Cell intrinsic heme synthesis supports glycolysis for activation of naive CD4+ T cell

Abstract Revealing the mechanism by which naive CD4+ (nCD4+) T cells are activated is vital to understanding how acquired immunity is orchestrated. Upon the activation of nCD4+ T cells, their energy metabolism undergoes a major switch from OXPHOS (oxidative phosphorylation) to glycolysis. However, little is known about whether nutrients actively support this metabolic switch. Interestingly, transferrin receptor 1, a major iron transporter, is significantly induced by the activation, suggesting that iron may play a significant role in this process. However, how iron is utilized intracellularly during their activation, and whether iron metabolism contributes to this metabolic switch, remains largely unknown. Therefore, we hypothesized that heme, one of the major iron metabolites, might play an indispensable role in the activation of nCD4+. To reveal the importance of heme synthesis during the nCD4+ T cell activation, we developed a T cell-specific heme synthase-deleted mouse (Coproporphyrinogen oxidase (Cpox) f/f: CD4-Cre, hereafter Cpox-KO). Numbers of the splenic nCD4+ T cells and effector memory CD4+T cells were decreased in Cpox-KO mice compared to control (Cpoxf/f) mice. Since the reduction of effector memory cells was more severe, we hypothesized that the heme synthesis is especially required during the activation of nCD4+ cells. In line with this notion, Cpox-KO nCD4+ T cells showed poorer proliferation during their activation (CD3/CD28 and IL-2) in vitro compared to control cells. Hemin addition in the culture medium rescued this activation disorder. In addition, this impairment was also validated by in vivo homeostatic lymphoproliferation models in which nCD4+ T cells were transferred to sublethally irradiated mice (CD45.1+) or Rag2-deficient mice. Therefore, these results indicate that the cell-intrinsic heme synthesis is indispensable for nCD4+ T cell activation. To elucidate the mechanism by which the heme synthesis supports the activation of nCD4+ T cells, we performed comprehensive transcriptomic analysis (RNA-seq) of control and Cpox-KO nCD4+ T cells with or without activation (day 0, day 2, and day 4). Interestingly, glycolysis-related gene expressions were enhanced through their activation in control cells, while their expressions were not adequately enhanced in Cpox-KO cells. Our extracellular flux analysis revealed that actual glycolysis, which is enhanced during the activation in control cells, was not adequately enhanced in Cpox-KO cells. Strikingly, hemin addition rescued not only key glycolysis-related gene expressions (Slc2a1 and Hk2), but also the actual glycolysis. Therefore, these results indicate that heme synthesis is essential to the proper induction of glycolysis during the nCD4+ T cell activation. To clarify how heme supports the expression of glycolysis-related genes, we examined the upstream factors of glycolysis: mTORC1, STAT5, and HIF1. Among them, only the STAT5 activation was significantly impaired in Cpox-KO cells and rescued by hemin, suggesting that heme might support the STAT5 signaling. Since, heme could regulate gene expressions by destabilizing the repressive transcription factor BACH2 in T cells, we merged our RNA-seq data with previous BACH2 or STAT5 ChIP-seq data, which revealed that Slc2a1 and Hk2 possess their own genomic loci on which BACH2 and STAT5 were commonly enriched. In addition, we identified several T cell activation-related genes, such as Tnfrsf8 and Il2rb, whose expression could be controlled by BACH2 and STAT5 at the same genomic loci and was repressed in Cpox-KO cells. Therefore, STAT5 and BACH2 may work competitively on their common target genes, and heme supports STAT5 signaling by inhibiting BACH2. Finally, to determine whether heme synthesis inhibition has a potency of clinical application, we took advantage of the CD4+T cell-derived graft versus host disease (GVHD) model. Cpox-KO nCD4+ T cell transplanted mice showed better GVHD score and overall survival compared to control cell transplanted mice. Pathological assessment revealed less infiltration of inflammatory cells in the colon and liver. Collectively, we revealed that the cell-intrinsic heme synthesis supports glycolysis for the activation of nCD4+ T cells. This finding will pave the way for a deeper understanding of T cell biology and the development of novel therapies for T cell-related disorders.