27: Treatment of experimental acute graft-versus-host disease using extracorporeal phototherapyA novel murine model

Extracorporeal phototherapy (ECP) is an emerging therapy for clinical graft-versus-host disease (GVHD) that exposes a patient’s peripheral white blood cells (WBCs) to photo-activatable 8-methoxypsoralen (8-MOP) and UVA light before re-infusing them. We have developed a novel murine model that closely mirrors ECP treatment of clinical GVHD to investigate its mechanism of action. C3H.SW mice were conditioned with 11Gy total body irradiation and injected with 5.0×106 bone marrow and 0.5×106 purified T cells from either syngeneic (C3H.SW) or allogeneic (B6-Ly5.2) donors. In order to model ECP as a treatment strategy rather than as prevention, animals were not treated until GVHD was clinically evident 7 days after BMT and then received 4 weekly infusions with 30.0×106 8-MOP+UVA treated splenocytes from similarly transplanted mice. Control mice were infused with untreated splenocytes, and did not show any differences from mice treated with L-15 (data not shown). Infusion of 8-MOP+UVA-treated splenocytes into allo-BMT recipients resulted in significantly better day +56 survival (74% vs 42%, p=0.0007), reduced GVHD clinical scores (2.9 vs 5.9, p<0.004) and GVHD histopathology in liver (7.3 vs 13.4), gut (11.4 vs 18.4) and skin (0.6 vs 1.2; all organs p<0.03) compared to mice infused with untreated splenocytes. ECP treated allo-BMT recipients also had significantly better immune reconstitution (p=0.01) on day +56, with both total thymocytes and distribution of thymocyte compartments indistinguishable from syngeneic controls. In vitro studies showed that >98% of cells were Annexin+ within 24h of ECP treatment and experiments using Ly5 congenic donors demonstrated that ECP treated cells are undetectable in the thymus and spleen following injection. Because apoptotic cells are known to induce tolerance in several systems, we hypothesized that ECP increased the number of T regulatory cells (Treg). The number of CD4+CD25+Foxp3+ Treg in ECP-treated allo-BMT recipients was significantly increased compared to mice infused with untreated splenocytes in the thymus (2.5 vs 1.0×104, p<0.02) and the spleen (1.8 vs 1.1×105, p=0.008). We conclude that 4 weekly infusions of ECP treated cells beginning after GVHD induction significantly decreases acute GVHD by all clinical, pathological and cellular parameters and is associated with increased numbers of Tregs.Tabled 1SYNALLO+ALLO++/- ECPNo ECP SplECP Splp-value⁎95confidence level, Allo+ECP Spl vs Allo+No ECP SplNumber151934Day +56 Analysis:Survival100%42%74%0.0007GVHD Clinical Score1.1 ± 0.45.9 ± 0.32.9 ± 0.40.004PathologyLiver2.1 ± 0.913.4 ± 5.57.3 ± 2.50.0001Intestine7.4 ± 2.818.4 ± 5.711.4 ± 3.10.0003Skin01.2 ± 0.70.6 ± 0.60.03Thymic reconstitutionThymocytes (×106)12.4 ± 9.92.2 ± 2.17.7 ± 7.20.01Thymic Treg (×104)5.1 ± 2.61.0 ± 0.62.5 ± 1.40.02Spleen Treg (×105)6.2 ± 2.91.1 ± 0.41.8 ± 0.10.008 95confidence level, Allo+ECP Spl vs Allo+No ECP Spl Open table in a new tab