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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.


Related in: MedlinePlus

CCL6 and CCL20 individually enhance the migrations of CD8+ and regulatory T lymphocytes in vitro. (A) CD8+CCR5− and CD8+CCR5+ T lymphocytes were isolated from TILs of ESCC patients using MoFlo XDP cell sorter (Purity >90%), counted and suspended in media containing 1% FBS. Then, cells were placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or supplemented with recombinant CCL4 (20 ng ml−1) in the bottom chambers. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in CD8+CCR5−/media alone group. (B) Purified CD4+CCR6− and CD4+CCR6+ T lymphocytes from TILs of ESCC patients were counted, suspended and placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or added with CCL20 (50 ng ml−1) in the bottom chambers as indicated. After incubation, FoxP3+ Treg cells that migrated through the transwell were counted as described in the Materials and Methods. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in FoxP3+CCR6−/media alone group. Data shown represent mean±s.d.
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fig3: CCL6 and CCL20 individually enhance the migrations of CD8+ and regulatory T lymphocytes in vitro. (A) CD8+CCR5− and CD8+CCR5+ T lymphocytes were isolated from TILs of ESCC patients using MoFlo XDP cell sorter (Purity >90%), counted and suspended in media containing 1% FBS. Then, cells were placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or supplemented with recombinant CCL4 (20 ng ml−1) in the bottom chambers. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in CD8+CCR5−/media alone group. (B) Purified CD4+CCR6− and CD4+CCR6+ T lymphocytes from TILs of ESCC patients were counted, suspended and placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or added with CCL20 (50 ng ml−1) in the bottom chambers as indicated. After incubation, FoxP3+ Treg cells that migrated through the transwell were counted as described in the Materials and Methods. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in FoxP3+CCR6−/media alone group. Data shown represent mean±s.d.

Mentions: To further validate the roles of CCL4 and CCL20 in T lymphocyte chemotaxis, we performed transwell experiments. First, CD8+CCR5−, CD8+CCR5+, CD4+CCR6− and CD4+CCR6+ T lymphocytes (the purities were over 90%) were purified from TILs. Then, the purified T lymphocytes were separately added into transwell chambers, whose bottom wells were filled of media containing 1% FBS alone or with corresponding chemokines (recombinant human CCL4 or CCL20). The migrated cells in bottom wells were collected, stained and calculated. As shown in Figure 3, CCL4 obviously enhanced the migrating ability of the CCR5-expressing subset in CD8+ T lymphocytes, whereas media alone had little impact on the movement of CD8+ T lymphocytes. Similarly, the directional movement of Treg cells was relying on CCL20 and CCR6 (Figure 3). These observations verify that CCL4–CCR5 and CCL20–CCR6 axes are critical for the migrations of CD8+ T and Treg lymphocytes, respectively.


CTL- vs T reg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma
CCL6 and CCL20 individually enhance the migrations of CD8+ and regulatory T lymphocytes in vitro. (A) CD8+CCR5− and CD8+CCR5+ T lymphocytes were isolated from TILs of ESCC patients using MoFlo XDP cell sorter (Purity >90%), counted and suspended in media containing 1% FBS. Then, cells were placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or supplemented with recombinant CCL4 (20 ng ml−1) in the bottom chambers. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in CD8+CCR5−/media alone group. (B) Purified CD4+CCR6− and CD4+CCR6+ T lymphocytes from TILs of ESCC patients were counted, suspended and placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or added with CCL20 (50 ng ml−1) in the bottom chambers as indicated. After incubation, FoxP3+ Treg cells that migrated through the transwell were counted as described in the Materials and Methods. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in FoxP3+CCR6−/media alone group. Data shown represent mean±s.d.
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Related In: Results  -  Collection

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fig3: CCL6 and CCL20 individually enhance the migrations of CD8+ and regulatory T lymphocytes in vitro. (A) CD8+CCR5− and CD8+CCR5+ T lymphocytes were isolated from TILs of ESCC patients using MoFlo XDP cell sorter (Purity >90%), counted and suspended in media containing 1% FBS. Then, cells were placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or supplemented with recombinant CCL4 (20 ng ml−1) in the bottom chambers. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in CD8+CCR5−/media alone group. (B) Purified CD4+CCR6− and CD4+CCR6+ T lymphocytes from TILs of ESCC patients were counted, suspended and placed in transwell inserts (5 μm pore size) with media containing 1% FBS alone or added with CCL20 (50 ng ml−1) in the bottom chambers as indicated. After incubation, FoxP3+ Treg cells that migrated through the transwell were counted as described in the Materials and Methods. Migration index was calculated by dividing the number of cells that migrated in each group by the number migrating in FoxP3+CCR6−/media alone group. Data shown represent mean±s.d.
Mentions: To further validate the roles of CCL4 and CCL20 in T lymphocyte chemotaxis, we performed transwell experiments. First, CD8+CCR5−, CD8+CCR5+, CD4+CCR6− and CD4+CCR6+ T lymphocytes (the purities were over 90%) were purified from TILs. Then, the purified T lymphocytes were separately added into transwell chambers, whose bottom wells were filled of media containing 1% FBS alone or with corresponding chemokines (recombinant human CCL4 or CCL20). The migrated cells in bottom wells were collected, stained and calculated. As shown in Figure 3, CCL4 obviously enhanced the migrating ability of the CCR5-expressing subset in CD8+ T lymphocytes, whereas media alone had little impact on the movement of CD8+ T lymphocytes. Similarly, the directional movement of Treg cells was relying on CCL20 and CCR6 (Figure 3). These observations verify that CCL4–CCR5 and CCL20–CCR6 axes are critical for the migrations of CD8+ T and Treg lymphocytes, respectively.

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.


Related in: MedlinePlus