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Vol 83, No 10 (2012)
ARTICLES
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Lymphocyte activation markers in patients with ovarian cancer

Jan Kotarski, Wanda Piekarczyk, Iwona Wertel, Bartłomiej Barczyński, Wiesława Bednarek, Ewelina Rogala, Aldona Nowicka
Ginekol Pol 2012;83(10).

open access

Vol 83, No 10 (2012)
ARTICLES

Abstract

Objectives: The aim of this study was to evaluate the phenotype and T cell activation markers: CD69, CD25 and HLA - DR in the peripheral blood and tumor tissue of ovarian cancer patients. Material and methods: The study group consisted of 26 patients operated due to ovarian cancer (FIGO IIb - IV). Mononuclear immune cells were isolated from peripheral blood and ovarian cancer tissue. To obtain peripheral blood lymphocytes, blood was collected into heparinized tubes and diluted 1:1 with PBS, then layered on Gradisol L and centrifuged 20 minutes at 2800 rpm. Mononuclear cells were washed twice with PBS and labeled with monoclonal antibodies. A small piece of tumor tissue (about 1cm3) was fragmented with a surgical blade. Minced tissue was suspended in PBS and layered on Gradisol L for mononuclear cells isolation. To assess the phenotype and activation status of peripheral blood and tumor infiltrating lymphocytes, we used FACS Canto cytometer and monoclonal antibodies conjugated with fluorochromes: anti-CD3-FITC, anti-CD4-PECy5, anti-CD8-APC, anti-CD25-PE, anti-CD69-PE-Cy7, anti-HLA-DR-PE-Cy7. Statistical analysis was performed using the Statistica 5.0 and Wilcoxon test. Results: In all cases we detected T helper CD3+CD4+ and cytotoxic CD3+CD8+ T lymphocytes in both blood samples and tumor tissues. We observed no statistically significant difference in the percentage of CD3+ CD4+ cells among the mononuclear cells present in peripheral blood and tumor tissue. The percentage of CD3+CD8+ cytotoxic T lymphocytes was higher among mononuclear cells isolated from the tumor tissue. The percentage of CD3+ lymphocytes expressing the very early activation marker CD69 was significantly higher among tumor infiltrating lymphocytes compared with peripheral blood lymphocytes. Similarly, the percentages of CD3+CD4+CD69+ T helper lymphocytes and CD3+CD8+CD69+ cytotoxic T lymphocytes were significantly higher on lymphocytes isolated from tumor tissue when compared to blood. The expression of an early activation marker – CD25 was significantly higher on the CD3+ and CD3+CD8+ peripheral blood lymphocytes compared to CD3+ and CD3+CD8+ tumor infiltrating lymphocytes. There were no statistically important differences between the percentages of, isolated from blood and tissue, CD3+CD4+ T helper lymphocytes. The expression of the late activation marker - HLA-DR was significantly higher on CD3+ lymphocytes isolated from tumor tissue compared with peripheral blood. Similarly, the percentages of CD3+CD4+ lymphocytes and CD3+CD8+ cytotoxic T cells expressing HLA-DR were significantly higher among tumor infiltrating lymphocytes when compared to peripheral blood ones. Conclusions: T cells obtained from ovarian cancer tissues are activated cells. The state of T cell activation may be the result of direct contact of these cells with tumor antigens. The low expression of CD25 may suggest abnormal clonal expansion of antigen-specific lymphocytes.

Abstract

Objectives: The aim of this study was to evaluate the phenotype and T cell activation markers: CD69, CD25 and HLA - DR in the peripheral blood and tumor tissue of ovarian cancer patients. Material and methods: The study group consisted of 26 patients operated due to ovarian cancer (FIGO IIb - IV). Mononuclear immune cells were isolated from peripheral blood and ovarian cancer tissue. To obtain peripheral blood lymphocytes, blood was collected into heparinized tubes and diluted 1:1 with PBS, then layered on Gradisol L and centrifuged 20 minutes at 2800 rpm. Mononuclear cells were washed twice with PBS and labeled with monoclonal antibodies. A small piece of tumor tissue (about 1cm3) was fragmented with a surgical blade. Minced tissue was suspended in PBS and layered on Gradisol L for mononuclear cells isolation. To assess the phenotype and activation status of peripheral blood and tumor infiltrating lymphocytes, we used FACS Canto cytometer and monoclonal antibodies conjugated with fluorochromes: anti-CD3-FITC, anti-CD4-PECy5, anti-CD8-APC, anti-CD25-PE, anti-CD69-PE-Cy7, anti-HLA-DR-PE-Cy7. Statistical analysis was performed using the Statistica 5.0 and Wilcoxon test. Results: In all cases we detected T helper CD3+CD4+ and cytotoxic CD3+CD8+ T lymphocytes in both blood samples and tumor tissues. We observed no statistically significant difference in the percentage of CD3+ CD4+ cells among the mononuclear cells present in peripheral blood and tumor tissue. The percentage of CD3+CD8+ cytotoxic T lymphocytes was higher among mononuclear cells isolated from the tumor tissue. The percentage of CD3+ lymphocytes expressing the very early activation marker CD69 was significantly higher among tumor infiltrating lymphocytes compared with peripheral blood lymphocytes. Similarly, the percentages of CD3+CD4+CD69+ T helper lymphocytes and CD3+CD8+CD69+ cytotoxic T lymphocytes were significantly higher on lymphocytes isolated from tumor tissue when compared to blood. The expression of an early activation marker – CD25 was significantly higher on the CD3+ and CD3+CD8+ peripheral blood lymphocytes compared to CD3+ and CD3+CD8+ tumor infiltrating lymphocytes. There were no statistically important differences between the percentages of, isolated from blood and tissue, CD3+CD4+ T helper lymphocytes. The expression of the late activation marker - HLA-DR was significantly higher on CD3+ lymphocytes isolated from tumor tissue compared with peripheral blood. Similarly, the percentages of CD3+CD4+ lymphocytes and CD3+CD8+ cytotoxic T cells expressing HLA-DR were significantly higher among tumor infiltrating lymphocytes when compared to peripheral blood ones. Conclusions: T cells obtained from ovarian cancer tissues are activated cells. The state of T cell activation may be the result of direct contact of these cells with tumor antigens. The low expression of CD25 may suggest abnormal clonal expansion of antigen-specific lymphocytes.
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Keywords

T cells, ovarian cancer, HLA-DR, CD26, CD25

About this article
Title

Lymphocyte activation markers in patients with ovarian cancer

Journal

Ginekologia Polska

Issue

Vol 83, No 10 (2012)

Bibliographic record

Ginekol Pol 2012;83(10).

Keywords

T cells
ovarian cancer
HLA-DR
CD26
CD25

Authors

Jan Kotarski
Wanda Piekarczyk
Iwona Wertel
Bartłomiej Barczyński
Wiesława Bednarek
Ewelina Rogala
Aldona Nowicka

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