Integrin α4 and CCR5 direct IFN-γ–activated MSCs to the gut and associated lymphoid tissue enabling effective GVHD prophylaxis

1. γMSC trafficking to GVHD tissues is essential for prevention, while monocytes/macrophages are needed for γMSC function, not for trafficking. 2. γMSC trafficking to GVHD tissues relies on integrin α4β1–MAdCAM-1 adhesion and CCR5-driven chemotaxis toward inflammatory chemokines. Interferon-γ-activated mesenchymal stromal cells (γMSCs) exhibit potent immunosuppressive properties; yet nonactivated MSCs remain the focus of clinical trials. Murine studies have shown that γMSCs can prevent acute graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT), but the site of immunomodulatory activity and the molecular mechanisms directing their distribution remain unclear. Using a mouse model of acute GVHD, human γMSC infused 24 hours after HCT localized primarily in the gut, gut-associated lymphoid tissue (GALT), liver, and spleen, but not skin draining lymph nodes (SDLN), sites comparable with the cellular initiation of GVHD. In vitro, γMSC adhered preferentially to spleen endothelial cells compared to SDLN endothelium. Integrin α4 knockdown and anti-MAdCAM-1 blocking antibody reduced binding to spleen endothelial cells by 94% and 48%, respectively. Activated T cells upregulated CCL3, CCL4, and CCL5, and blocking these chemokines together abolished γMSC migration. γMSC expressed CCR5 was upregulated upon exposure to activated T cell media and CCR5 knockdown reduced in vitro migration by 90%. In vivo , knockdown of α4 or CCR5 impaired γMSC trafficking to GALT and significantly reduced GVHD prophylaxis, although T cell suppression in vitro was retained. Finally, depletion of monocytes/macrophages by liposomal clodronate did not affect γMSC trafficking but abolished the GVHD-suppressive activity of γMSC, indicating that macrophages are not required for γMSC trafficking, but essential for their therapeutic function. Our study reveals that the coordinated expression of integrin α4 and CCR5 on γMSC governs their trafficking where they exert their immunosuppressive effect and significantly reduce GVHD in a murine model.