Intravital Multiphoton Microscopy Research Articles

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors.

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.

Analysis of bone tissues by using fluorescent imaging

Rapid development of fluorescent imaging techniques enables us to understand cellular dynamics in vivo . We have originally established an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy. By means of the system we have recently succeeded in visualization of mature osteoclasts in live bones and revealed that RANKL regulates bone-resorptive functions of mature osteoclasts in vivo . We also developed new image analysis software for tracking morphological changes of mature osteoclasts and generated pH-sensing chemical fluorescent probes for detecting bone resorption at local sites on the bone surface. Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Insights into cell-to-cell and cell-to-blood-vessel communications in the brain: in vivo multiphoton microscopy

A complex and reciprocal communication of cells with each other and with relevant parts of the tissue stroma governs many biological processes in both health and disease. However, in the past, the study of these anatomical and molecular interactions has suffered from a lack of appropriate experimental models. An imaging methodology aimed at changing this should allow intravital display and quantification in an intact non-traumatized organ, imaging over a wide range of time spans including extended periods (i.e., months), many repetitive measurements of the same cell or area to permit the study of the cause and consequence of biological processes, the display of various cell types and their reciprocal interaction with each other in three dimensions, the co-registration of relevant physiological parameters and reporters for selected molecular pathways and as high as possible resolution to visualize sub-cellular structures such as organelles. Remarkably, intravital multiphoton microscopy (in vivo MPLSM) through a chronic cranial window allows us to do all these things, making the brain the inner organ of choice for this technology. Here, we give an overview of the application of in vivo MPLSM to study the choreography of cellular, vascular and molecular interactions in the healthy brain and in neurological diseases. We focus on brain tumor formation, progression and response to therapies. This review further aims at demonstrating that we stand at the beginning of full exploitation of the opportunities provided by this technology and gives clues to future directions that appear most promising.

Anatomically Restricted Synergistic Antiviral Activities of Innate and Adaptive Immune Cells in the Skin

Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci and numerous migratory dermal inflammatory monocytes that outlie the foci. We observed Ly6G(+) innate immune cells infiltrating and controlling foci, while CD8(+) T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8(+) T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8(+) T cells into foci, modestly reducing viral titers. Depletion of Ly6G(+) and CD8(+) cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.

Abstract PR5: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

Abstract Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. We have used intravital multiphoton microscopy to visualize the in vivo migration and invasion of human breast tumor cells in live primary tumors. We used two different tumors for this study: a. orthotopic xenografts of the highly metastatic breast cancer cell line MDA-MB-231, and b. low passage orthotopic xenografts of breast tumor cells isolated from a patient pleural effusion sample (TN1). The TN1 tumors were only passaged in vivo and never cultured. Both tumor cells were engineered to stably express GFP (green fluorescence protein) and were visualized in vivo based on their fluorescence emission. Collagen I matrix was visualized by second harmonic generation, while injected fluorescent dyes were used for visualization of blood vessels and stromal cells in the primary tumors. High-resolution 4D imaging of these two human tumors in vivo showed that they both shared common patterns of high-speed migration: a. cells moving as single entities irrespective of their neighboring cells, and b. multiple cells following each other in the same direction and in a single file but without cohesive cell junctions (i.e. multicellular streaming). We found that although the two tumors were derived from diverse genetic backgrounds, they exhibited almost identical proportions of either single or streaming cells, suggesting our observations may be generally applied to human breast cancer in vivo cell migration. Cells moving as multicellular streams were overall more frequent in both tumors, with approximately 60% of total motile cells moving as streams. Critically, we found that only multicellular streaming and not random migration of single cells was significantly associated with proximity to blood vessels. Macrophages were co-migrating with the tumor cells in the multicellular streams in both human tumors analyzed, and functional inhibition of macrophages in vivo by clodronate liposomes significantly decreased the frequency of multicellular streams, while increasing the frequency of single moving cells. Macrophage inhibition also significantly abrogated intravasation, as measured by the number of circulating tumor cells in the bloodstream of the treated mice. Since we found that macrophage inhibition reduced streaming migration while enhancing single cells migration, we deduce that multicellular streaming is the type of in vivo migration that most likely directly contributes to intravasation in human breast tumors. Finally, we analyzed the gene expression profile of the migratory tumor cells from both the MDA-MB-231 and the TN1 tumors and found that they both exhibited multiple gene expression changes in genes regulating cell motility. These gene expression changes were largely common in the two tumors and coordinated to regulate the basic actin motility machinery, leading to the same end phenotype of activated actin polymerization and enhanced migration. Our data is the first direct visualization of in vivo migration of live patient-derived breast tumor cells and the first assessment of the type of cell motility involved in tumor cell dissemination in a human tumor cell derived breast tumor. This abstract is also presented as Poster A51. Citation Format: Antonia Patsialou, Jose Javier Bravo-Cordero, Yarong Wang, Huiping Liu, Michael F. Clarke, John S. Condeelis. Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR5.

Use of imaging to study leukocyte trafficking in the central nervous system

The migration of leukocytes from the bloodstream into the central nervous system (CNS) is a key event in the pathogenesis of inflammatory neurological diseases and typically involves the movement of cells through the endothelium of post-capillary venules, which contains intercellular tight junctions. Leukocyte trafficking has predominantly been studied in animal models of multiple sclerosis, stroke and infection. However, recent evidence suggests that immune cells and inflammation mechanisms play an unexpected role in other neurological diseases, such as epilepsy and Parkinson's disease. Imaging leukocyte trafficking in the CNS can be achieved by epifluorescence intravital microscopy (IVM) and multiphoton microscopy. Epifluorescence IVM is ideal for the investigation of leukocyte-endothelial interactions, particularly tethering and rolling, signal transduction pathways controlling integrin activation, slow rolling, arrest and adhesion strengthening in CNS vessels. Multiphoton microscopy is more suitable for the investigation of intraluminal crawling, transmigration and motility inside CNS parenchyma. The mechanisms of leukocyte trafficking in the CNS are not well understood but the use of in vivo imaging techniques to unravel the underlying regulatory pathways will provide insight into the mechanisms of brain damage and may contribute to the development of novel therapeutic strategies. In this review, we discuss recent work in this field, highlighting the development and use of in vivo imaging to investigate leukocyte recruitment in the CNS.

Dynamic visualization of RANKL and Th17-mediated osteoclast function

Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static - bone resorptive (R) to moving - nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.

The Transcription Factor NFAT Exhibits Signal Memory during Serial T Cell Interactions with Antigen-Presenting Cells

Interactions with antigen-presenting cells (APCs) interrupt Tcell migration through tissues and trigger signaling pathways that converge on the activation oftranscriptional regulators, including nuclear factor of activated Tcells (NFAT), which control Tcell function and differentiation. Both stable and unstable modes of cognate Tcell-APC interactions have been observed invivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t(1/2 max)∼1min), nuclear export was slow (t(1/2)∼20min) in Tcells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the Tcell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of Tcell tolerance.

Extravasating Neutrophil-derived Microparticles Preserve Vascular Barrier Function in Inflamed Tissue

Emerging evidence suggests that gap formation and opening of the endothelial junctions during leukocyte extravasation is actively controlled to maintain the integrity of the vascular barrier. While the role for endothelial cells to this process has been well defined, it is not clear whether leukocytes are also actively contributing to endothelial barrier function. We have recently showed that extravasating leukocytes deposit microparticles on the subendothelium during the late stages of extravasation, which is LFA-1 dependent. Using multiphotonintravital microscopy (MP-IVM) of mouse cremaster muscle vessels in the current work, we show that microparticle formation and deposition maintains the integrity of the microvascular barrier during leukocyte extravasation. Inhibition of neutrophil-derived microparticle formation resulted in dramatically increased vascular leakage. These findings suggest that deposition of microparticles during neutrophil extravasation is essential for maintaining endothelial barrier function and may result in temporal difference between neutrophil extravasation and an increase in vascular leakage.

Non-Invasive Multimodal/Multi-Scale Imaging of Bone Marrow Microenvironment Reveals Structural and Functional Heterogeneity in Different Bone Marrow Compartments: Functional Implications On Mouse and Human Hematopoietic Stem Cell Niches in Health and Disease

AbstractAbstract 25 Introduction:Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) microenvironments called stem cell niches (HSCNs). Two types of HSCNs have been reported, involving osteoblasts (osteoblastic niche) and blood sinusoids (vascular niche). Various hematopoietic and non-hematopoietic cell types are contributing to the regulation of HSCs and HSCNs, however, the bone marrow microenvironment has not yet been precisely characterized and the fine localization, composition and regulation of the niches remain highly controversial.Intravital microscopy of the calvarium (IVMC) is the only non-invasive option for high resolution imaging of bone marrow HSCNs. However it is unclear if IVMC provides data representative of all BM compartments. Indeed it has been shown that endochondral ossification, the process used for long bones growth, is required for the formation of HSCNs. By contrast, the calvarium is a flat bone which develops through intra-membranous ossifications, therefore questioning the presence of HSCNs in this bone (Chan CK et al, Nature 2009). Methods:Combined confocal and multiphoton excitation intravital microscopy was used for multiparametric observation of live bone marrow in intact bones after in-vivo contrasting procedures. Bioluminescence imaging was used to quantify the systemic trafficking and proliferation of murine HSCs and human leukemia. Flow cytometry and histology were used to sort specific populations of cells, analyze the frequency of mouse and human stem cells or leukemic cells at steady state or after transplantation, quantify perfusion efficiency and hypoxia and cross-validate in-vivo imaging procedures. Results:Thanks to advanced imaging modalities we realized a thorough study of potential niche markers and HSCs distribution at homeostasis and during hematopoietic reconstitution in calavia, epiphyses and diaphyses. We report important heterogeneity between these compartments in terms of bone remodelling activity (BRA) and blood vessel fraction (BVF). Although BVF was surprisingly high in any compartment, including in very close proximity to any endosteal surface, we found that compartments displaying the highest BVF and BRA were preferentially seeded and engrafted following mouse and human HSC transplantation. Unexpectedly, the macro-anatomical distribution of mouse and human HSCs at steady state is homogeneous across the skeleton and independent of these 2 markers. These data suggest the existence of “reconstituting niches” which would be distinct from “homeostatic niches”. Importantly, this study provides the first evidence that both types of niches are fully functional in the calvarium, including for mouse and human HSCs, indicating that endochondral ossification is dispensable for adult HSCNs.The model is currently being exploited to analyze leukemia/microenvironment interactions in live bone marrow. Conclusions:This study confirms and extends our recent statement considering the critical need for multimodal imaging (Lassailly F et al, Blood 2010). Further more, it demonstrates that combination of different imaging modalities for in-vivo and ex-vivo analysis is a powerful strategy allowing to shed a new light on the structure of the bone marrow microenvironment and improve our understanding of stem cells/niches interactions in health and disease. Disclosures:No relevant conflicts of interest to declare.

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

Improved intravital microscopy via synchronization of respiration and holder stabilization

A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy.

HIV-infected T cells are migratory vehicles for viral dissemination

After host entry through mucosal surfaces, HIV-1 disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during early stages of transmission, and systemically during subsequent dissemination are not known1. In vitro studies suggest that formation of virological synapses (VSs) during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer2. It is unclear, however, if T cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial3 and lymphoid tissues4. Here, using multiphoton intravital microscopy (MP-IVM), we examined the dynamic behavior of HIV-infected T cells in lymph nodes (LNs) of humanized mice. We found that most productively infected T cells migrated robustly, resulting in their even distribution throughout the LN cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope (Env)-dependent cell fusion. Both uncoordinated motility of syncytia as well as adhesion to CD4+ LN cells led to the formation of long membrane tethers, increasing cell lengths to up to 10 times that of migrating uninfected T cells. Blocking the egress of migratory T cells from LNs into efferent lymph, and thus interrupting T cell recirculation, limited HIV dissemination and strongly reduced plasma viremia. Thus, we have found that HIV-infected T cells are motile, form syncytia, and establish tethering interactions that may facilitate cell-to-cell transmission through VSs. While their migration in LNs spreads infection locally, T cell recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.

Quantitative intravital microscopy of hepatic transport

Intravital microscopy has a long history of application to studies of the liver. Here we demonstrate how intravital multiphoton microscopy may be combined with digital image analysis to quantitatively evaluate the effects of drugs on hepatocyte transport in vivo.

Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates-only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

Role of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers

This study investigated the role of inducible nitric oxide synthase (iNOS) in failure of ethanol-induced fatty liver grafts. Rat livers were explanted 20h after gavaging with ethanol (5g/kg) and storing in UW solution for 24h before implantation. Hepatic oil red O staining-positive areas increased from ∼2 to ∼33% after ethanol treatment, indicating steatosis. iNOS expression increased ∼8-fold after transplantation of lean grafts (LG) and 25-fold in fatty grafts (FG). Alanine aminotransferase release, total bilirubin, hepatic necrosis, TUNEL-positive cells, and cleaved caspase-3 were higher in FG than LG. A specific iNOS inhibitor 1400W (5μM in the cold-storage solution) blunted these alterations by >42% and increased survival of fatty grafts from 25 to 88%. Serum nitrite/nitrate and hepatic nitrotyrosine adducts increased to a greater extent after transplantation of FG than LG, indicating reactive nitrogen species (RNS) overproduction. Phospho-c-Jun and phospho-c-Jun N-terminal kinase-1/2 (JNK1/2) were higher in FG than in LG, indicating more JNK activation in fatty grafts. RNS formation and JNK activation were blunted by 1400W. Mitochondrial polarization and cell death were visualized by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. After implantation, viable cells with depolarized mitochondria were 3-fold higher in FG than in LG and 1400W decreased mitochondrial depolarization in FG to the levels of LG. Taken together, iNOS is upregulated after transplantation of FG, leading to excessive RNS formation, JNK activation, mitochondrial dysfunction, and severe graft injury. The iNOS inhibitor 1400W could be an effective therapy for primary nonfunction of fatty liver grafts.

If You Don’t Look, You Won’t See: Intravital Multiphoton Imaging of Primary and Metastatic Breast Cancer

A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interactions.

The role of a cx3cr1+/egfp mucosal resident dendritic cell network in mucosal homeostasis (49.10)

Abstract Cxcr1 is expressed in all CD11c+ mucosal dendritic cells (DC). Mucosal DCs are perivascular and are interconnected to form a network. However, the importance of the network in maintaining mucosal homeostasis is poorly understood. We first aimed to gain insight on the structure of the DC network. Using multi-photon intravital microscopy, we demonstrate that the cx3cr1+/egfp DC network interconnects DCs between villi and throughout small and large bowel. The network overlaps a radiation resistant non-hematopoietic cell network. We next aimed to examine the role of DCs in vascular permeability. cx3cr1+/egfp DCs were selectively depleted by targeting depletion with CD11c-diptheria toxin receptor (DTR) transgenic intercross. Preliminary data suggests that DCs are not necessary to maintain vascular integrity, as measured by permeability of 70kDa dextran fluor. Critical for maintaining adaptive immunity is spatial temporal regulation of T cell migration as T cells survey antigen present cells (APC). Since DCs are a critical APC, T cells patrolling was examined following depletion of the mucosal DC network. Adoptive transfer studies demonstrate that CD4 and CD8 T cell migration remains intact despite selective depletion of DCs. In summary, the mucosal DC network represents a higher order of organization of DCs than previously appreciated. Resident DCs may play a role in maintaining vascular permeability but surprisingly do not play a role in regulating mucosal T cell migration.

Antigen availability is a critical determinant of in vivo CD8+ T cell-dendritic cell interaction kinetics and initiates effector/memory fate decisions (110.21)

Abstract Naïve CD8+ T cells are activated through interactions with antigen (Ag) bearing dendritic cells (DCs) in lymph nodes (LNs). Multiphoton intravital microscopy (MP-IVM) of LNs has demonstrated that T cell activation occurs in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely related to Ag dose. It had been assumed that the second phase, characterized by stable DC-T cell contacts, is necessary for T cell activation, however, here we show that this is not always the case. T cells interacting with DCs presenting Ag at low abundance and with a short half-life did not transition from phase one to phase two, whereas a higher dose of the same Ag yielded robust phase two transition. T cells exposed to either antigenic constellation proliferated and developed equivalent effector functions, including cytokine production and cytotoxicity, over the first two days after priming. There are profound differences in the transcriptome at 12h and 24h after activation between CD8 T cells exposed to antigenic constellations that did or did not promote phase two interactions. Only T cells that had undergone stable contacts gave rise to memory, whereas activating conditions that did not support stable contacts yielded immunological amnesia. T cells can make fate decisions in vivo within a few hours after Ag exposure that result in long-term memory or abortive effector responses and correlate with the kinetics of T cell-DCs interactions.

Multiphoton intravital microscopy analysis of signaling in T cells reveals persistent NFAT activation during dynamic APC scanning. (121.21)

Abstract The Ca2+ dependent activation and nuclear shuttling of the cytoplasmic transcription factor NFAT is a hallmark of productive T cell receptor (TCR) triggering. However, the kinetics of NFAT activation and the relationship to the Ca2+ dependent migratory stop signal have not been explored in vivo. We used Multiphoton Intravital Microscopy (MP-IVM) to visualize the subcellular dynamics of a fluorescent reporter of NFAT signaling (NFAT-eGFP) in cytotoxic T cells during their antigen-dependent interactions with various APC in lymph nodes (LNs) and in tumor tissue in vivo. During interactions with peptide-loaded B cells representing optimal APC in LNs, t1/2 of NFAT nuclear translocation was 1 minute. Relocation to the cytoplasm upon contact cessation occurred with a slower t1/2 of ~20 minutes. During its delayed nuclear export, NFAT continued to drive transcription of IFN-gamma. We therefore hypothesized that NFAT may facilitate the integration of TCR signals received through serial APC contacts. To test this in vivo, we performed MP-IVM to visualize contacts between cytotoxic T cells and tumor cells, representing suboptimal APCs. We observed that among T cells with continuously nuclear NFAT, ~30% remained motile while engaging in transient, serial contacts with multiple tumor cells. Hence, NFAT slow off-kinetics may constitute a mechanism of short-term biochemical memory enabling the integration of intermittent TCR signaling into a continuous transcriptional response.