Soluble Lymphotoxin Plays a Critical Role in Acute Graft-Versus-Host Disease

Abstract

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies.We have employed multiple well established preclinical models of GVHD to investigate the roles of these molecules. We first used a functional bioassay to confirm that a human TNFR:Fc construct was capable of preventing physiological activity of both recombinant murine TNF and LTα3. Irradiated B6D2F1 recipients were then transplanted with allogeneic B6.TNF−/− bone marrow and T cells, then treated with control Ig or the TNFR:Fc. Suprisingly, the TNFR:Fc provided significant protection from GVHD mortality in the absence of donor derived TNF (median survival 25.5 vs 35 days, P<0.0002). Having observed a potentially TNF-independent but TNFR dependent pathway in the generation of GVHD we next examined mRNA expression by real-time PCR. This demonstrated that the majority of LTα mRNA was within donor CD4 T cells and that expression was unbalanced (> 3 logs more LTα than LTβ). We subsequently developed the first reported ELISA to assess murine LTα3 protein and were able to clearly demonstrate LTα3 production by wild-type (WT) CD4 T cells seven days after BMT. To further examine this molecule, we performed experiments using WT, LTα−/− or LTβ−/− donor grafts in two BMT systems (B6 → B6D2F1 and B6 → BALB/c). Recipients of LTα−/− grafts were protected from GVHD (P<0.01) in both systems while LTβ−/− grafts induced GVHD that was equivalent to, or more severe than in recipients of WT grafts. This data confirms soluble LTα3 but not membrane bound LTα1β2 as an additional pathogenic molecule in GVHD. Using a luciferase-expressing, host-type leukemia (P815) in the B6 → B6D2F1 system, relapse occurred at an identical rate in recipients of WT and LTα−/− grafts confirming that GVL is maintained in the absence of donor derived soluble LTα3. Thus the combined neutralization of both LTα3 and TNF represents an important and logical therapeutic intervention in transplant medicine.