(b) Lethally irradiated CB6F1 mice received BMC and splenocytes from syngeneic donors (closed squares, = 10), C57BL/6 mice and antibodies PIP (open circles, = 15), or 6A6 (closed circles, = 15). cell) and allowed for graft-versus-tumor (GVT) effects as well as robust responses to pathogens. These results suggest that BTLA agonism rebalances Tirabrutinib T cell expansion in lymphopenic hosts after aHSCT, thereby preventing GVHD without global immunosuppression. Thus, targeting BTLA with a Tirabrutinib monoclonal antibody at the initiation of aHSCT therapy might reduce limitations imposed by histocompatibility and allow broader application to treatment of nonClife-threatening diseases. Replacement of an abnormal lymphohematopoietic system by allogeneic hematopoietic stem cell transplantation (aHSCT) from a healthy donor is an effective treatment for many disorders of the hematopoietic system (Sykes and Nikolic, 2005; Copelan, 2006). Induction of a mixed hematopoietic donor-host chimerism can induce long-lasting tolerance to foreign tissues without the need for life-long immunosuppressive therapy (Kawai et al., 2008). aHSCT therapy has been improved by better donor identification (Petersdorf et al., 2004), more tolerable conditioning regimens (McSweeney et al., 2001), and enhanced supportive care. However, significant treatment-related morbidity and mortality from chemotherapy, radiotherapy, infections, and graft-versus-host disease (GVHD) remain significant clinical problems. Therefore, aHSCT is commonly indicated only for treatment of conditions where other treatment options are far inferior or lacking. Costimulatory molecules of the CD28 and TNF families regulate GVHD, with inhibitory and activating receptors either decreasing or increasing its severity (Tamada et al., 2000; Blazar et al., 2003; Xu et al., 2007). B and T lymphocyte associated (BTLA) is an inhibitory immunoglobulin superfamily receptor, whose ligand is the TNF receptor herpesvirus entry mediator (HVEM) and which has only been examined in a nonirradiated model of chronic allostimulation without classical GVHD where donor cells lacking BTLA failed to persist (Hurchla et al., 2007). The role of BTLA in aHSCT using irradiated recipients, in which clinical symptoms and pathology similar to human GVHD develop, has not been examined. RESULTS AND DISCUSSION To determine the role of BTLA in the development of GVHD, we first examined Tirabrutinib WT and BTLA?/? donor mice (Watanabe et al., 2003) using a nonlethal parent-into-irradiated F1 model of aHSCT (Stelljes et al., 2008). In this model, GVHD results from partial MHC mismatch between H-2b haplotype donor cells and lethally irradiated H-2b/d haplotype recipients. BM and splenocytes from WT or BTLA?/? mice on the C57BL/6 background were transferred into lethally irradiated CB6F1 recipients (Fig. 1 a). Transplantation of WT donor cells into CB6F1 recipients caused body weight loss of 30% and clinical scores (Cooke et al., 1996) of 3 that persisted for 40 d. BTLA?/? and WT donor cells caused similar GVHD, suggesting that BTLA does not normally regulate GVHD in this model. To test whether BTLA expressed by recipient mice might Tirabrutinib regulate GVHD in this model, we used BTLA?/? CB6F1 hosts as recipients of BTLA?/? BM and splenocytes (Fig. S1 a). BTLA?/? donor cells induced similar GVHD in BTLA+/? and BTLA?/? hosts, which is comparable to GVHD by WT donor cells (Fig. 1 a). Collectively, these data suggest that BTLA Tirabrutinib does not normally regulate GVHD. Open Smoc1 in a separate window Figure 1. Anti-BTLA treatment permanently prevents GVHD. (a) Lethally irradiated CB6F1 mice received BMC and splenocytes from C57BL/6 WT (closed squares, = 5) or BTLA?/? (open squares, = 5) donors. (b) Lethally irradiated CB6F1 mice received BMC and splenocytes from syngeneic donors (closed squares, = 10), C57BL/6 mice and antibodies PIP (open circles, = 15), or 6A6 (closed circles, = 15). Shown are cumulative data from three independent experiments. (c) Lethally irradiated CB6F1 mice received BMC and splenocytes from C57BL/6 mice plus control antibody PIP (open circles, = 5) or 6A6 (closed circles, = 5) on the day of BMT or 6A6 14 d after BMT (open squares, = 5)..