Re-activating Memory T Cells in the Microenvironment of Human Tumors

Tumor-associated CD4+ and CD8+ T cells with an effector memory phenotype (Tem) are present within the microenvironment of human non-small cell lung tumors and ovarian carcinomas, but fail to control tumor progression. Our data have established that the non-responsiveness of these cells to the tumor is due in part to their failure to respond to activation signals via the T cell receptor (TCR). By elucidating the site of the arrest in the TCR pathway and by defining the molecular and cellular events that initiate this arrest it should be possible to design and test strategies to re-activate the Tem in situ. The local re-activation of the Tem within the treated tumor microenvironment is expected to result in, (a) T cell mediated killing of tumor cells in situ, (b) release of tumor antigens into the circulation, (c) generation of a systemic anti-tumor immunity, and (d) T cell recognition and eradication of existing tumors at sites that are adjacent to or distant from the initially treated tumor site. Using a combination of multispectral immaging flow cytometry, confocal microscopy, western blot and rtPCR our first aim is to determine where in the TCR pathway the transduction signal is blocked. In a related second aim the cells and molecules that are causally linked to triggering the TCR arrest are determined by cell depletion and add back protocols and by monitoring the effects of selected molecules on the initiation of this regulatory signaling checkpoint. Our preliminary studies have localized the site of the signaling checkpoint to occur somewhere upstream of PLC-γ and the TCR signal arrest has been causally linked to TGF-β1 thereby demonstrating the feasibility and viability of our experimental protocols. The results obtained from these mechanistic studies will be utilized in aims 2 and 3 to identify biologically active factors that act directly or indirectly on the T cells to reverse their non-responsiveness, and to develop and test liposome formulations that are designed to deliver these factors in a local and sustained fashion in vivo. In the final aim the therapeutic efficacy of each factor for re-activating Tem in situ and for inducing a local and systemic anti-tumor response is evaluated. The latter is to be accomplished using an established xenograft model in which nondisrupted pieces of human tumor are surgically implanted into SCID or NOD-SCID/IL2 receptor γ chainnull mice. In these xenografts the tumor microenvironment is preserved and the tumor- associated leukocytes remain viable and predictably responsive to cytokine signals for prolonged periods. Following the inoculation of the factor loaded liposomes into the xenografts tumor killing and Tem response patterns are monitored to determine the therapeutic efficacy of each liposomal preparation. These studies are expected to lay the foundation for the design of strategies that can be used to enhance the efficacy of our current cancer vaccination clinical trials.