Real-time Two-photon Microscopy Research Articles

Real-Time Two-Photon Microscopy Reveals Spatially Heterogeneous T Lymphocyte Trafficking in the GI Microenvironment Influenced By GI Tissue Damage Level

Introduction: The trafficking of immune cells in the GI is crucial for tissue immunity and can be targeted for the treatment of immune-related GI damage. Flow cytometry, as the primary technique to identify immune cells in organs such as the GI, has made substantial progress, offering valuable insights into a variety of immune cell subtypes and their microenvironments. However, one major limitation is the destruction of tissue samples during processing, lacking spatial information on immune cells in their microenvironment. Furthermore, there is a lack of information about how stress-induced tissue damage affects T-cell trafficking. In particular, the pre-HCT conditioning regime is essential for identifying the extent of organ damage and donor immune cell trafficking to the GI microenvironment and the subsequent occurrence of Graft vs Host Disease (GVHD). To address this need, it is necessary to develop a real-time imaging method for characterizing immune cells in the GI in an allogeneic bone marrow transplant (BMT) system. Here, we report developing a temperature-controlled Two-photon microscopy (TPM) setup investigating the pattern of T cell trafficking in the GI in real time and how modulating radiation dose affects immune cell trafficking in the preclinical mouse GVHD model of allogeneic BMT. Method: BALB/c Host mice were treated with 8 Gy TBI in 2 fractions, 6 h apart. For the GI dose reduction study (GI-4 Gy), Total Marrow Irradiation (TMI 8:4; 8 Gy to Bone marrow, 4 Gy to rest of the body including GI) was used as described (Darren et al. 2021). Donor B6 T cell Depleted bone marrow cells along with 1 million enriched tdtomato+ T cells were transplanted, and 7 days later, live T cell trafficking in GI was monitored by TPM (Prairie Ultima microscope). Vessels were labeled with QTracker 655, and TPM parameters were set as described (J Brooks et al. 2021). For imaging, a temperature-controlled externalized intestine's intravital imaging (EIII) window was developed (Fig 1.A) and was first evaluated to visualize the three-dimensional basis of Jejunum. It allows continuous blood flow while effectively controlling peristalsis, cardiac and respiratory motion artifacts with minimal tissue damage, delay in tissue degradation. A section of the intestine (Jejunum) was externalized from anesthetized mice and fixed on the EIII window's surface using n-butyl cyanoacrylate veterinary adhesive. The luminal surface was exposed by making an incision along the intestine, carefully avoiding the vascular plexus. The spatial distribution of vessels and tdtomato+ T-cells (green) at different z-stacks was evaluated by imaging through both serosa and luminal sides. GI samples were fixed in 10% NBF, OCT embedded, cryosection (5-10 m), and Anti-MAdCAM1 immunofluorescence (IF) performed using a standard protocol. Results: EIII window usage significantly reduced peristaltic movement, enabling T cells imaging at different depths from serosa to villi crypt (Fig 1.A-B). T cells reside preferentially in the lower crypt region and are affected by the radiation dose given to GI (Fig 1.D-G). T-cell localization at the crypt base region (~ 100µ from serosa) was significantly higher in TBI (8 Gy GI dose) mice than in TMI (4 Gy GI dose). Fig. 1 (F-G) shows T cell distribution at the top, middle, and base of the villi, and in the crypt, during luminal side imaging. Further, microvascular structures were affected at higher radiation dose, as seen by reduced vessel density in 8 Gy over 4 Gy treatments or control (Fig 1.C). Thus, ~50% dose reduction improved vasculature and reduced T cell trafficking. Histological analysis of intestinal tissue with lysozyme and anti-MAdCAM1 staining for confocal imaging further shows reduced crypt damage and T cell trafficking in lower radiation (Fig 1.H). Summary: TPM enabled us to visualize T cells in real-time within the GVHD mouse intestine. Radiation-induced tissue damage-dependent T-cell trafficking was evident in the mouse GI with GVHD and validated through the target receptor of MAdCAM1 endothelial cell expression. The spatial heterogeneity of T cells emphasizes the preferential niche of immune trafficking and associated GI environment and thus, the need for cautious interpretation of GVHD biopsies and warrants further studies on their spatial localization in immune cell-mediated GI diseases.

Abstract 2475: Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells

Abstract The efficacy of chimeric antigen receptor (CAR) T-cell therapy in solid tumors is limited by inefficient T-cell infiltration, localization and hypofunctionality within the tumor microenvironment. Prior studies demonstrated that stromal cell targeted CAR T cells, in particular CAR T cells that target fibroblast activation protein (FAP), can effectively infiltrate, and inhibit tumor growth in mouse models of pancreatic ductal adenocarcinomas (PDAC) due to their capacity to deplete stromal cells and extracellular matrix (ECM) that otherwise present a barrier to adoptive cell therapies. However, the kinetics of tumor infiltration by FAP-CAR T cells, and the cell-cell and cell-matrix interactions that contribute to their anti-tumor activity are poorly understood. A better understanding FAP-CAR T-cell kinetics and intratumoral interactions should provide insights into the requirements for successful adoptive cell therapies in solid tumors. By combining immunostaining and real-time two-photon microscopy in tumor fragments of PDAC, we found that stromal cell targeted FAP-CAR T cells can efficiently traffic into tumors. Initially (days 1-3) FAP-CAR T cells were enriched in the stroma surrounding tumor nests but excluded from the tumor nests per se. While associated with the stroma rich region, the CAR T cells appeared activated and T cell motility was observed in loose collagen regions, but T cells migrated poorly in dense matrix areas. Aligned collagen fibers in the tumor boarder dictated the migratory trajectory of T cells and restricted them from entering tumor nests. By day 5 we observed depletion of immunosuppressive stromal cells, dissolution of the fibrillar collagen network surrounding tumor nests and infiltration of FAP-CAR T cells into tumor nests. Treatment with hyaluronidase increased the ability of T cells to infiltrate the tumor nest. Thus, FAP-CAR T cell-mediated ablation of immunosuppressive FAP+ stromal cells and disruption of the desmoplastic stroma they generate, can enhance accumulation and functionality of CAR-T cell therapy in the context of highly desmoplastic solid tumors. Citation Format: Zebin Xiao, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L. Barrett, Ellen Puré. Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2475.

Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets

Detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of blood-brain barrier (BBB). The present work describes two novel single-domain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology.

Monitoring the Oxygen Dynamics of Brain Tissue In Vivo by Fast Acousto-Optic Scanning Microscopy: A Proposed Instrument.

The function of the brain neural circuit is highly dependent on oxygen supply. Imaging the precise oxygen distribution and dynamics are critical for understanding the relationship between neuronal activity and oxygen dynamics of the nearby capillaries. Here, we develop fast acousto-optic scanning two-photon microscopy. Combined with oxygen probes, such as PtP-C343, we can monitor oxygen dynamics at the submicron level by this real-time microscopy. In this fast acousto-optic scanning microscopy, an acousto-optic deflector (AOD), an inertia-less scanner, is used to scan the femtosecond laser. A cylindrical lens is used to compensate the 'cylindrical lens effect' of AOD and a prism is used to compensate the chromatic dispersion of AOD. An electro-optical modulator (EOM) and a sCMOS camera are gated to measure the phosphorescence lifetime. With a 40× water objective lens, this set-up can image a 100 μm × 100 μm field of view at a speed of 20 frames per second and a 25 μm × 8 μm field of view at a speed of 500 frames per second. This real-time two-photon microscopy is expected to be a good tool for observing and recording the precise rapid oxygen dynamics in the cerebral cortex, which will facilitate studies of oxygen metabolism in neurosciences.

Phenotypic and Morphological Properties of Germinal Center Dark Zone Cxcl12-Expressing Reticular Cells.

The germinal center (GC) is divided into a dark zone (DZ) and a light zone (LZ). GC B cells must cycle between these zones to achieve efficient Ab affinity maturation. Follicular dendritic cells (FDCs) are well characterized for their role in supporting B cell Ag encounter in primary follicles and in the GC LZ. However, the properties of stromal cells supporting B cells in the DZ are relatively unexplored. Recent work identified a novel stromal population of Cxcl12-expressing reticular cells (CRCs) in murine GC DZs. In this article, we report that CRCs have diverse morphologies, appearing in open and closed networks, with variable distribution in lymphoid tissue GCs. CRCs are also present in splenic and peripheral lymph node primary follicles. Real-time two-photon microscopy of Peyer's patch GCs demonstrates B cells moving in close association with CRC processes. CRCs are gp38(+) with low to undetectable expression of FDC markers, but CRC-like cells in the DZ are lineage marked, along with FDCs and fibroblastic reticular cells, by CD21-Cre- and Ccl19-Cre-directed fluorescent reporters. In contrast to FDCs, CRCs do not demonstrate dependence on lymphotoxin or TNF for chemokine expression or network morphology. CRC distribution in the DZ does require CXCR4 signaling, which is necessary for GC B cells to access the DZ and likely to interact with CRC processes. Our findings establish CRCs as a major stromal cell type in the GC DZ and suggest that CRCs support critical activities of GC B cells in the DZ niche through Cxcl12 expression and direct cell-cell interactions.

Intravital microscopic interrogation of peripheral taste sensation.

Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

Visualization of Helios-expressing T cells in the skin (179.12)

Abstract The majority of Foxp3(+)CD4(+) T cells develops in thymus as thymus-derived Tregs. On the other hand, naive T cells can express Foxp3 to become "induced" Tregs under certain conditions where TGF-b plays a key role. Helios is potentially a specific marker for thymus-derived Tregs. However characteristics of Helios(+) and Helios(-) Tregs remain elusive because it is still difficult to sort these cells alive. To solve the above problem and to delineate difference between Helios(+) and Helios(-) Tregs in the skin, we established a gene-targeted mouse in which Helios-expression can be monitored by Venus, a derivative of green fluorescent protein. Venus was expressed in a subset of double negative thymocytes, intra-thymic Tregs, and 80% of peripheral Tregs in accordance with endogenous Helios expression. Characteristics of the cutaneous Tregs are currently under investigation. Our Helios/Venus mouse is a versatile tool for studying Treg-subsets in various tissues including the skin not only with flow cytometer but also under real-time two-photon microscopy. Additionally, this system would provide a novel scope for screening factors that control Helios expression and alter epigenetic state of Tregs.

Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells

The mechanism of B cell-antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells. Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune complexes by a complement receptor-dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore, cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an important site of B cell encounter with immune complexes and show that intrafollicular B cell migration facilitates the transport of immune complexes as well as encounters with cognate antigen.

Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity

Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8(+) T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15-20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC-T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance.

Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes.

The cellular dynamics underlying activation of CD8+ T cells by dendritic cells (DCs) in the lymph node are not known. Here we have tracked the behavior of T cells and DCs by subjecting intact lymph nodes to real-time two-photon microscopy. We show that DCs scan at least 500 different T cells per hour in the absence of antigen. Antigen-bearing DCs are highly efficient in recruiting peptide-specific T cells and can engage more than ten T cells simultaneously. The duration of these interactions is of the order of hours, not minutes. The overall avidity of the interaction influences the probability that T cells will be stably captured by DCs, providing a possible basis for T cell competition. Taken together, our results identify the cellular behaviors that promote an efficient CD8+ T cell response in the lymph node.

Real-Time Two-Photon Microscopy and Its Application for In Situ Imaging.

The recent developments in fluorescent-probes and laser technologies have provided us opportunities to observe the biological functions in living organs and tissues under a microscope. Although single-photon excitation confocal microscopy is an indispensable tool for observing these specimens, the observable depth of this microscopy is still not enough to analyze the functions of whole parts of organs in living organisms. To observe the deeper parts, we have attempted to develop new real-time two-photon excitation microscopy equipped with a microlens-arrayed multipinhole scanning device. Using this real-time two-photon microscope, we examined the dynamics of the intracellular calcium concentration ([Ca2+] i) in the cardiac myocytes in the rat whole heart to understand their physiological functions and mechanisms. We could observe Ca 2+ waves on the surface in the whole heart. However, we still need higher-energy laser power for two-photon excitation and visualization of [Ca2+]i dynamics in the deeper parts of the whole heart.