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
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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

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Figure 4

6-Aminonicotinamide (6AN), a small-molecule inhibitor of 6PGD, reduces GvHD severity in a fully allogeneic murine model and a xenogeneic GvHD model.

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6-Aminonicotinamide (6AN), a small-molecule inhibitor of 6PGD, reduces G...
(A–C) BALB/c (H-2d) mice were lethally irradiated (6 Gy) on day –1 and transplanted i.v. with 1 × 107 BM cells with or without 1 × 106 splenic T cells from WT C57BL/6 (H-2b) mice on day 0. Mice were injected i.p. with 0.5 mg/kg 6AN or vehicle (1% DMSO) daily. Mouse survival (A), GvHD clinical score (B), and body weight loss (C) are reported. n = 18–20 mice per group combined from 3 independent repeats (2-way ANOVA). (D) Splenic and lymph node T cells from WT C57BL/6 mice were activated in vitro with plate-bound anti-CD3 and anti-CD28 (10 μg/mL each) plus rmIL-2 (100 ng/mL) for 72 hours and examined by Vybrant DyeCycle (catalog V35003, Thermo Fisher Scientific) for cell cycle determination. (E and F) T cells were treated as in D and examined for Ki67 proliferation marker (E) and apoptosis rate (F). n = 3–6 data points per group. Data representative of 3 independent repeats (Student’s t test). (G) T cells were treated as in D after CFSE labeling and proliferation of CD4+ (top) and CD8+ (bottom) T cells was examined after 72 hours. n = 3 data points per group. Data representative of 3 independent repeats (Student’s t test). (H and I) NSG mice were sublethally irradiated (2.5 Gy) on day –1 and transplanted with 2 × 106 human PBMCs on day 0. Mice were injected i.p. with 1 mg/kg 6AN or vehicle every 2 days. Mouse survival (H) and GvHD clinical scores (I) are reported. n = 12 mice per group from 2 independent repeats (2-way ANOVA). (J and K) Mice were transplanted as in H and I and numbers of splenic CD4+ (J) and CD8+ (K) T cells were examined on day +14 after allo-HCT (Student’s t test). (L and M) Human T cells from healthy donors were activated with anti-CD3 and anti-CD28 (10 μg/mL each) plus rhIL-2 (100 ng/mL) and treated with 6AN or vehicle for 72 hours. Cell cycle status was determined by Vybrant DyeCycle (L) and apoptosis rate by Annexin V staining (M). n = 3 data points per group. Data representative of 2 independent repeats (Student’s t test). (N) Human T cells were isolated and treated as in L after CFSE labeling. The proliferation of CD4+ (top) and CD8+ (bottom) T cells was examined after 72 hours. n = 3 data points per group. Data representative of 2 independent repeats (Student’s t test). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.