P0088 3D tissue-level analysis of intestinal T cell dynamics reveals dual effects of etrasimod on recruitment and tissue exit in experimental colitis

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Abstract Background Sphingosin-1-phosphate (S1P) receptor (S1PR) agonists are a novel class of anti-trafficking agents approved for the treatment of ulcerative colitis. These compounds induce T lymphocyte sequestration in secondary lymphoid organs, reducing their availability in peripheral blood and presumably in inflamed intestinal tissue. However, direct evidence for their effects on T cell dynamics within intestinal tissue is lacking. Moreover, while S1PR-dependent mechanisms regulate T cell egress from the gut, it remains unclear whether S1PR agonists modulate this process. This limited understanding of tissue-specific effects presents a crucial barrier to comprehending their full therapeutic mechanism. Methods We developed a comprehensive volumetric imaging platform to investigate tissue-level mechanisms of colitis within the complex three-dimensional architecture of the intestine. Using the T cell transfer colitis model, mice were treated with etrasimod or vehicle control. Full-thickness colon samples underwent tissue clearing and whole-mount immunofluorescence for T cell markers. Advanced light-sheet and volumetric confocal microscopy, combined with machine learning-based image analysis, enabled three-dimensional reconstruction of T cell distributions and lymphatic networks, allowing for quantitative histocytometry and spatial mapping of immune cell dynamics. Results T cell transfer colitis induced substantial lymphocyte infiltration in the colon, with T cells forming both distinct clusters and diffuse distributions throughout the tissue. The presence of numerous T cells within lymphatic vessels indicated active tissue egress, suggesting that T cell accumulation reflects a dynamic balance between recruitment and exit (Figure 1). Etrasimod treatment not only reduced overall T cell infiltration but also markedly altered their spatial distribution pattern. Notably, we observed a decreased proportion of T cells within lymphatic vessels, coupled with increased accumulation of T cells in proximity to lymphatic structures, indicating a previously unrecognized effect on tissue exit dynamics. Conclusion Our advanced volumetric imaging approach revealed novel insights into a potential dual mechanism of etrasimod in inflammatory bowel disease. Beyond reducing lymphocyte recruitment, etrasimod appears to create a tissue exit block, effectively trapping T cells within the intestinal compartment. These findings raise important questions about how enforced tissue residency influences local inflammatory circuits and may guide optimization of therapeutic strategies.