- Correlation of immunological and clinical responses to F16-IL2 immunotherapy
- Correlation of systemic and intratumoral immune responses
- Identification and characterization of immune escape mechanisms of MCC
- Establish the impact of the microenvironment on the immune mediated tumor eradication
Previous trials of immunotherapy of cancer unequivocally demonstrated the feasibility of therapeutic induction of tumor-specific immune responses in the circulating blood; the clinical benefit of the majority of these trials, however, was very limited. The discussed reasons for this gap are immune escape mechanisms of the tumor, impaired homing of induced T cells to the tumor site, or negative regulatory circuits impairing the induced immune response. By detailed comparative analysis of patients treated by F16-IL2 administration within IMMOMEC mounting systemic immune responses but experiencing either a clinical benefit or not we will establish the etiological factors governing whether a systemic MCC-specific T-cell response is able to eradicate the tumor. These analyses will employ and extend the results obtained in WP2, WP3 and WP4. Particular endeavor will be given to the in situ analysis of regressing and non-regressing MCC metastases.
Analysis of T cell functional capacity
The functional capacity of induced specific T cells will be measured by in vitro assays. In this regard, the general capacity determined by mitogenic stimulation as well as specific towards MCC cell lines, either autologous were applicable or otherwise HLA-matched MCC cell lines which have been already established within the consortium will be determined. These analyses will comprise classical cytotoxicity assays, flow cytometric ICS assays as well as immunofluorescence staining using confocal microscopy for functional morphology. Similarly, if applicable the immunogenicity of MCC cell lines generated from treated but not responding patients will betested in cytotoxicity assays using WP3 generated MCC-specific T cell lines or clones. Moreover, analysis of these MCC specific T cell lines will reveal whether T cell function is intrinsically or extrinsically hampered.
In situ detection of MCC specific T cells
In situ peptide/MHC-multimer staining to detect specific T cells in tissue has been previously established by members of the consortium. The innovative technique will be applied not only to visualize MCC-specific T cells in MCC lesions, but also to characterize their functional capacity. These results will be correlated with the presence of immune modulatory cells, such as regulatory T cells or myeloid-derived suppressor cell (MDSCs) populations in the tumor microenvironment.
T cell characterization
As the primary physical barrier between blood and tissue compartments within the body, blood vessel endothelial cells and integrity of the cell junctions connecting them must be carefully regulated to support leukocyte transendothelial migration only when necessary. Integrins and chemokines mediate critical steps for T cell trafficking and extravasation into the tumor microenvironment. In this regard, chemokines have been shown to trigger firm adhesion of leukocytes to vascular endothelium under flow condition by enhancing integrin–ligand-binding affinity. Thus, the integrin, chemokine and chemokine receptor expression profile will be established on circulating MCC-specific T cells, those infiltrating the tumor as well as the tumor endothelium. Notably, since Tenascin C is present in the extracellular matrix of newly formed blood vessels, and IL2 has been demonstrated to impact both the migration capacity of lymphocytes as well as the functional state of endothelialcells, the obtained results will provide further insight into the function of F16-IL2 immunotherapy.
The ultimate goal of WP5 is to deliver sufficient understanding of the immune escape mechanisms preventing tumor eradication by MCC-specific T cells to design strategies to overcome these.