described28,29, 6 weeks after the transplant of hematopoietic stem cells (HSCs) at the time of concomitant tumor implantation and every 10 days thereafter in the humanized tumor model described above. The transferred Tregs were labeled with CFSE (Figure 7A). After transfer, Foxp3+ Tregs accounted for about 4% of human PB CD4+ T cells (Figure 7B and 7C). Most of the PB Tregs were CFSE labeled, indicating that they were mainly adoptively transferred. When the tumors became palpable, the mice were injected intraperitoneally with PBS, CD4-AsiC-con or CD4-AsiC-PI. AsiC treatment did not affect the number of PB Tregs (Figure 7C). In the control mice given PBS or CD4-AsiC-con, Tregs accounted for about 30% of TI CD4+ T cells and most of these were CFSE− (Figure 7D and 7E). The proportion of TI Tregs in the adoptively transferred mice was similar to that in the humanized mice that did not receive transferred Tregs (compare Figure 6F, Figure 7D-7E). These data, together with the paucity of CFSE-stained Tregs in the tumors from adoptively transferred mice, strongly suggest that TI Tregs were mainly derived from recruited naive CD4+ T cells, rather than from circulating Tregs. In fact, in the adoptively transferred tumor-bearing mice, knockdown of PITPNM3 by CD4-AsiC-PI in CD4+ T cells markedly reduced CFSE− TI Tregs, but had no effect on the small number of TI CFSE+ Tregs (Figure 7D and 7E). As before, CD4-AsiC-PI treatment dramatically reduced tumor growth and lung metastasis in the adoptively transferred tumor-bearing mice (Figure 7F and 7G). Together, these results suggest that Tregs are generated from naive CD4+ T cells responding via PITPNM3 to CCL18 produced in the tumor. They also suggest that interfering with PIPTNM3 or CCL18 could be used therapeutically to enhance anti-tumor immunity 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