Graft-versus-host disease (GVHD) is a major obstacle to allogeneic hematopoietic cell transplantation. GVHD is induced when donor mature T cells encounter host antigens and get activated. Activated T cells require high metabolic flux through growth-promoting pathways to support clonal expansion. It is generally thought that glycolysis is employed in this process. However, conflicting data have been reported by different groups regarding the role of glycolysis in GVHD. Since glucose transporter 1 (Glut1) is essential for T cell glycolysis, we attempted to define the role of glycolysis in GVHD using donor T cells that are deficient of Glut1.
One million T cells from Glut1-knockout (CD4CreGlut1fl/fl) or wild-type (control) C57BL/6 donors were transplanted into lethally irradiated (9.5 Gy) C3H/HeJ mice along with 1×107 donor T-cell-depleted bone marrow (TCDBM) cells. While all recipients of control T cells developed lethal GVHD and died within 20 days after transplantation, eight out of ten CD4CreGlut1fl/fl T cell recipients survived more than 60 days after transplantation (Figure 1, P<0.01). Body weights were slightly lower and clinical scores were slightly higher (P=not significant) in the surviving CD4CreGlut1fl/fl T cell recipients at day +56 when compared to those in the TCDBM alone group, suggesting that CD4CreGlut1fl/fl T cell recipients developed very mild GVHD if any. We further studied the role of Glut1 in CD4 and CD8 T cell mediated GVHD separately using C57BL/6 into BALB/c model. Similar to the results from the total T cell experiment, all CD4CreGlut1fl/fl CD4 and CD8 T cell recipients survived more than 60 days after transplantation. By contrast, 100% control CD4 and 50% CD8 T cell recipients died of lethal GVHD within 60 days after transplantation. These data demonstrate that Glut1 is required for alloresponses and glycolysis is indispensasble in the development of lethal GVHD.
To understand the mechanism by which CD4CreGlut1fl/fl T cells mediate less GVHD, we first studied their ability to respond to alloantigens in mixed lymphocyte reaction. The results indicated that CD4CreGlut1fl/fl T cells have dramatically decreased ability to respond to BALB/c antigens when compared with the control T cells. Consistent with the in vitro data, T cell expansion was 59.4% less in CD4CreGlut1fl/fl T cell recipients than that in control T cell recipients when measured at day +7 after GVHD induction. Apoptosis was increased in alloantigen-activated (CD69+) CD4 but not in CD8 T cells from CD4CreGlut1fl/fl mice when compared with that from control mice when measured 16 hours after the initiation of culture. However, more cell death was observed in both activated CD4 and CD8 T cells from CD4CreGlut1fl/fl mice than that from control mice. In addition, alloreactive CD4CreGlut1fl/fl T cells failed to sustain activated mTORC1 signaling, as assessed by phosphorylation of the downstream p70 S6 kinase target, small ribosomal subunit S6. These results indicate that, upon response to alloantigens, T cells are prone to apoptosis and cell death and lead to decreased clonal expansion in the absence of Glut1, suggesting a mechanism by which CD4CreGlut1fl/fl T cells have decreased ability to induce GVHD.
To determine whether absence of Glut1 in T cells affects graft-versus-tumor (GVT) effects, we transplanted 1×106 T cells from Glut1-knockout (CD4CreGlutfl/fl) C57BL/6 donors into lethally irradiated (8.5 Gy) BALB/c mice along with 1×107 donor TCDBM cells. At the time of transplantation, the recipients were challenged with 1×105 BCL1 cells, a BALB/c-derived leukemia/lymphoma cell line. While all TCDBM alone recipients died of leukemia/lymphoma and all control T cell recipients died of lethal GVHD, all CD4CreGlut1fl/fl T cell recipients survived more than 100 days after transplantation without leukemia/lymphoma (Figure 2), indicating that T cells deficient of Glut1 preserve GVT effect.
The results from this study definitely demonstrate that glycolysis is essential for the development of GVHD. Inhibition of glycolysis in T cells increases apoptosis and cell death and suppresses clonal expansion of allospecific T cells. Interestingly, GVT effect is preserved despite the inhibition of glycolysis. These data suggest that glycolysis could potentially be targeted for prevention and treatment of GVHD.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.