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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.


CCR5 and CCR6 show differential expressions on CD8+ and regulatory T lymphocytes in PBLs and matched TILS. Paired PBL and TIL samples were stained with antibodies specific for CD3, CD4, CD8, FoxP3, CCR4 and CCR6. The CD8+ T lymphocytes (CD3+CD8+) and Treg lymphocytes (CD3+CD4+FoxP3+) were separately gated, and then the expressions of CCR5 and CCR6 were determined with multicolour flow cytometry. (A) Representative plots of CCR5 staining on CD8+ T lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR5 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel). (B) Representative plots of CCR6 staining on Treg lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR6 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel).
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fig2: CCR5 and CCR6 show differential expressions on CD8+ and regulatory T lymphocytes in PBLs and matched TILS. Paired PBL and TIL samples were stained with antibodies specific for CD3, CD4, CD8, FoxP3, CCR4 and CCR6. The CD8+ T lymphocytes (CD3+CD8+) and Treg lymphocytes (CD3+CD4+FoxP3+) were separately gated, and then the expressions of CCR5 and CCR6 were determined with multicolour flow cytometry. (A) Representative plots of CCR5 staining on CD8+ T lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR5 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel). (B) Representative plots of CCR6 staining on Treg lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR6 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel).

Mentions: As specific receptors are critical for T lymphocytes moving towards tumour tissues in response to the recruitment of chemokines, monitoring the expression differences of chemokine receptors is helpful for elucidating the roles of specific chemokines. First, we detected the frequencies of CCR5 (CCL4-specific receptor) and CCR6 (CCL20-specific receptor) on peripheral CD8+ T and Treg lymphocytes. As shown in Figure 2A, the proportions of CCR5-positive cells in CD8+ T lymphocytes were significantly greater than in CD4+FoxP3+ Treg lymphocytes (21.60%±15.83% vs 11.61%±14.34% P<0.01). The percentages of CCR6-positive cells in regulatory T lymphocyte groups were 38.83%±22.76%, and the counterparts in CD8+ T lymphocytes were 3.95%±5.03% (P<0.001; Figure 2B). These observations indicate that CCR5 preferentially expresses on CD8+ T lymphocytes, whereas CCR6 shows biased expression on Treg lymphocytes. Following that, we examined tumour-infiltrating T lymphocytes. Compared with 34.38%±13.35% in peripheral blood, the percentages of CD8+ T lymphocytes were obviously increased to 42.84%±15.24% (t=3.156, P<0.005; Supplementary Figure 2). Similarly, the portions of Treg lymphocytes were sharply elevated to 16.65%±9.77% in TILs from 6.67%±6.71% in PBLs (Supplementary Figure 2). The difference is statistically significant (t=6.42, P<0.0001). These observations indicated that CD8+ T and Treg lymphocytes are efficiently recruited into ESCC sites. In tumour-infiltrating CD8+ T lymphocytes, CCR5 expressed on greater portions of cells (Figure 2A). The difference between CCR5-expressing CD8+ T lymphocytes in TILs and PBLs is statistically significant (45.90%±25.28% vs 21.60%±15.82%, P<0.001; Figure 2A). In Treg lymphocytes in tumour sites, CCR5 expression was also observed. However, the intensities of CCR5+ Treg lymphocytes in TILs were not obviously enhanced, compared with the counterparts in PBLs (19.32%±17.41% vs 11.61%±14.34%, P>0.05; Figure 2A). Moreover, CCR5+ Treg lymphocytes were less frequent than CCR5+CD8+ T lymphocytes in tumours (P<0.0001). Conversely, CCR6 expression was robustly increased in tumour-infiltrating Treg lymphocytes (Figure 2B). The portions of CCR6+ Treg lymphocytes in TILs were much greater than that in PBLs (57.49%±29.58% vs 38.83%±22.76%, P<0.05), or CCR6+CD8+ T lymphocytes in TILs (57.49%±29.58% vs 10.77%±10.04%, P<0.0001; Figure 2B). Together with the RT–PCR data, these results indicate that CCL4 plays major roles in recruiting CD8+ T lymphocytes into malignancies, whereas CCL20 mainly directs the tumoural movement of Treg lymphocytes.


CTL- vs T reg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma
CCR5 and CCR6 show differential expressions on CD8+ and regulatory T lymphocytes in PBLs and matched TILS. Paired PBL and TIL samples were stained with antibodies specific for CD3, CD4, CD8, FoxP3, CCR4 and CCR6. The CD8+ T lymphocytes (CD3+CD8+) and Treg lymphocytes (CD3+CD4+FoxP3+) were separately gated, and then the expressions of CCR5 and CCR6 were determined with multicolour flow cytometry. (A) Representative plots of CCR5 staining on CD8+ T lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR5 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel). (B) Representative plots of CCR6 staining on Treg lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR6 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel).
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Related In: Results  -  Collection

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fig2: CCR5 and CCR6 show differential expressions on CD8+ and regulatory T lymphocytes in PBLs and matched TILS. Paired PBL and TIL samples were stained with antibodies specific for CD3, CD4, CD8, FoxP3, CCR4 and CCR6. The CD8+ T lymphocytes (CD3+CD8+) and Treg lymphocytes (CD3+CD4+FoxP3+) were separately gated, and then the expressions of CCR5 and CCR6 were determined with multicolour flow cytometry. (A) Representative plots of CCR5 staining on CD8+ T lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR5 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel). (B) Representative plots of CCR6 staining on Treg lymphocytes in matched PBLs and TILs (left panel). Paired analysis of CCR6 expressions on CD8+ T and Treg lymphocytes in paired PBLs and TILs (right panel).
Mentions: As specific receptors are critical for T lymphocytes moving towards tumour tissues in response to the recruitment of chemokines, monitoring the expression differences of chemokine receptors is helpful for elucidating the roles of specific chemokines. First, we detected the frequencies of CCR5 (CCL4-specific receptor) and CCR6 (CCL20-specific receptor) on peripheral CD8+ T and Treg lymphocytes. As shown in Figure 2A, the proportions of CCR5-positive cells in CD8+ T lymphocytes were significantly greater than in CD4+FoxP3+ Treg lymphocytes (21.60%±15.83% vs 11.61%±14.34% P<0.01). The percentages of CCR6-positive cells in regulatory T lymphocyte groups were 38.83%±22.76%, and the counterparts in CD8+ T lymphocytes were 3.95%±5.03% (P<0.001; Figure 2B). These observations indicate that CCR5 preferentially expresses on CD8+ T lymphocytes, whereas CCR6 shows biased expression on Treg lymphocytes. Following that, we examined tumour-infiltrating T lymphocytes. Compared with 34.38%±13.35% in peripheral blood, the percentages of CD8+ T lymphocytes were obviously increased to 42.84%±15.24% (t=3.156, P<0.005; Supplementary Figure 2). Similarly, the portions of Treg lymphocytes were sharply elevated to 16.65%±9.77% in TILs from 6.67%±6.71% in PBLs (Supplementary Figure 2). The difference is statistically significant (t=6.42, P<0.0001). These observations indicated that CD8+ T and Treg lymphocytes are efficiently recruited into ESCC sites. In tumour-infiltrating CD8+ T lymphocytes, CCR5 expressed on greater portions of cells (Figure 2A). The difference between CCR5-expressing CD8+ T lymphocytes in TILs and PBLs is statistically significant (45.90%±25.28% vs 21.60%±15.82%, P<0.001; Figure 2A). In Treg lymphocytes in tumour sites, CCR5 expression was also observed. However, the intensities of CCR5+ Treg lymphocytes in TILs were not obviously enhanced, compared with the counterparts in PBLs (19.32%±17.41% vs 11.61%±14.34%, P>0.05; Figure 2A). Moreover, CCR5+ Treg lymphocytes were less frequent than CCR5+CD8+ T lymphocytes in tumours (P<0.0001). Conversely, CCR6 expression was robustly increased in tumour-infiltrating Treg lymphocytes (Figure 2B). The portions of CCR6+ Treg lymphocytes in TILs were much greater than that in PBLs (57.49%±29.58% vs 38.83%±22.76%, P<0.05), or CCR6+CD8+ T lymphocytes in TILs (57.49%±29.58% vs 10.77%±10.04%, P<0.0001; Figure 2B). Together with the RT–PCR data, these results indicate that CCL4 plays major roles in recruiting CD8+ T lymphocytes into malignancies, whereas CCL20 mainly directs the tumoural movement of Treg lymphocytes.

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.