The expression of tumor antigens elicits adaptive immune responses, which in favorable situations can lead to tumor rejection in cancer patients. However, several biological characteristics of malignant growths more frequently lead to the development of tumor-specific immunological tolerance. This tolerance is induced and maintained by the complex interplay of a multitude of immunoregulatory factors. T regulatory cells (Treg), myeloid-derived suppressor cells, as well as tumor-infiltrating macrophages and myeloid dendritic cells are among the most prominent cellular constituents of these regulatory networks. It is becoming apparent that local crosstalk between Treg and myeloid antigen-presenting cells (APC) in the tumor microenvironment is critical for their immunosuppressive function on anti-tumor effector cells, most prominently CD8+ cytotoxic T lymphocytes (CTL). Indirect evidence suggests that the crosstalk between Treg and myeloid APC, as well as the suppression of CTL is conferred by direct cell-cell contacts that allows for information exchange through soluble factors and membrane proteins in the context of antigen-dependent interactions. However, the physical nature, the kinetics, and stoichiometry of T cell-APC interactions in the tumor environment are unexplored. In preliminary work for this project, we have developed a multiphoton intravital microscopy approach to study the dynamic behavior and intercellular signal exchange of tumor-associated APC and T cell receptor (TCR) transgenic tumor-antigen-specific Treg and CTL in intact subcutaneous tumors of anesthetized mice. This imaging approach produces 3D time-lapse movies of interacting T cells and APC at subcellular resolution and will be used to address the following three specific aims: 1.) To characterize the local impact of tumor- infiltrating myeloid APC and Treg on CTL accumulation and function in tumors;2.) To test if Treg-mediated suppression of CTL manifests itself in an impairment of TCR signal transduction;and 3.) To investigate the cellular interactions of myeloid APC with CTL and Treg in the tumor environment. The proposed experiments will generate a comprehensive, mechanism-oriented survey of the local interplay between immunoregulatory cells of the myeloid and the lymphoid lineage. This information may lead to improved strategies for clinical immunomodulation, not only for tumor therapy, but also for the treatment of infectious, inflammatory and autoimmune diseases. PUBLIC HEALTH RELEVANCE: Immunotherapy has emerged as a powerful treatment modality for cancer patients. In order to optimize its effectiveness and to minimize side effects through systemic immune activation, a more profound mechanistic understanding of immune responses against tumors is required. The proposed studies will generate insight into the interplay of immunosuppressive cell populations, which will enhance our capacity for the rational design of targeted therapies for cancer.