PD-L1/CD80 and PD-L1/PD-1 Signaling Reciprocally Regulate Alloreactive CD8+ T Cell Glycolysis, Proliferation, Apoptosis and Gvhd-Inducing Capacity

Graft versus host disease (GVHD) remains the major obstacle for widespread application of allogeneic hematopoietic cell transplantation (HCT), a curative therapy for hematological malignancies. We and others have reported that while sorted CD4⁺ T cells induce severe acute GVHD, the same numbers of CD8⁺ T cells induce no acute GVHD. CD8⁺ T cells can facilitate donor cell engraftment and mediate graft versus leukemia (GVL) effect in the absence of GVHD, but the mechanisms remain largely unknown. Programmed death-ligand 1 (PD-L1) interacts with both CD80 and PD-1. We have recently reported that simultaneous PD-L1/CD80 and PD-L1/PD-1 signaling augments activated alloreactive CD4⁺ T cell proliferation and apoptosis and ameliorates acute GVHD. However, the impact of this PD-L1-mediated signaling on alloreactive CD8⁺ T cells has not been investigated. In addition, T cell glycolytic metabolism has been recently reported to be able to regulate acute GVHD, and PD-L1/PD-1 signaling inhibits CD4⁺ T cell glycolytic flux, but how PD-L1/CD80 modulates T cell metabolism remains unstudied. In the current studies, we evaluated the role of PD-L1/CD80 and PD-L1/PD-1 signaling on alloreactive CD8⁺ T cell glycolysis, proliferation, apoptosis and GVHD-inducing capacity, using a MHC-mismatched murine HCT model of C57BL/6 (H-2b) donor to BABL/c recipient (H-2d). We have observed the following early (3-5 days) after HCT: 1) In wild-type (WT) recipients, PD-1^-/- CD8⁺ T cells significantly upregulate expression of Glucose Transporter I (Glut1) and exhibit much higher rates of glycolytic flux; PD-1^-/- CD8⁺ T cells also show markedly enhanced proliferation and reduced apoptosis as well as up-regulation of gut tissue homing and chemokine receptors (i.e. α4β7 and CCR9) and induce lethal GVHD, as compared to WT CD8⁺ T cells that induce little signs of GVHD. 2) In PD-L1^-/- recipients, PD-1^-/- CD8⁺ T cells reduce expression of Glut1 and no longer exhibit enhanced glycolytic flux or reduced apoptosis; PD-1^-/- CD8⁺ T cells transplanted into PD-L1^-/- recipients also induce little GVHD, similar to WT CD8⁺ T cells.  3) After injection of anti-PD-L1 mAb (clone 43H12) that specifically blocks PD-L1/CD80 interaction and preserves PD-L1/PD-1 interaction, WT CD8⁺ T cells in WT recipients show drastically reduced expansion, as compared to control recipients treated with rat-IgG. Blockade of PD-L1/CD80 interaction reduces phosphorylation of key elements in the TCR signaling cascade and CD28 co-stimulatory pathway including ZAP-70, AKT, mTOR and rpS6. Taken together, these studies indicate that 1) PD-L1/CD80 signaling alone augments alloreactive CD8⁺ T cell expansion and GVHD-inducing capacity; 2) PD-L1/CD80 and PD-L1/PD-1 signaling reciprocally regulate CD8⁺ T cell glycolysis, proliferation, and apoptosis as well as their GVHD-inducing capacity, and a balance of the two signaling pathways would be required to allow donor CD8⁺ T cells to facilitate engraftment and mediate GVL effect without causing GVHD. This work was supported by Institutional Funds of The Beckman Research Institute of City of Hope.