Regulation der anti-Tumor-Immunantwort bei Patienten mit kolorektalem Karzinom

Abstract

Despite radical surgical resection there is a high rate of tumor recurrence in patients with colorectal carcinoma. During an observation period of 3 years 18% of the patients in our collective (n = 74) suffered a tumor relapse with locally or distinct metastases after initial R0-resection. There is evidence that this can be explained, at least in part, by a modulated anti-tumor immune response inhibiting effective tumor destruction. Regulatory T cells seem to play a major role for this phenomenon. Thus, it was the aim of our study to investigate the tumor-specific immune response in patients with colorectal cancer dependent on their UICC stage and to characterize the cellular immune responses against defined antigens that are overexpressed in most of the patients with colorectal cancer. For this purpose, the tumor suppressor gene p53 was chosen as tumor associated antigen (TAA) as it can be found mutated and subsequently overexpressed in up to 60% of colorectal carcinoma. 21% of the 74 patients with colorectal cancer were of UICC stage I, 26% of UICC II, 26% of UICC III and 27% of UICC stage IV. Peripheral blood lymphocytes (PBLs) of the patients isolated from preoperatively taken blood samples were stimulated with single overlapping synthetic peptides encompassing the complete wildtype-p53 protein sequence and subsequently characterized for their cytokine expression pattern (IL-10, IFN-γ) using ELISPOT and ELISA. Both, PBL cytospin preparations and native tumor tissue were analyzed using immunohistochemistry in single- and doublestaining techniques. Specific p53 gene mutations and expression of T_Reg genes in the tumor tissue were analyzed by using RTq-PCR. After stimulation of the PBLs with p53 peptides distinct residues were found that induced IFN-γ or IL-10 expression. Thereby the UICC stage played a crucial role for the IL-10 secretion independent from specific mutations within the p53 gene. T cells from patients in UICC III and IV expressed significant higher IL-10 levels than T cellsfrom patients of UICC I and II. In contrast, no correlation was observed between IFN-γ production and UICC stage. Additionally, immunohistochemical examination showed comparably more CD4+CD25+ cells, as well as a stronger p53 accumulation within the tumor in patients of higher disease stages (UICC III and IV) than in patients of UICC stages I and II. As Real Time PCR analysis showed an increased T_Reg specific gene expression (CD4, CD25, Foxp3, GITR, GATA-3) at the tumor site that also positively correlated with the UICC stage, this indicates that the CD4+CD25+ cells seen immunohistologically indeed comprised an increased population of regulatory tumor specific T cells. This data indicates, that p53 induces both IFN-γ and IL-10 secretion. The predominance of the IL-10 production indicates that T_Reg directly participate in modulating the anti tumor immune response. IL-10 levels in the blood as well as expression of T_Reg-specific genes at the tumor site correlate with the UICC stage of the disease. This delivers a possible explanation for a poor prognosis and an increased recurrence rate in patients with advanced carcinoma. In addition, our results implicate new therapeutical options. A specific anti-IL-10 antibody treatment or depletion of tumor-specific T_Reg could possibly shift an anti-tumor immune response back toan effective immunological destruction of the tumor.