We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.

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The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1-dependent migration was regulated by the PI3K and ERK/ MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be IntroductionMultiple myeloma (MM) is the second most prevalent hematologic malignancy; it remains incurable, and the median survival time is 3 to 5 years. 1,2 It is characterized by the presence of multiple lytic lesions and widespread involvement of the bone marrow at diagnosis, implying a continuous (re)circulation of MM cells in the peripheral blood and (re)entrance into the bone marrow. 1 Studies have demonstrated the presence of circulating malignant plasma cells in more than 70% of patients diagnosed with MM. 3,4 Migration of cells through the blood to the bone marrow niches requires active navigation, a process termed homing.Chemokines are small chemoattractant cytokines that bind to specific G-protein-coupled 7-span transmembrane receptors present on the plasma membranes of target cells. [5][6][7] Chemokines play a central role in lymphocyte trafficking and homing. [8][9][10][11] One of the most extensively studied chemokines in migration is SDF-1 and its receptor, CXCR4. 12,13 SDF-1 is primarily produced by stromal cells. CXCR4 is expressed on the surfaces of normal cells such as hematopoietic stem cells and T and B lymphocytes and on malignant cells such as breast cancer cells and lymphoid malignancies. 6,11,[14][15][16] To date, the role of CXCR4 in homing of MM cells to the bone marrow has not been fully elucidated. Inhibitors of CXCR4, such as AMD3100 (AnorMED, Toronto, ON, Canada), have been shown to induce the mobilization of stem cells. 17,18 AMD3100 (AnorMED) is a bicyclam molecule that reversibly blocks the binding of CXCR4 with SDF-1. 19 Because SDF-1/CXCR4-dependent signaling differs between cell types and between malignant and normal counterparts, 20 it is critical to investigate the unique role of CXCR4/SDF-1 in MM. In this study, we sought to determine the effect of CXCR4 and its specific inhibitor, AMD3100, on the migration and in vivo ...

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Transfer of T cells to freshly irradiated allogeneic recipients leads to their rapid recruitment to nonlymphoid tissues, where they induce graft-versus-host disease (GVHD). In contrast, when donor T cells are transferred to established mixed chimeras (MCs), GVHD is not induced despite a robust graft-versus-host (GVH) reaction that eliminates normal and malignant host hematopoietic cells. We demonstrate here that donor GVH-reactive T cells transferred to MCs or freshly irradiated mice undergo similar expansion and activation, with similar up-regulation of homing molecules required for entry to nonlymphoid tissues. Using dynamic two-photon in vivo microscopy, we show that these activated T cells do not enter GVHD target tissues in established MCs, contrary to the dogma that activated T cells inevitably traffic to nonlymphoid tissues. Instead, we show that the presence of inflammation within a nonlymphoid tissue is a prerequisite for the trafficking of activated T cells to that site. Our studies help to explain the paradox whereby GVH-reactive T cells can mediate graft-versus-leukemia responses without inducing GVHD in established MCs.

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Here we present methods to longitudinally track islet allograft—infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, ‘color-coded’ proteins enabling in vivo detection and identification of effector T cells (Teff cells) and discrimination between natural and induced regulatory T cells (nTreg and iTreg cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance.