Erythropoietin Promotes GvHD Prevention through Type 1 Conventional Dendritic Cells

Allogeneic hematopoietic cell transplantation (HCT) is a proven treatment for hematologic malignancies; however, it is associated with significant morbidity and mortality due to complications such as graft-versus-host disease (GVHD). GVHD occurs when donor T cells react against recipient antigens (Ags) presented by Ag-presenting cells (APCs), with dendritic cells (DCs) playing a crucial role in this process. Treatment with apoptotic cells (ACs) induced by extracorporeal photopheresis (ECP) has been successful in reducing acute and chronic GVHD, especially in patients unresponsive to standard immunosuppressive therapies. Type 1 conventional dendritic cells (cDC1s), which acquire apoptotic cells by a process known as efferocytosis, possess unique antigen processing and presentation capabilities that determine the direction and intensity of the T cell immune response. The Engleman laboratory recently discovered that erythropoietin (Epo) mediated signaling in cDC1s is a key mechanism regulating cell-associated Ag-specific T cell tolerance, linked to efferocytosis-induced cDC1 tolerogenic maturation. On this basis, we hypothesized that combined treatment with Epo and ECP could further reduce GVHD severity by inducing cDC1 dependent T cell-mediated immune tolerance, thereby reducing GVHD induction and severity.

Splenocytes were incubated with 8-MOP and exposed to UVA light before injection into mice two days before HCT. Each mouse received 1x10⁷ ECP treated splenocytes. Epo was administered intraperitoneally daily at a dose of 50 IU/animal from day -3 to day +1. Mice were conditioned with total body irradiation (TBI: BALB/c: 880 cGy, C57BL/6: 1100 cGy) and received T-cell-depleted bone marrow (TCD-BM; 5x10⁶/animal) plus conventional T cells (Tcon; BALB/c, 1x10⁶/animal; C57BL/6, 4x10⁶/ animal) from allogeneic donors. First, to explore the potential of targeting the EpoR tolerogenic pathway in cDC1s to prevent GVHD, we utilized EpoR-tdTomato-Cre mice as recipients to enable analysis of EpoR expression levels following TBI, with ECP or ECP+Epo. We found that TBI by itself had only a minor effect on the percentage of cDC1s among DCs in the spleen (p=0.2546) but significantly increased EpoR expression on cDC1s, (p=0.0002), which was further enhanced by ECP plus Epo treatment (p=0.0059). This raises the possibility that EpoR⁺ cDC1s undergo tolerogenic activation following TBI, which could be amplified by administering ECP+Epo. In line with our previous study, recipients (BALB/c) that received Tcon (C57BL/6) alone died from GVHD (median 41.5 days), while animals that received ECP had prolonged survival (median 63.5 days; p=0.0088) as did animals that received Epo (median 57.5 days; p=0.0522). Remarkably, recipients treated with ECP+Epo showed superior survival outcomes (median not reached; p<0.0001) compared to both the ECP alone and Epo alone treated groups. Similar results were observed using C57BL/6 mice as recipients and BALB/c mice as donors. Next, to investigate the role EpoR expression on cDC1s in mediating the GVHD preventing effect of ECP+Epo, we utilized cDC1-specific conditional knock out EpoR^ΔXCR1 (XCR1^Cre/+EpoR^flox/flox; H-2Kb⁺) by cross breeding EpoR^flox/flox with Xcr1^Cre-mTFP1 mice. The benefit of Epo in preventing GVHD was diminished in EpoR^ΔXCR1 mice (p=0.0943), and that of ECP was also reduced.

Taken together, our results indicate that ECP+Epo serves as a novel therapeutic regimen in preventing GVHD post-HCT, which is dependent on cDC1 EpoR expression. Further studies are underway to identify the underlying tolerogenic mechanisms.