Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

BACKGROUND: Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma. METHODOLOGY/PRINCIPAL FINDINGS: Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25(high)FOXP3⁺CD127(low) putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4⁺ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8⁺granzyme B⁺ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4⁺ T cells in the tumor, while frequencies of CD4⁺CCR4⁺ lymphocytes were significantly increased. CONCLUSIONS/SIGNIFICANCE: This study shows that CCR4⁺CTLA4(hi) Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols

Chemokine receptor expression on T cells from tumor and unaffected mucosa.

<p>The expression of CXCR3 (A) and CCR4 (B) was evaluated on conventional CD4⁺ and CD8⁺ T cells and Treg from the tumor and unaffected mucosa using flow cytometry. The frequencies of positive cells are given as the percentage of all CD4⁺ or CD8⁺ cells, respectively. Graphs show paired individual values from 9 to 10 patients. * p<0.05, ** p<0.01, Wilcoxon signed rank test.</p

Immunofluorescence detection of T cell subsets in tumor and unaffected mucosa.

<p>The expression of surface CD4 (A and B) and CD8 (C and D) in combination with intracellular FOXP3 was determined in unaffected (A and C) and tumor mucosa (B and D). E and F show isotype control staining of the respective tissues, and G a close-up of FOXP3⁺ cells (green nucleus) in close association with CD8⁺ conventional T cells (red surface staining) in a section of tumor mucosa.</p

Expression of β7 integrins on T cells from tumor and unaffected mucosa.

<p>The expression of α4β7 (A) and αEβ7 (B) was evaluated on CD4⁺ and CD8⁺ T cells and Treg from the tumor and unaffected mucosa using flow cytometry. The frequencies of positive cells are given as the percentage of all CD4⁺ or CD8⁺ cells, respectively. Graphs show paired individual values from 7 to 16 patients. * p<0.05, ** p<0.01, Wilcoxon signed rank test.</p

Activation and differentiation state of T cells from tumor and unaffected mucosa.

<p>Left hand panels in A–B show CD4⁺ T cells and right hand panels show CD8⁺ T cells. The expression of surface CD69 (A) and intracellular Granzyme B (B) was evaluated on conventional CD4⁺ FOXP3⁻ and CD8⁺ T cells from the tumor and unaffected mucosa using flow cytometry. The frequencies of positive cells are given as the percentage of all CD4⁺ or CD8⁺ cells, respectively. The expression of intracellular CTLA-4 (C) was evaluated in conventional CD4⁺ FOXP3⁻ T cells and the frequencies of positive cells are given as the percentage of all CD4⁺ T cells. C. (D) MFI for CTLA4 on conventional CD4⁺FOXP3⁻ T cells and putative Tregs isolated from tumors and unaffected tissue. Graphs show paired individual values from 9 to 15 patients. (E) Ratios of CD8⁺Granzyme B⁺ cells to CD4⁺CD25^hi Treg cells among cells isolated from tumor and unaffected mucosa * p<0.05, ** p<0.01, *** p<0.001, Wilcoxon signed rank test.</p

Treg frequencies in tumor and unaffected mucosa.

<p>The frequencies of CD4⁺CD25^high putative Treg in tumor associated and unaffected mucosa from colon cancer patients were investigated using flow cytometry. (A) Paired individual frequencies of CD4⁺CD25^hiputative Tregs among all CD4⁺ T cells from 17 patients, and an example of CD4 and CD25 staining in one individual. (B) Intracellular FOXP3 and surface CD127 staining of CD25^hi cells in the tumor from one individual. (C) Frequencies of CD4⁺CD25^int T cells among all CD4⁺ T cells in unaffected and tumor mucosa. Connected lines represents data from the same individual. (D) Methylation status of the FOXP3 promoter region in FOXP3⁺ and FOXP3⁻ CD4⁺ T cells sorted by flow cytometry from lamina propria cells isolated from unaffected and colon tumor tissue, as determined by Ms-SNUPE. Each symbol represents data from one individual. *** p<0.001, Wilcoxon signed rank test.</p