A Novel Role for AMP-Activated Protein Kinase (AMPK) in Controlling T Cell Sensitivity to IL-6

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and many non-malignant hematologic disorders. However, the use of alloHSCT as a therapy 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 alloreactive T cells increase phosphorylation of the intracellular energy sensor AMP-activated protein kinase (AMPK) as early as day 3 post-transplant (levels 8-fold higher in alloreactive T cells compared to naive or syngeneic controls (p=0.0003)) and donor T cells lacking AMPK (AMPK KO) cause significantly less GVHD in both major- and minor-histocompatibility mismatch models of GVHD. At the same time, AMPK KO T cells equivalently clear leukemia cells and extend recipient survival in leukemia models with high tumor burden. Transplantation of cells lacking AMPK lowers 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 AMPKKO respectively) but interestingly, increases both the percentage and total number of regulatory T cells (T_reg) (0.85 +/- 0.32x10⁴ vs. 1.69 +/- 0.34x10⁴, wt vs. KO, p=0.004).

To understand these findings mechanistically, we sought to study the role of AMPK in donor T_reg expansion and stabilization post-alloHSCT. Canonically, interleukin-2 (IL-2) signaling, through phosphorylation of Stat5, promotes T_reg generation and stability, while inflammatory signals (e.g. IL-6 signaling through Stat3) are known to decrease T_reg stability post-transplant. Studies testing IL-2 sensitivity in KO cells, or the degree of Stat5 phosphorylation post-transplant, were unrevealing. However, AMPK KO T_regs significantly decrease their sensitivity to IL-6, with a 50% reduction in phosphorylated-Stat3 levels following intermediate levels of cytokine stimulation (Figure 1A). To demonstrate the functional consequences of this difference in cytokine sensitivity, we exposed in vitro generated T_reg to increasing concentrations of IL-6. As expected, the percentage of wt FoxP3+ cells decreased sharply with increasing IL-6 concentrations (decreasing from 50% to 14%), while AMPK KO T_regs were minimally affected over this dose range (47% versus 42%; Figure 1B, p<0.02).

To determine if increased T_reg stability was the predominant reason for improvements in GVHD seen with AMPK KO cells, CD25⁺ cells were removed from the donor inoculum of AMPK KO or wt mice by magnetic selection (to eliminate >90% of T_reg), and then either CD25-replete or CD25-depleted sets of cells were transplanted into B6xDBA2 F1 recipients in a major MHC mismatch model of GVHD. Despite a decrease in AMPK KO T_reg numbers on day 7 to levels commiserate with wt donors (Figure 2A), elimination of AMPK KO T_reg had only a modest effect on GVHD severity (Figure 2B), implicating a substantial role for AMPK in the non-T_reg compartment.

To explore the role of AMPK in non-T_regs further, we investigated the sensitivity of unmanipulated wt and AMPK KO T cells to IL-6 stimulation. Similar to FoxP3⁺ cells, both naïve CD4 and CD8 T cells lacking AMPK experienced lower levels of Stat3 phosphorylation in response to graded concentrations of IL-6 (Figure 3A). Furthermore, if IL-6 was added to a 4-day culture with anti-CD3 stimulation, the number of wt T cells recovered after 96 hours increased >2.2-fold. In contrast, AMPK KO cells experienced minimal changes in T cell number over a wide range of IL-6 concentrations (Figure 3B).

In conclusion, we define in these studies a novel role for AMPK in controlling donor T cell sensitivity to IL-6, both at the level of Stat3 phosphorylation, and in terms of functional consequences. Not only does this striking result elegantly explain our previous observation of increased T_reg percentages, and decreased donor T cell numbers, following transplantation of AMPK KO cells, it continues to make the case that inhibition of AMPK represents an attractive clinical target for the amelioration of GVHD.

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