Integrin α4 and CCR5 on IFNγ-activated MSC determine biodistribution to critical sites for aGVHD prophylaxis in mice

Abstract

Background & Aim

Mesenchymal stromal cells (MSC) activated with interferon-γ (γMSC) exhibit potent immunosuppressive properties, yet nonactivated MSC remain the focus of clinical trials. Extensive mouse studies show that γMSC can prevent acute Graft versus Host Disease (GVHD) after hematopoietic cell transplantation, but the molecular mechanisms affecting their distribution post-IV infusion and the sites of activity remain unclear. This study aims to identify the site(s) of activity and elucidate the molecular and cellular mechanisms governing γMSC trafficking.

Methodology

A mouse model of fully MHC mismatched hematopoietic cell transplantation (HCT) was used. Human γMSC were infused 24 hours post-HCT, and their tissue localization was assessed. CRISPR/Cas9 was used to modify γMSC, and in vitro analyses conducted using IncuCyte or fluorescence microscopy. Macrophages depletion with clodronate was performed to study their role in trafficking and GVHD prevention.

Results

We observed that γMSC were enriched in gut-associated lymphoid tissue (GALT) 16 hours post-IV infusion, critical site for the development of aGVHD. In vitro, γMSC exhibited significantly greater adhesion to spleen endothelial cells compared to other SLO endothelial cells, and integrin α4 was crucial for this interaction, as α4 knockdown reduced binding by 94%. We identified CCL3, CCL4, and CCL5 as key chemokines upregulated in murine activated T cell conditioned media and blocking these chemokines collectively abolished γMSC migration. γMSC expressed CCR5 knockdown in γMSC reduced migration by 90%. In vivo, knockdown of α4 or CCR5 in γMSC impaired their trafficking to GALT and significantly the prophylactic effects against GVHD, although T cell suppression by γMSC was retained. Furthermore, depletion of monocytes/macrophages by liposomal clodronate did not affect γMSC trafficking to the spleen but abolished the GVHD-suppressive activity of γMSC, indicating that macrophages are not required for γMSC trafficking, but essential for their therapeutic function.

Conclusion

Our study reveals that the coordinated expression of integrin α4 and CCR5 on γMSC governs their trafficking to GALT, where they exert their immunosuppressive effect and significantly reduce GVHD in a mouse model. These findings highlight the importance of GALT localization in γMSC efficacy, offering insights that could inform strategies to enhance the therapeutic potential of γMSC in GVHD prevention.