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CTL- vs T reg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Tumoural infiltration of T lymphocytes is determined by local patterns of specific chemokine expression. In this report, we examined the roles of CCL4 and CCL20 in the accumulation of CD8+ cytotoxic T lymphocytes (CTLs) and regulatory T (Treg) lymphocytes in oesophageal squamous cell carcinoma (ESCC), and determined the correlations between chemokine expressions and ESCC patients' survival.

Methods:: Reverse transcriptase–PCR and immunohistochemistry (IHC) staining were performed to examine the expressions of interested genes. Flow cytometry was adopted to check the expressions of CCL4- and CCL6-specific receptors, CCR5 and CCR6, on CTLs and Treg cells. In addition, transwell assay was carried on.

Results:: The CCL4 expression was significantly correlated with the expression of CTL markers (CD8 and Granzyme B), whereas CCL20 was positively correlated with Treg markers (FoxP3 and IL-10). Consistently, CCR5 was found to be mainly expressed on CD8+ T lymphocytes, while CCR6 showed prevalence on Treg lymphocytes and the frequencies of CCR5+CD8+ CTLs and CCR6+ Treg cells were higher in TIL compared with PBMC. Respectively, CCL4 and CCL20 recruited CD8+ and regulatory T cells in vitro. Importantly, high levels of CCL4 in the lesions of ESCC patients predicted prolonged survival. Furthermore, CCL4high/CCL20low group demonstrated better overall survival, whereas CCL4low/CCL20low and CCL4low/CCL20high groups showed the worst overall survival.

Conclusions:: Our data showed that CCL4 and CCL20 recruit functionally different T lymphocyte subsets into oesophageal carcinoma, indicating CCL4 and CCL20 are potential predictors of ESCC patients' survival.

No MeSH data available.


Intratumoural expression levels of CCL4 and CCL20 predict patients' survival. (A) Kaplan–Meier survival curve comparing the survival of patients with or without lymph node metastasis. (B and C) Kaplan–Meier survival curve comparing the high and the low CCL4 (B) or CCL20 (C) expression groups. (D) Kaplan–Meier survival curve according to the expressions of both CCL4 and CCL20.
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fig5: Intratumoural expression levels of CCL4 and CCL20 predict patients' survival. (A) Kaplan–Meier survival curve comparing the survival of patients with or without lymph node metastasis. (B and C) Kaplan–Meier survival curve comparing the high and the low CCL4 (B) or CCL20 (C) expression groups. (D) Kaplan–Meier survival curve according to the expressions of both CCL4 and CCL20.

Mentions: First, we checked the associations of the patterns of chemokine expression with clinical and pathologic parameters (Supplementary Table 2). The expressions of CCL4 were not associated with patients' gender, age, clinical stage, T stage, lymph node metastasis and tumour length. Similarly, the CCL20 levels were not associated with these factors, except for lymph node invasion (Supplementary Table 2). Increased expressions of CCL20 were observed in patients with lymph node metastases (P<0.05). The lymph node invasion was significantly associated with patients' survival (P<0.05; Figure 5A and Table 1). Prompted by these data, we investigated the effects of CCL4 and CCL20 on clinical outcomes. For CCL4, increased expression showed a significant association with prolonged overall survival (Figure 5B and Table 1). The medium survival time of CCL4high group was 53 months, whereas it was only 26.5 months in CCL4low group (P<0.001). Between low and high CCL20-expressing patients, the difference in survival was not significant (P>0.05; Figure 5C and Table 1). Nevertheless, further classification of patients into four groups, according to both expressions of CCL4 and CCL20, revealed a prognostic role of CCL20. As shown in Figure 5D, the CCL4high/CCL20low group demonstrated best overall survival compared with other three groups (P<0.005), whereas the CCL4low/CCL20high and CCL4low/CCL20low groups showed poor survival. To determine whether chemokines were predicting markers for patients' survival, multivariate analysis was performed. The results revealed that CCL4 expression is a strong predictive factor for increased survival (P<0.005; Table 1). Lymph node invasion was an independent predictive factor for survival as well (P<0.05; Table 1).


CTL- vs T reg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma
Intratumoural expression levels of CCL4 and CCL20 predict patients' survival. (A) Kaplan–Meier survival curve comparing the survival of patients with or without lymph node metastasis. (B and C) Kaplan–Meier survival curve comparing the high and the low CCL4 (B) or CCL20 (C) expression groups. (D) Kaplan–Meier survival curve according to the expressions of both CCL4 and CCL20.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4559838&req=5

fig5: Intratumoural expression levels of CCL4 and CCL20 predict patients' survival. (A) Kaplan–Meier survival curve comparing the survival of patients with or without lymph node metastasis. (B and C) Kaplan–Meier survival curve comparing the high and the low CCL4 (B) or CCL20 (C) expression groups. (D) Kaplan–Meier survival curve according to the expressions of both CCL4 and CCL20.
Mentions: First, we checked the associations of the patterns of chemokine expression with clinical and pathologic parameters (Supplementary Table 2). The expressions of CCL4 were not associated with patients' gender, age, clinical stage, T stage, lymph node metastasis and tumour length. Similarly, the CCL20 levels were not associated with these factors, except for lymph node invasion (Supplementary Table 2). Increased expressions of CCL20 were observed in patients with lymph node metastases (P<0.05). The lymph node invasion was significantly associated with patients' survival (P<0.05; Figure 5A and Table 1). Prompted by these data, we investigated the effects of CCL4 and CCL20 on clinical outcomes. For CCL4, increased expression showed a significant association with prolonged overall survival (Figure 5B and Table 1). The medium survival time of CCL4high group was 53 months, whereas it was only 26.5 months in CCL4low group (P<0.001). Between low and high CCL20-expressing patients, the difference in survival was not significant (P>0.05; Figure 5C and Table 1). Nevertheless, further classification of patients into four groups, according to both expressions of CCL4 and CCL20, revealed a prognostic role of CCL20. As shown in Figure 5D, the CCL4high/CCL20low group demonstrated best overall survival compared with other three groups (P<0.005), whereas the CCL4low/CCL20high and CCL4low/CCL20low groups showed poor survival. To determine whether chemokines were predicting markers for patients' survival, multivariate analysis was performed. The results revealed that CCL4 expression is a strong predictive factor for increased survival (P<0.005; Table 1). Lymph node invasion was an independent predictive factor for survival as well (P<0.05; Table 1).

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Tumoural infiltration of T lymphocytes is determined by local patterns of specific chemokine expression. In this report, we examined the roles of CCL4 and CCL20 in the accumulation of CD8+ cytotoxic T lymphocytes (CTLs) and regulatory T (Treg) lymphocytes in oesophageal squamous cell carcinoma (ESCC), and determined the correlations between chemokine expressions and ESCC patients' survival.

Methods:: Reverse transcriptase&ndash;PCR and immunohistochemistry (IHC) staining were performed to examine the expressions of interested genes. Flow cytometry was adopted to check the expressions of CCL4- and CCL6-specific receptors, CCR5 and CCR6, on CTLs and Treg cells. In addition, transwell assay was carried on.

Results:: The CCL4 expression was significantly correlated with the expression of CTL markers (CD8 and Granzyme B), whereas CCL20 was positively correlated with Treg markers (FoxP3 and IL-10). Consistently, CCR5 was found to be mainly expressed on CD8+ T lymphocytes, while CCR6 showed prevalence on Treg lymphocytes and the frequencies of CCR5+CD8+ CTLs and CCR6+ Treg cells were higher in TIL compared with PBMC. Respectively, CCL4 and CCL20 recruited CD8+ and regulatory T cells in vitro. Importantly, high levels of CCL4 in the lesions of ESCC patients predicted prolonged survival. Furthermore, CCL4high/CCL20low group demonstrated better overall survival, whereas CCL4low/CCL20low and CCL4low/CCL20high groups showed the worst overall survival.

Conclusions:: Our data showed that CCL4 and CCL20 recruit functionally different T lymphocyte subsets into oesophageal carcinoma, indicating CCL4 and CCL20 are potential predictors of ESCC patients' survival.

No MeSH data available.