The Intestinal Stem Cell Compartment is the Initial Target of T Cell Invasion in GI Gvhd

Crypt apoptosis is a hallmark of GI GVHD pathology, which can be prevented by inhibition of T cell homing to the gut. However, lymphocytic infiltration is frequently not apparent in clinical GI GVHD biopsies. Given the limitations of traditional 2-D histologic imaging, there is thus little understanding of the key sites within the intestinal mucosa where allogeneic T cells exert their effector functions leading to GVHD pathology and damage to the intestinal stem cell (ISC) compartment. Using 3-D microscopy of intact intestinal tissue to accurately preserve spacial relationships (Figure 1A), we investigated T cell infiltration in the villus and crypt regions after C57BL/6 (B6) into BALB/c experimental GVHD. The lower crypt region, where the ISC compartment is located, was the only area demonstrating increased CD3+ T cell density early post-transplant, suggesting specific invasion of the ISC compartment by alloreactive T cells (Figure 1B). Allogeneic bone marrow transplantation (BMT) with purified GFP+ donor T cells confirmed donor T cell invasion of the crypt base ISC compartment four days after transplant, which was associated with a significant loss of crypt base columnar ISCs (Figure 1C). Assessment of T cell localization day 7 post-BMT demonstrated substantial increase in T cell density throughout the intestinal mucosa, but the lower crypt region remained the most densely infiltrated area. Surprisingly, this T cell migration pattern directly contrasted with the density of the intestinal vasculature, which was significantly greater in the villi than in the crypt region. However, evaluation of vascular integrin ligand expression demonstrated clustering of MAdCAM-1+ vessels around the crypt base, which appeared to mirror the pattern of T cell infiltration of the lower crypt region (Figure 2). To test the significance of this MAdCAM-1 expression pattern, donor T cell localization was imaged after treatment with a monoclonal antibody targeting MAdCAM-1. Anti-MAdCAM-1 was administered starting day 2 post-BMT to allow for initial allo-activation in secondary lymphoid organs. Treatment with anti-MAdCAM-1 specifically reduced crypt invasion by donor T cells without impacting migration to the villi, resulting in protection of crypt base ISCs from GVHD (Figure 3).Figure 2MAdCAM-1* vasculature clusters around the crypt compartment. (A) 3-D imaging of small intestine vasculature with MAdCAM-1 vessels (red) and MAdCAM-1 staining (light blue) demonstrates greater vascular density in the villus region (above the dash line) compared to the crypt region (below the dash line), but MAdCAM-1 vasculature was predominantly localized arount the crypt compartment; scale bars, 150 µm. (B) Quantification of MAdCAM-1 density; **P < .01;. ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Treatment with anti-MAdCAM-1 reduced crypt region invasion by donor T cells, resulting in ISC protection. (A) Quanfication of donor T cell densities indicate the reduction of donor T cells infiltration in the lower crypt region with anti-MAdCAM-1 treatment; **P < .01. (B) Representative nuclear images of crypt base columnar ISCs (light blue) and Paneth cells (dark blue) in 3-D after treatment with isotype antibody or anti-MAdCAM-1; scale bars, 25 µm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our results reveal that the crypt base region where the ISC compartment is located is the first site of donor T cell migration into the GI tract. This T cell invasion of the ISC compartment occurs early after BMT resulting in loss of ISCs, and MAdCAM-1, which is thought to contribute to intestinal T cell homing in general, actually specifically directs T cell recruitment to the stem cell compartment. These findings provide new mechanistic understanding of how the ISC compartment is targeted post-transplant and how this can be prevented by inhibition of the α4β7: MAdCAM-1 axis.