Cancer - T Cell Immunity
Cancer patients can harbor high numbers of activated tumor-specific T cells that exhibit tumoricidal activity in vitro but in most cases fail to eradicate tumors in vivo. CD4+CD25+ regulatory T (Treg) cells are thought to contribute to immune evasion in cancer by repressing tumor-specific CD8 T cytotoxic (CTL) and CD4 T helper (Th) responses and to characterize the main obstacle tempering successful cancer immunotherapy. Yet, where and how Treg cells act to restrain effector T cells remain under intense investigation. Because the complexity of in vivo environments is not easy to faithfully reproduce in vitro, our goal is to employ noninvasive in vivo imaging techniques to objectively monitor tumor-specific T cell responses. Specifically, we aim to improve our understanding of Treg-cell mediated suppression of CTL immunity and to define the in vivo effects of therapeutic interventions that modulate such suppression. We have recently validated an in vivo approach for the study of tumor-specific CTL, Treg and Th cell responses in nearly physiological conditions [collaboration with Khashayarsha Khazaie and Harald von Boehmer, Dana Farber Cancer Institute, Harvard Medical School, Boston]. The model recapitulates observations made in cancer patients in that antigen-experienced anti-tumor T cells are restrained by Treg-cell mediated tolerance mechanisms and fail to control tumor progression. Our program currently has several areas of emphasis including (1) Treg cell-mediated suppression of CTL effector functions, (2) Treg cell-mediated suppression of CTL trafficking.
CTL Effector Functions
The rare naive T cells that recognize cognate peptide presented by dendritic cells in lymph nodes undergo a program of proliferation and differentiation. The progeny of naive T cells comprises effector and memory cells that are equipped with distinct functional and homing capacities. Effector cells enter nonlymphoid tissues and display immediate function to destroy abnormal cells, whereas memory cells travel to secondary lymphoid organs and generate a new wave of effector cells upon secondary challenge. We are employing intravital multiphoton microscopy coupled with a novel assay for visualization in vivo of effector T cell and Treg cell activity [collaboration with Thorsten R Mempel and Ulrich H von Andrian, the CBR Institute for Blood Research, Harvard Medical School, Boston]. We show that Treg cells can reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill (Immunity, 2006, 25:129).
CTL's kiss of death. Interaction between a CTL (green) and a target B cell (purple) is initiated at 3:30 min and characterized by co-migration of the conjugate. The B cell stops to migrate at 12:00 min and undergoes a sudden color change at 12:45 min, indicative of loss of structural integrity. [collaboration with Thorsten R Mempel and Ulrich H von Andrian, the CBR Institute for Blood Research, Harvard Medical School, Boston].
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Effector CTL are key players in the struggle for immune protection because they migrate to nonlymphoid tissues where they specifically kill abnormal cells and generate local inflammation. Investigations so far have been mainly performed with immune cells isolated from peripheral blood or lymphoid tissues. The CIP employs novel, non-invasive imaging modalities that allow quantitative, three-dimensional reconstructions of tumor-specific T cells over time in the whole body of living mice, including nonlymphoid tissues.
A first approach exploits biocompatible and physiologically inert nanoparticles (CLIO-HD) for highly efficient intracellular labeling that allows in vivo MRI tracking of systemically injected cells at near single cell resolution. This noninvasive and nontoxic technique permits the concomitant monitoring of tumor nodules and tumor-specific T cells in longitudinal studies up to 48 hr following adoptive transfer. Such technology may provide clinical applications for evaluation of both cell delivery in adoptive transfer cell therapy and therapeutic effectiveness in patients (Cancer Res., 2003, 63:6838)
Accumulation of tumor-specific T cells in tumors visualized by serial magnetic resonance imaging (MRI). Serial MRI was performed following adoptive transfer into a mouse carrying both B16F0 (left side) and B16-OVA (right side) melanomas. a-d, axial slices through the mouse thighs at a, prior to adoptive transfer; b, 12h; c, 16 h; d, 36 h after adoptive transfer of CLIO-HD labeled OT-I CD8+ T cells. e-h, 3D color-scaled reconstructions of B16F0 (left) and B16-OVA (right) melanomas at e, 0 h; f, 12 h; g, 16 h; h, i, 36 h following adoptive transfer. Numbers of cells/voxel are color-coded as shown in scale, j, axial; k, sagittal; l, coronal plane slices through the 3D reconstruction shown in i.
Another imaging modality, called fluorescent protein tomography (FPT), allows quantitative, three-dimensional reconstruction of tumor-specific T cells genetically modified to stably express enhanced green fluorescence protein (EGFP). This approach permits detection of cells that extensively divide in vivo, i.e. effector or memory T cells derived from naive EGFP+ T cell precursors. FPT collects photons emitted by EGFP+ T cells that propagated through tissues, and combines measurements obtained after stimulation from different sources. Reconstruction of T cell distribution is achieved with specific algorithm and algebraic reconstruction techniques, developed at CMIR, that take into account the nature of photon propagation in tissues.
Residence of tumor-specific CTL within tumor lesions visualized by fluorescence protein tomography (FPT). Naive EGFP+ HA-specific CD8 T cells were adoptively transferred into BALB/c mice further challenged with CT26 (HA-, right footpad) and CT44 (HA+, left footpad) tumors. Mice were anesthetized on d 14 and subjected to imaging. Non-invasive FPT shows selective homing of HA-specific CD8 T cells to CT44 tumors (color signal). Pictures depict EGFP signals obtained at different depths (Z = 0.529,1.029 cm). Note the selective rejection of CT44 tumors at this time point. White light and FPT images and superimposed.