Abstract
Allogeneic hematopoietic stem cell transplantation (alloHSCT) represents a curative treatment for high-risk leukemia and a number of non-malignant hematologic disorders. However, the therapeutic use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that the alloreactive T cells responsible for GVHD increase their dependence on the oxidation of fat relative to either syngeneic or naive T cells. To explore this adaptation mechanistically, we studied the role of AMPK, an intracellular energy sensor and known driver of fat oxidation, in donor T cells during GVHD. Alloreactive T cells increased phosphorylation of AMPK as early as day 3 post-transplant, with up-regulation in pathways both up- and downstream of AMPK. Changes in phosphorylation were up to 8-fold higher in alloreactive T cells compared to naive T cells or syngeneic controls (p=0.0003). We then investigated the role of AMPK during GVHD pathogenesis using donor cells deficient in AMPK (from AMPKα1fl/flα2fl/fl x CD4-Cre mice). AMPK-/- T cells caused significantly less GVHD in both major-MHC and minor-histocompatibility mismatch models of GVHD (Figure 1A), with a coordinated decrease in the number of donor T cells recovered on day 7 post-transplant (3.15 +/- 0.49x106 versus1.87 +/- 0.53x106, p=0.0006, wildtype (wt) versus AMPK-/- respectively). Importantly, expansion of syngeneic T cells was unaffected by AMPK deficiency (Figure 1B).
We next investigated the ability of AMPK-/- T cells to mount effective cytotoxic and anti-leukemia responses. AMPK-/- T cells demonstrated equivalent cytotoxicity against MHC-mismatched targets both in vitro and in vivo and differentiated in similar proportions into cytokine-producing cells (IFN-γ, TNFα, IL-17, and IL-4). We then assessed graft-versus-leukemia (GVL) potential in AMPK-/- cells using a GVL model with high tumor burden. AMPK-/- T cells exhibited equivalent clearance of p815 leukemia cells on day 13 post-transplant (Figure 2A), and extended survival of recipient mice similarly to wt T cells (Figure 2B). To elucidate possible mechanisms underlying this separation of GVL and GVHD responses, we evaluated metabolic pathways in wt and AMPK-/- T cells recovered on day 7. To our surprise, rates of fatty acid oxidation were identical between wt and AMPK-/- T cells and loss of AMPK did not impact alloreactive T autophagy, nor impair signaling downstream of mammalian target of rapamycin.
To define the mechanism underlying AMPK-/- benefits, we quantitated levels of regulatory T cells (Treg) on day 7 post-transplant. In contrast to expectation, both the percentage and total number of Treg increased in mice receiving AMPK-/- T cells (0.85 +/- 0.32x104 vs. 1.69 +/- 0.34x104, wt vs. AMPK-/-, p=0.004). Loss of AMPK facilitated donor Treg expansion, as elimination of FoxP3+ cells prior to transplantation abrogated differences between wt and AMPK-/- donors on day 7. Importantly, Treg levels were equal in wt versus AMPK-/- donors prior to transplantation. Finally, we assessed the ability of AMPK-/- T cells to infiltrate into GVHD target organs. As shown in Figure 3, peri-portal infiltration of AMPK-/- cells was significantly reduced compared to wt T cells, and infiltrates in recipients of AMPK-/- cells contained many fewer CD3+ T cells per high-powered field, with CD3+ cells representing a lower percentage of cells overall. Decreased hepatic infiltration correlated with a lower percentage of circulating CD4+ cells and lower levels of the integrin pair α4β7 (55.2 +/- 1.4% versus 47.2 +/- 2.8% α4β7Hi cells, p=0.0017, wt vs. AMPK-/-).
In conclusion, deletion of AMPK in donor T cells decreases GVHD severity but spares anti-leukemia responses and preserves homeostatic immune reconstitution. Mechanistically, this occurs through a decrease in pathogenic T cell numbers, an increase in the number and percentage of Treg cells, fewer circulating CD4+ cells, and decreased infiltration of donor cells into target organs. From these findings, we conclude that AMPK represents a clinically relevant target in donor T cells pre-transplant and are actively exploring ways to translate this exciting therapy into clinical practice.
Disclosures
No relevant conflicts of interest to declare.
Metabolism plays the key roles in Th cells differentiation
