Targeting the pentose phosphate pathway mitigates graft-versus-host disease by rewiring alloreactive T cell metabolism
Saeed Daneshmandi, Eun Ko, Qi Yan, Jee Eun Choi, Prashant K. Singh, Richard M. Higashi, Andrew N. Lane, Teresa W.M. Fan, Jingxin Qiu, Sophia Hani, Keli L. Hippen, Jianmin Wang, Philip L. McCarthy, Bruce R. Blazar, Hemn Mohammadpour
Saeed Daneshmandi, Eun Ko, Qi Yan, Jee Eun Choi, Prashant K. Singh, Richard M. Higashi, Andrew N. Lane, Teresa W.M. Fan, Jingxin Qiu, Sophia Hani, Keli L. Hippen, Jianmin Wang, Philip L. McCarthy, Bruce R. Blazar, Hemn Mohammadpour
View: Text | PDF
Research Article Immunology Oncology

Targeting the pentose phosphate pathway mitigates graft-versus-host disease by rewiring alloreactive T cell metabolism

Abstract

Glycolysis fuels cytotoxic allogeneic T cells in acute graft-versus-host disease (aGvHD), but the downstream role of glucose metabolism in modulating aGvHD remains unclear. Targeting glycolysis or glucose receptors is toxic. Therefore, we explored alternative glucose-dependent pathways, focusing on the pentose phosphate pathway (PPP). Single-cell RNA sequencing revealed PPP upregulation in allogeneic T cells during allogeneic hematopoietic cell transplantation (allo-HCT). We showed that donor T cell deficiency in 6-phosphogluconate dehydrogenase (6PGD), the second rate-limiting enzyme in the PPP, significantly reduced aGvHD severity and mortality in murine models. Functional assays demonstrated that PPP blockade led to proliferation arrest without inducing apoptosis. PPP blockade shifted T cell metabolism away from T cell dependency on glycolysis for rapid T cell proliferation. Pharmacological inhibition of the PPP through 6PGD blockade with 6-aminonicotinamide (6AN) effectively reduced aGvHD severity, like donor 6PGD-deficient T cells in an allogeneic aGvHD model. Similarly, 6AN reduced xenogeneic GvHD lethality. 6PGD inhibition preserved the graft-versus-tumor (GvT) effect, with the generation of a small subset of granzyme Bhi effector T cells with potent antitumor activity. These findings highlight the PPP as a key regulator of allogeneic T cell proliferation and differentiation and identify 6PGD as a promising therapeutic target to mitigate aGvHD severity while preserving beneficial GvT effects.

Authors

Saeed Daneshmandi, Eun Ko, Qi Yan, Jee Eun Choi, Prashant K. Singh, Richard M. Higashi, Andrew N. Lane, Teresa W.M. Fan, Jingxin Qiu, Sophia Hani, Keli L. Hippen, Jianmin Wang, Philip L. McCarthy, Bruce R. Blazar, Hemn Mohammadpour

×

Figure 5

6PGD blockade in T cells induces a metabolic reprogramming evident by reduced glycolysis and higher mitochondrial respiration.

Options: View larger image (or click on image) Download as PowerPoint
6PGD blockade in T cells induces a metabolic reprogramming evident by re...
(A) Splenic naive T cells from WT C57BL/6 mice were activated in vitro with plate-bound anti-CD3 and anti-CD28 mabs (10 μg/mL each) plus rmIL-2 (100 ng/mL) for 4 days with 6AN (10 μM) or vehicle (DMSO) control, and then exposed to 10 mM D7-D-glucose. The 6-phosphogluconate (6PG) and ribose-5-phosphate (R5P) quantities generated from glucose were determined at 8 hours. n = 3 data points per group (Student’s t test). (B) Levels of NADPH and NADP+ in in vitro–activated T cells after 6AN or vehicle treatment for 72 hours are shown. n = 3 data points per group (Student’s t test). (C and D) The metabolic profiles of 6AN- and vehicle-treated T cells were analyzed by SCENITH single-cell translation profiling (C). 6AN-treated T cells showed lower glycolysis and higher mitochondrial function when compared with vehicle (D). n = 4–5 data points per group. Data representative of 2 independent repeats (2-way ANOVA). (E) In vitro–activated T cells in presence of 6AN or vehicle were examined for expression of the plasma membrane glucose transporter Glut1. n = 4–5 data points per group. Data representative of 2 independent repeats (Student’s t test). (F and G) Splenic naive T cells from Pgdfl/fl or Pgdfl/flCd4Cre mice were activated with plate-bound anti-CD3 and anti-CD28 (10 μg/mL each) plus rmIL-2 (100 ng/mL) for 4 days. For the pharmacologic experiment, naive T cells were isolated from WT mice and stimulated as above in the presence of 6AN (10 μM) or vehicle control. The harvested cells were examined for extracellular acidification rate (ECAR) (F) or oxygen consumption rate (OCR) (G) using the Seahorse assay. n = 4 data points per group. Data representative of 2 independent repeats. (H) T cells were prepared and activated as in F and G. The mitochondrial mass was determined by MitoTracker Deep Red staining. n = 4 data points per group. Data representative of 3 independent repeats (Student’s t test). (I and J) T cells were prepared as in F and G and examined by transmission electron microscopy. The mitochondria in the cells are highlighted by black arrows and mitochondrial number was counted in 20 random fields for each group (I). Scale bar: 1.5 μm. Mitochondrial activity was examined by evaluation of mitochondrial potential (ΔΨm) by tetramethylrhodamine ester (TMRE) (J). n = 4 data points per group. Data representative of 3 independent repeats (Student’s t test) and are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.