Donor Pretreatment with Agonistic Anti-DR3 Antibody Prevents Chronic GvHD in a Murine Sclerodermatous Model

Background: DR3 is a cell surface receptor of the TNF superfamily and is expressed preferentially by activated T cells. The agonistic antibody of DR3 (aDR3) can stimulate regulatory T cell (Treg) expansion and ameliorate acute GVHD in a major-mismatch murine model. However, it is still unclear whether the aDR3 is effective in other GVHD models. A murine sclerodermatous GVHD model features skin fibrosis and shares many clinical characteristics with human chronic GVHD, for which currently available therapies have demonstrated limited efficacy.

Methods: The αDR3 or isotype control antibody (0.5 mg/kg) was intraperitoneally injected into B10.D2 mice four days before transplantation. To generate a sclerodermatous GVHD model, lethally irradiated BALB/c recipient mice were transplanted with T cell-depleted bone marrow cells from non-treated B10.D2 mice and splenocytes isolated from the αDR3 or isotype-treated B10.D2 mice. Some Balb/c mice were transplanted with only the T cell-depleted bone marrow cells (omitting splenocytes) and served as the non-GVHD control (the control recipients). Recipient mice were monitored for six weeks to assess body weight, GVHD score, and survival or sacrificed to harvest skin, spleen, and colon on 7, 14, and 42 days after transplantation. Bronchoalveolar lavage (BAL) was performed on days 7 and 14.

Results: Recipients of the aDR3-treated splenocytes (the aDR3 recipients) experienced significantly less weight loss and showed decreased GVHD scores than recipients of isotype-treated splenocytes (the isotype recipients). The severity of cutaneous GVHD was considerably reduced in the aDR3 recipients than in the isotype recipients. Pathologic examinations such as the ratio of the dermis to subcutaneous tissue in the skin and histologic scores of the colon performed on days 7, 14, and 42 post-transplant confirmed the clinical results. Flow cytometric analysis suggested that the reconstitution of B and T cells in the spleen of isotype recipients was severely disrupted during the later phase of the transplant. Although the number of CD4 T cells and B cells in the control recipients and the aDR3 recipients had been increasing until day 42, those in the isotype recipients slightly increased on day 14 but markedly decreased on day 42. Therefore, the number of CD4 T cells in the isotype recipients was significantly lower than that of the other groups on day 42, and the number of B cells in the isotype showed a significant difference on days 14 and 42. In contrast, the reconstitution of CD11b monocytes was not disrupted in the spleen of the isotype recipients. The reconstitution of splenic Treg showed a similar pattern to CD4 T cells. Interestingly, the number of Treg in the aDR3 recipients was consistently higher than that of the other groups. These findings suggest that GVHD may disrupt lymphocyte reconstitution after hematopoietic cell transplantation but treating donors with αDR3 can prevent it.

Next, we performed qPCR analysis to quantitate the mRNA levels of key cytokines and chemokines in the skin and colon. The expression level of TGF-β was remarkably higher in the skin than in the colon, and the reduction of TGF-β in the αDR3 recipients was more pronounced in the skin than in the colon, confirming its primary role in skin fibrosis. In addition, the αDR3 recipients had decreased levels of IFNg, IL-6, and IL-10 in the skin and the colon than the isotype recipients. Moreover, the αDR3 recipients had diminished expression of various chemokines (CCL1, CCL2, CCL3, CCL4, CCL8, CCL17, and CCL22) in the skin compared to the isotype recipients. Chemokine expression profile in the colon was almost identical to the skin except for CCL17 and CCL22. These two cytokines were similarly reduced in the colon of the αDR3 recipients and the isotype recipients. Next, we examined inflammatory cell content in the BAL fluid. The numbers of total cells and macrophages were significantly lower in the αDR3 recipients than in the isotype recipients on days 7 and 14, but the numbers of lymphocytes and neutrophils were different between these two groups only on day 14. The αDR3 recipients had significantly fewer CD4 T cells, B cells, and CD11b monocytes in BAL fluid than the isotype recipients.

Conclusions: Treating donors with αDR3 before transplantation could alleviate sclerodermatous GVHD by preventing disruption of hematopoiesis, fostering immune reconstitution, and reducing skin fibrosis and lung inflammation.

No relevant conflicts of interest to declare.