Three-Dimensional Visualization of Immunological Processes of Entire Organs in Subcellular Resolution.

Abstract 61

In vivo models of complex immune processes like stem cell engraftment, host-pathogen interactions or anti-tumor responses face the challenge to either provide dynamic information in low resolution (e.g. in vivo bioluminescence Imaging, BLI) or provide high resolution information with a limited field of view (e.g. multi-photon laser scanning microscopy/confocal microscopy).

To overcome these limitations we applied a novel selective plane illumination microscopy (SPIM) technique (also termed ultramicroscopy), which enabled us to visualize structural and cellular changes in intact organs in high resolution. Here we investigated dynamic shifts at sites of graft-versus-host disease (GVHD) initiation in mice after allogenic hematopoietic cell transplantation (allo-HCT).

For in vivo and ex vivo imaging we transplanted 1,2×10⁶ luciferase+ DsRed+ transgenic C57Bl/6 T cells (H-2^b, Thy1.1⁺) plus 5×10⁶ bone marrow (BM) cells (H-2^b, Thy1.2⁺) into myeloablative conditioned allogeneic Balb/c recipients (H-2^d, Thy1.2⁺, 8 Gy) to induce aGVHD. Utilizing BLI we followed the development of GVHD in vivo. At the transition from GVHD initiation phase to effector phase (day+3 and day+4) we prepared the small bowel and Peyer‘s Patches (PPs) of allo-HCT recipients for whole organ microscopy and compared these to organs of untreated mice.

Staining for T cell populations (CD4) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) allowed us to visualize MAdCAM-1 expression in relation to T cell areas in whole PPs and surrounding intestinal mucosa (> 8mm³) in a high throughput format by creating approx. 1000 optical sections using multiple lasers and a sensitive CCD camera within 10 minutes. Adding another color channel revealed structural details through measurement of intrinsic autofluorescence. The individual color stacks were overlaid via computational image processing for three dimensional tissue reconstruction, volume measurements and quantification of protein expression.

Ultramicroscopy exposed microanatomical structures like intestinal villi, crypts and PPs with its subepithelial dome regions and follicles by giving detailed information about MAdCAM-1 expressing blood vessels and T cell areas. The 3D reconstruction of small intestines in non-conditioned mice revealed that the MAdCAM-1 expression averaged at 1-2% of the entire PP and was predominantly restricted to the high endothelial venules (HEV). During the transition from the initiation to the effector phase of acute GVHD (day3-4) the overall MAdCAM-1 expression in PPs increased by 50fold. At this point MAdCAM-1 expression was also found in parafollicular and subepithelial cell populations, which still need to be characterized further concerning their lineage differentiation.

MAdCAM-1 is well known as a vascular addressin molecule. Because of its importance in homing of α4β7⁺ T and B cell into the gastrointestinal tract we investigated the dynamic expression of MAdCAM-1 during acute GVHD within intact PPs. It is well established that MAdCAM-1 is an important regulator in the homing of α4β7⁺ T cell to PPs via HEVs. Additionally our data lead to the hypothesis that MAdCAM-1 might contribute to retain proliferating α4β7⁺ T cells during the initiation of an adaptive immune response and might support a synchronized exit of alloreactive T cells in the aGVHD effector phase.

Conventional histological sections are labor intensive, time consuming and often restricted to a very limited region of interest and therefore, can result in missing rare events and under- or overestimation of biological processes. Ultramicroscopy helped us to overcome these limitations and enabled us to visualize immunological processes with subcellular resolution in whole organs.

CB and MF, GSH and AB contributed equally to this work.