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.