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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 cell abundance within breast tumors is associated with increased numbers of Tregs and poor patient prognosis. (A) IHC staining of CD3 (red) and CD45RA (brown) in a representative breast cancer sample. Naive T cells were defined as CD3+CD45+ cells. Scale bar, 50 μm. (B) Correlation of TI naive CD4+ T cell numbers and TI Treg numbers in breast cancer samples (n = 626, Pearson correlation coefficient R and P-value are shown). (C) Representative immunofluorescent staining for naive T cells (CD3 (red), CD45RA (green) and CD4 (purple); upper panels) or Tregs (Foxp3 (green) and CD4 (purple); lower panels) in serial sections from a human breast cancer sample. Arrows indicate CD3+CD4+CD45RA+ naive CD4+ T cells (upper) and CD4+Foxp3+ Tregs (lower). Scale bar, 50 μm. The localization of naive CD4 T cells and Tregs relative to the perivascular space or tumor parenchyma for 626 tumor samples is provided in Supplementary information, Figure S3C. (D) Representative immunofluorescent staining of CD3 (red), CD45RA (green), CD4 (purple) and DAPI (blue) in breast cancer samples with high (upper panel) or low (lower panel) number of naive CD4+ T cells, which are indicated by arrows. Scale bar, 50 μm. (E) Kaplan-Meier survival curve of breast cancer patients with low and high numbers of TI naive CD4+ T cells. BV, blood vessel; TN, tumor nest.
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fig2: Naive CD4+ T cell abundance within breast tumors is associated with increased numbers of Tregs and poor patient prognosis. (A) IHC staining of CD3 (red) and CD45RA (brown) in a representative breast cancer sample. Naive T cells were defined as CD3+CD45+ cells. Scale bar, 50 μm. (B) Correlation of TI naive CD4+ T cell numbers and TI Treg numbers in breast cancer samples (n = 626, Pearson correlation coefficient R and P-value are shown). (C) Representative immunofluorescent staining for naive T cells (CD3 (red), CD45RA (green) and CD4 (purple); upper panels) or Tregs (Foxp3 (green) and CD4 (purple); lower panels) in serial sections from a human breast cancer sample. Arrows indicate CD3+CD4+CD45RA+ naive CD4+ T cells (upper) and CD4+Foxp3+ Tregs (lower). Scale bar, 50 μm. The localization of naive CD4 T cells and Tregs relative to the perivascular space or tumor parenchyma for 626 tumor samples is provided in Supplementary information, Figure S3C. (D) Representative immunofluorescent staining of CD3 (red), CD45RA (green), CD4 (purple) and DAPI (blue) in breast cancer samples with high (upper panel) or low (lower panel) number of naive CD4+ T cells, which are indicated by arrows. Scale bar, 50 μm. (E) Kaplan-Meier survival curve of breast cancer patients with low and high numbers of TI naive CD4+ T cells. BV, blood vessel; TN, tumor nest.

Mentions: We next evaluated by immunohistochemistry (IHC) whether we could detect naive T cells within clinical breast cancer tissues. CD3+CD45RA+ naive T cells were detected in 74% (462 of 626 cases) of clinical breast cancer tissues, predominantly in the perivascular space (Figure 2A). The number of naive CD4+ T cells positively correlated with the numbers of Tregs in tumors (P < 0.0001; Figure 2B). In contrast, the number of memory CD4+ T cells (CD4+CD45RO+Foxp3−) in tumors negatively correlated with the number of Tregs and naive CD4+ T cells in the slides (Supplementary information, Figure S3A). We also used immunofluorescence microscopy to analyze serial breast tumor sections stained for CD3 and CD4, and either CD45RA (naive CD4+ T cells) or Foxp3 (Tregs). Naive CD4+ T cells were more prominent in the perivascular space, whereas the majority of Tregs were far from blood vessels and localized close to the tumor parenchyma (Figure 2C, Supplementary information, Figure S3B and S3C). These data are consistent with the possibility that naive CD4+ T cells entering from the blood differentiate into Tregs after they migrate toward the tumor.


Blocking the recruitment of naive CD4 + T cells reverses immunosuppression in breast cancer
Naive CD4+ T cell abundance within breast tumors is associated with increased numbers of Tregs and poor patient prognosis. (A) IHC staining of CD3 (red) and CD45RA (brown) in a representative breast cancer sample. Naive T cells were defined as CD3+CD45+ cells. Scale bar, 50 μm. (B) Correlation of TI naive CD4+ T cell numbers and TI Treg numbers in breast cancer samples (n = 626, Pearson correlation coefficient R and P-value are shown). (C) Representative immunofluorescent staining for naive T cells (CD3 (red), CD45RA (green) and CD4 (purple); upper panels) or Tregs (Foxp3 (green) and CD4 (purple); lower panels) in serial sections from a human breast cancer sample. Arrows indicate CD3+CD4+CD45RA+ naive CD4+ T cells (upper) and CD4+Foxp3+ Tregs (lower). Scale bar, 50 μm. The localization of naive CD4 T cells and Tregs relative to the perivascular space or tumor parenchyma for 626 tumor samples is provided in Supplementary information, Figure S3C. (D) Representative immunofluorescent staining of CD3 (red), CD45RA (green), CD4 (purple) and DAPI (blue) in breast cancer samples with high (upper panel) or low (lower panel) number of naive CD4+ T cells, which are indicated by arrows. Scale bar, 50 μm. (E) Kaplan-Meier survival curve of breast cancer patients with low and high numbers of TI naive CD4+ T cells. BV, blood vessel; TN, tumor nest.
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fig2: Naive CD4+ T cell abundance within breast tumors is associated with increased numbers of Tregs and poor patient prognosis. (A) IHC staining of CD3 (red) and CD45RA (brown) in a representative breast cancer sample. Naive T cells were defined as CD3+CD45+ cells. Scale bar, 50 μm. (B) Correlation of TI naive CD4+ T cell numbers and TI Treg numbers in breast cancer samples (n = 626, Pearson correlation coefficient R and P-value are shown). (C) Representative immunofluorescent staining for naive T cells (CD3 (red), CD45RA (green) and CD4 (purple); upper panels) or Tregs (Foxp3 (green) and CD4 (purple); lower panels) in serial sections from a human breast cancer sample. Arrows indicate CD3+CD4+CD45RA+ naive CD4+ T cells (upper) and CD4+Foxp3+ Tregs (lower). Scale bar, 50 μm. The localization of naive CD4 T cells and Tregs relative to the perivascular space or tumor parenchyma for 626 tumor samples is provided in Supplementary information, Figure S3C. (D) Representative immunofluorescent staining of CD3 (red), CD45RA (green), CD4 (purple) and DAPI (blue) in breast cancer samples with high (upper panel) or low (lower panel) number of naive CD4+ T cells, which are indicated by arrows. Scale bar, 50 μm. (E) Kaplan-Meier survival curve of breast cancer patients with low and high numbers of TI naive CD4+ T cells. BV, blood vessel; TN, tumor nest.
Mentions: We next evaluated by immunohistochemistry (IHC) whether we could detect naive T cells within clinical breast cancer tissues. CD3+CD45RA+ naive T cells were detected in 74% (462 of 626 cases) of clinical breast cancer tissues, predominantly in the perivascular space (Figure 2A). The number of naive CD4+ T cells positively correlated with the numbers of Tregs in tumors (P < 0.0001; Figure 2B). In contrast, the number of memory CD4+ T cells (CD4+CD45RO+Foxp3−) in tumors negatively correlated with the number of Tregs and naive CD4+ T cells in the slides (Supplementary information, Figure S3A). We also used immunofluorescence microscopy to analyze serial breast tumor sections stained for CD3 and CD4, and either CD45RA (naive CD4+ T cells) or Foxp3 (Tregs). Naive CD4+ T cells were more prominent in the perivascular space, whereas the majority of Tregs were far from blood vessels and localized close to the tumor parenchyma (Figure 2C, Supplementary information, Figure S3B and S3C). These data are consistent with the possibility that naive CD4+ T cells entering from the blood differentiate into Tregs after they migrate toward the tumor.

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