Purpose of review Controlling T cell activity through metabolic manipulation has become a prominent feature in immunology and practitioners of both adoptive cellular therapy (ACT) and haematopoietic stem cell transplantation (HSCT) have utilized metabolic interventions to control T cell function. This review will survey recent metabolic research efforts in HSCT and ACT to paint a broad picture of immunometabolism and highlight advances in each area. Recent findings In HSCT, recent publications have focused on modifying reactive oxygen species, sirtuin signalling or the NAD salvage pathway within alloreactive T cells and regulatory T cells. In ACT, metabolic interventions that bolster memory T cell development, increase mitochondrial density and function, or block regulatory signals in the tumour microenvironment (TME) have recently been published. Summary Metabolic interventions control immune responses. In ACT, efforts seek to improve the in-vivo metabolic fitness of T cells, while in HSCT energies have focused on blocking alloreactive T cell expansion or promoting regulatory T cells. Methods to identify new, metabolically targetable pathways, as well as the ability of metabolic biomarkers to predict disease onset and therapeutic response, will continue to advance the field towards clinically applicable interventions.
Graft-versus-host disease (GVHD) is the most common cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation. Using a pre-clinical model of GVHD, previous work has found that CD8 T cells recovered on day 7 post-transplant upregulate the transcription factor, peroxisome proliferator activated receptor delta (PPARd). While PPARd has been shown to be a key regulator of fatty acid oxidation (FAO) in other tissues, it’s role in T cells is not well studied. We hypothesize that PPARd drives FAO in alloreactive CD8 T cells and that CD8 T cells lacking PPARd will be unable to oxidize fat and thus unable to cause GVHD. To investigate this hypothesis, ex vivo FAO was measured in day 7 WT versus PPARd KO CD8 T cells by quantitating conversion of 3H-palmitate to 3H2O. Production of 3H2O decreased by 50% in KO CD8 T cells, suggesting an inability to fully oxidize fat. To understand the mechanism underlying this decrease, we performed RNA sequencing and identified thirty genes that were differentially expressed in WT versus KO CD8s including 8 genes related to FAO. Strikingly, despite a decreased ability to oxidize fat, PPARd KO CD8 T cells were recovered in equal numbers on day 7 post transplantation, suggesting that CD8 T cells lacking PPARd adopt alternative metabolic pathways to generate sufficient energy for in vivo proliferation and activation. Ultimately, our analyses indicate that PPARd is required for FAO in alloreactive CD8 T cells, but this process is dispensable for short term proliferation and/or survival of CD8 T cells during GVHD. Future directions will identify compensatory metabolic pathways utilized by PPARd KO T cells through a combination of in vitro nutrient drop out experiments and metabolomic flux analyses.
Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and multiple non-malignant hematologic disorders. However, the routine 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 GVHD-causing T cells increase fat oxidation compared to both syngeneic and naive T cells. To explore this adaptation mechanistically, we studied the role of the transcription factor Peroxisome Proliferator Activated Receptor delta (PPAR-δ) in alloreactive donor T cells during the initiation of GVHD. By day 7 post-transplant, alloreactive T cells up-regulated PPAR-δ >5-fold compared to pre-transplant naive T cells (p<0.0001, Figure 1A). Furthermore, PPAR-δ was necessary for maximally severe GVHD, as major-MHC mismatched B6xDBA2 F1 mice receiving donor T cells deficient in exon 4 of PPAR-δ (PPAR-δ KO) survived longer than mice receiving wildtype (WT) T cells (p<0.007, Figure 1B). We next investigated the mechanism underlying this observed decrease in GVHD severity. As a transcription factor, PPAR-δ controls expression of multiple genes involved in fat transport and oxidation. To determine its role in alloreactive cells, RNA was collected from CD4 and CD8 T cells on day 7 post-transplant and levels of 8 known PPAR-δ targets quantitated by RT-PCR. These 8 targets were selected from a longer list of genes known to be up-regulated in alloreactive cells. Transcript levels of both carnitine palmitoyl transferase-1a (CPT-1a) and CD36 decreased in PPAR-δ KO CD8 T cells (Figure 2A), with decreases in CD36 protein levels confirmed by immunoblot (Figure 2B). Interestingly, changes in CPT-1a and CD36 did not occur in PPAR-δ KO CD4 T cells. To assess the functional consequence of these changes, day 7 WT versus PPAR-δ KO CD8 T cells were plated with 3H-palmitate and fat oxidation measured ex vivo. Consistent with a decrease in expression of genes involved in fat transport and mitochondrial fat import, fat oxidation decreased by >75% in PPAR-δ KO CD8 cells (Figure 2C). However, despite these decreases, the number of PPAR-δ KO CD8 T cells recovered on day 7 post-transplant was equivalent to WT T cells (Figure 3A, left panel). In contrast, PPAR-δ KO CD4 T cell numbers decreased by 30% on day 7, despite equivalent levels of CD36 and CPT1a (Figure 3A, right panel). Finally, we addressed whether pharmacologic inhibition of PPAR-δ might also effectively mitigate GVHD. Administration of the PPAR-δ inhibitor GSK3787 on days 3-6 post-transplant substantially decreased the number of donor T cell recovered on day 7 (Figure 3B), with PPAR-δ impairment corroborated by a decrease in CPT1a gene transcription. However, instead of improving recipient health, GSK3787 treatment instead worsened weight loss and increased rates of post-transplant morbidity and mortality. From these data, we conclude that PPAR-δ is necessary in alloreactive T cells to cause maximally severe GVHD and that mechanistically, an absence of PPAR-δ impairs fat oxidation in CD8 T cells without impacting CD8 T cell numbers. In contrast, PPAR-δ deficiency decreases the number of CD4 T cells post-transplant, but does so without impacting CPT1a or CD36 levels, highlighting clear differences in metabolic reprogramming between CD4 and CD8 alloreactive cells. Finally, our data suggest that systemic inhibition of PPAR-δ post-transplant is not feasible given a sharp increase in toxicity. Future work will elucidate the mechanism of PPAR-δ in CD4 T cells, define the additional metabolic adaptations of CD8 cells which lack PPAR-δ, and determine if similar changes occur in human T cells. Together, these studies will test whether cellular inhibition of PPAR-δ represents a clinically-relevant, future therapy for GVHD. Disclosures No relevant conflicts of interest to declare.
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