Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative therapy for life-threatening disorders including high-risk leukemia and bone marrow failure. However, the benefits of alloHSCT remain limited by acute graft-versus-host disease (GVHD), where alloreactive donor T cells destroy host tissues in the skin, liver and gastrointestinal tract. New therapies to distinguish pathogenic T cells from T cells mediating beneficial immune responses are necessary to improve the safety and applicability of alloHSCT. Our previous publications and current preliminary data suggest that targeting alloreactive T cell metabolism may allow for this selective intervention. Specifically, our data demonstrate that deletion of AMPK in donor cells mitigates GVHD but still preserves lymphopenia-driven immune reconstitution and T cell-driven graft-versus-tumor (GVT) effects. Mechanistically, our data further suggest that lower rates of GVHD result from decreased sensitivity of AMPK knock-out (KO) cells to the effects of pro-inflammatory cytokines. From this, we form the following central hypothesis: AMPK is activated early post-transplant in a tissue-specific fashion, increasing local T cell sensitivity to pro-inflammatory cytokines. In the absence of AMPK, inflammatory signals are blunted, stabilizing regulatory T cell (Treg) development and decreasing effector responses. These changes mitigate GVHD, while GVT responses are unaffected because increased cytokine sensitivity is unnecessary for inducing leukemia-directed cytotoxicity and because leukemia clearance occurs at sites where AMPK activation is less pronounced. We will test this hypothesis through three specific aims.
In Aim 1, we will determine the location and temporal necessity of AMPK by eliminating AMPK in T cells at defined times post- transplant and quantitating AMPK activation in cells recovered from multiple tissues simultaneously. We will also define the relationship between AMPK activation and cytokine sensitivity by measuring cytokine responses following stimulation with an array of AMPK agonists.
In Aim 2, we will elucidate mechanisms linking AMPK deficiency to improved GVHD and decreased cytokine sensitivity by comparing the GVHD potential of single KO cells, to cells lacking both AMPK and the IL-6 receptor. In addition, we will use mass spectrometry to measure phosphorylation of novel AMPK target proteins in cytokine-stimulated and alloreactive T cells.
In Aim 3 we will determine the GVHD and GVT potential of AMPK-deficient human T cells after decreasing AMPK levels using CRISPR/Cas9 gene editing and short hairpin RNA transduction, followed by transplantation of modified cells into xenogeneic models of GVHD and immunodeficient models of GVT. These studies will deepen our understanding of AMPK activation, how this activation impacts cytokine sensitivity, and whether these findings can be translated into human cells. If successful, our studies will define a novel mechanism linking energy sensing to T cell effector function that will likely extend beyond GVHD to include the robust and sustained activation of any T cell, including during autoimmunity and following solid organ transplantation.

Public Health Relevance

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative therapy for many life-threatening conditions, but the benefits of alloHSCT are often overshadowed by the risks of acute graft-versus-host disease (GVHD), where donor T cells attack and destroy tissues in the host. Targeting the specialized metabolism of alloreactive T cell represents one way to overcome the risks of GVHD, but still retain the benefits of immune reconstitution and graft-versus-tumor effects. In this proposal, we investigate the mechanisms of why alloreactive T cells lacking AMP-activated protein kinase cause less GVHD, and will translate these findings into human T cells, in an effort to design better therapies for GVHD and comparable T cell-mediated immune disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL144556-01
Application #
9637009
Study Section
Transplantation, Tolerance, and Tumor Immunology (TTT)
Program Officer
El Kassar, Nahed
Project Start
2019-02-01
Project End
2024-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pediatrics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213