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Turqueti Neves, Adriana (2011): Recognition of renal cell carcinoma by CD8+ and CD4+ TCR-engineered T lymphocytes. Dissertation, LMU München: Fakultät für Biologie



Immunotherapy using T cells is a new approach that is being explored for the treatment of metastatic melanoma. For renal cell carcinoma (RCC), adoptive T cell therapy (ATT) is currently hampered by the lack of T cells expressing suitable T cell receptors (TCR). A tumor-infiltrating T cell population (TIL) was identified in our group (TIL-53) that showed a pattern of tumor recognition consistent with the requirements of a TCR applicable for immunotherapy. With the advent of genetic TCR engineering it was possible to further define the TIL specificity which was previously precluded by the failure to cultivate TIL-53. To achieve high expression levels and functionality, the TCR53 required the exchange of the human TCR constant region by the TCR murine constant region (TCR53m). The B3Z T cell hybridoma which stably expressed TCR53m after retroviral transduction was used to analyze a large panel of tumor lines and non-malignant cell cultures for expression of the TCR53 ligand. The analysis included 34 RCC cell lines, 55 tumor cell lines of different histologies and 30 non-tumor cell lines. 65 % of the HLA-A2+ RCC cells and 25 % of other HLA-A2+ tumor lines were recognized by the B3Z-TCR53m cells. Among the non-RCC tumors, the TCR53 ligand was frequently found in malignant B cell lines and EBV-transformed B-lymphoblastoid cell lines (5/13, 38 %). Of 25 HLA-A2+ non-tumor cells only 2 were marginally recognized. The TCR53 ligand expression could be increased with IFN-a but not IFN-g treatment on cell lines that already had some TCR53 ligand expression. De novo induction in cell lines that had no prior expression of the TCR53 ligand was not observed. The B3Z-TCR53m cell line could detect the TCR53 ligand on fresh tumor material and, if used for therapy, B3Z-TCR53m could be used to identify those patients whose tumors are positive for the TCR53 ligand and thus could benefit from the therapy. To achieve high expression levels and functionality of the TCR53m on human PBLs, the TCR53 a and b chain sequences had to be optimized for codon usage. PBLs expressing these recombinant TCR sequences (TCR53mc) showed very low formation of hybrid TCRs between the TCR53mc b chain and endogenous TCR a chains. TCR53mc-expressing T cells of RCC patients and healthy donors showed specific killing of tumor cell lines and had a polyfunctional profile, defined by the detection of T cells that simultaneously secreted cytokines (IFN-g, TNF-a or IL-2) and performed granule exocytosis when recognizing targets. The functional response of TCR53mc-expressing T cells depended on the expression of HLA-A2 on the target cells. Analysis of RCC tumors using multicolor fluorescence immunohistology allowed the detection and localization of CD8+ T cells in relation to blood vessels. The majority of CD8+ T cells were found extra-luminal, indicating strong extravasation of T cells into RCC tumors. The majority of the T cells in the lumen of the blood vessel had perforin (~ 90 %), while T cells that were outside the blood vessels were to a large percentage perforin negative (~ 60 %). Thus, CD8+ T cells apparently arrive at the tumor being perforin-positive and lose perforin when outside the blood vessels. The three-dimensional growth of cells in spheroids was used to mimic the tumor milieu in vitro and to evaluate the functional capacity of T cells with transgenic RCC specific TCR expression. T cells infiltrated the spheroids and preferentially accumulated in the rim of the spheroid (~ 100 μm). The killing capacity of TCR53mcexpressing T cells in the 3-D environment in a 4 h assay was similar to that observed in a standard 4 h chromium release assay with RCC cells in suspension. However, after being cultured for 24 h in the spheroids, the T cells were no longer able to secrete cytokines upon stimulation with target cells and were negative for perforin, granzyme B and CD28. The presence of CD4+ T cells in the spheroids significantly increased the number of CD8+ T cells infiltrating the 3-D tumors. Moreover, the CD8+ T cell response was enhanced with more degranulating T cells and T cells secreting cytokines, which was not seen in the absence of CD4+ T cells. The functional improvement of the CD8+ T cell response required the CD4+ T cells to be activated, as it was not observed when CD4+ T cells were used that lacked TCRs specific for the spheroid tumor cells.