Understanding the regulation of T cell responses in complex in vivo environments may help at developing better anti-tumor therapies. Two non-invasive, three-dimensional imaging modalities are presented here that permit the in vivo study of (1) trafficking of tumor-specific CD8 cells within the whole-body of tumor-bearing mice, and (2) killing of targeted cells by tumor-specific effector T cells in tumor-draining LN. We provide new insights on the mechanisms of suppression of CD8 cells by CD4+CD25+ Treg in vivo. First, we present a novel imaging modality called fluorescent protein tomography (FPT) that allows quantitative, three-dimensional reconstruction of tumor-specific EGFP CD8 cells in the course of an immune response. FPT collects photons emitted by EGFP CD8 cells that propagated through tissues and combines measurements obtained after stimulation from different sources. Reconstruction of CD8 cell distribution is achieved by using a specific algorithm and algebraic reconstruction techniques that take into account the nature of photon propagation in tissues. In our model, antigen-primed CD8 cells expand >200 fold in tumor draining LN, home to and selectively accumulate at tumor sites, where they efficiently eliminate tumor cells. The presence of tumor-specific CD4+CD25+ Treg completely abrogates CD8 tumor immunity, however, the CD8 cells expand to the same extent in the absence or presence of Treg, which is in marked contrast to previous studies reporting that Treg suppress CD8 proliferation in vitro. Preliminary experiments suggest that Treg do not interfere with CD8 cell trafficking to tumor sites, in line with the reported presence of high numbers of tumor-specific CD8 cells in tumor lesions from cancer patients. Second, we employ multiphoton microscopy coupled with a novel assay for the visualization of CD8 cytotoxicity in vivo. Intravital LN imaging reveals a profound control by tumor-specific CD4+CD25+ Treg on the functional behaviour of effector CD8 cells. In the absence of Treg, the CD8 cells selectively form long-lasting interactions with cells presenting cognate tumor Ag. The CD8 cells induce a complete loss of motility of their targets, then initiate target cell apoptosis. The latter event coincides with the disengagement of CD8 cells from their targets. In the presence of Treg, the CD8 cells also form long-lasting contacts with their targets, however, CD8 cell/target cell conjugates maintain a high motility and target cell apoptosis is never initiated. As a result, CD8 cells ultimately disengage from still unharmed targets. The molecular mechanism for such functional tolerance possibly involves TGF-_eta since expression of a dominant-negative TGF-_eta receptor by CD8 cells renders them resistant to suppression and is associated with tumor rejection and unimpaired cytotoxicity. The potential implications of this study for the development of improved immunization strategies will be discussed.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]