CD47 Upregulation Inhibits Phagocytic Clearance of Alloreactive Donor T Cells after Allo-HCT

Acute graft-versus-Host disease (aGvHD) is a life-threatening complication that frequently occurs after allogeneic hematopoietic cell transplantation (allo-HCT). Resolving inflammation is a key therapeutic goal in aGvHD. Macrophage-mediated phagocytosis contributes substantially to resolve inflammatory processes. However, the role of phagocytosis after allo-HCT still remains unexplored. Therefore, we analyzed the expression of the “don´t eat me” signal CD47 on allogeneic T cells from aGvHD patients and in two MHC major mismatch aGvHD mouse models (FVB à C57BL/6, 9 Gy TBI and C57BL/6 à Balb/C, 8 Gy TBI). We evaluated the effect of in vitro and in vivo administration of an anti-CD47 antibody on antibody-dependent cellular phagocytosis (ADCP) of alloreactive T cells using flow cytometry and confocal microscopy, bioluminescence imaging, cytokine measurements and histopathology. Initial analysis of a global dataset revealed increased CD47 expression on intestinal T cells in patients with chronic gastrointestinal inflammation. Subsequently, we examined CD47 levels on T cells in the allo-HCT setting. Notably, intestinal T cells in patients with severe GvHD (Lerner grade III) significantly upregulated CD47 (p = 0.0079). There were no significant changes in CD24 (another “don´t eat-me”-signal) or in the “eat-me”-signals calreticulin and ATP10A. This upregulation of CD47 correlated with reduced macrophage-mediated phagocytosis. Similarly, intestinal alloreactive donor T cells highly upregulated CD47 in mice developing aGvHD after allo-HCT (p = 0.0317). In vitro studies revealed that CD47 expression is regulated by NF- kB in T cells after TCR activation and primed T cells inhibit ADCP in a CD47-dependent fashion. Transfer of CD47 knock-out T cells and bone marrow resulted in swift clearance of donor T cells after allo-HCT resulting in significantly improved clinical scores and survival compared to controls (p = <0.0001). Crucially, application of an anti-CD47 antibody significantly antagonized the impaired ADCP of alloreactive T cells. In anti-CD47 treated mice, we observed increased phagocytosis of T cells in the gastrointestinal tract (GIT) and induction of immunosuppressive responses in vivo. Besides an increase of the myeloid-derived suppressor cell population, systemic pro-inflammatory cytokine levels such as TNF, IL-1β and IFN-β decreased in the serum. In conclusion, our data suggest that high CD47 expression impairs phagocytic clearance of alloreactive T cells after allo-HCT, potentially contributing to exaggerated inflammatory responses in the GIT of GvHD patients. Thus, CD47 emerges as potential therapeutic target to eliminate alloreactive T cells, with anti-CD47 treatment aiding the resolution of inflammation in GvHD.