Control of regulatory T cell fitness and function by heme synthesis

/Abstract: Despite advances in targeted immunotherapies, many treatments for autoimmune diseases remain ineffective and can cause severe side effects, such as increased susceptibility to infections and cancer. Foxp3+ regulatory T (Treg) cells play a critical role in preventing harmful immune responses, but their dysfunction can lead to autoimmune diseases. The mechanisms behind Treg dysfunction are poorly understood, hindering the development of effective Treg-based therapies. This proposal aims to investigate the novel role of heme synthesis, identified through a CRISPR screen, in controlling Treg fitness and function. Preliminary data from our lab show that Effector T cell Reprogrammed Tregs (ER-Tregs), which exhibit superior fitness and function compared to naturally occurring Tregs (nTregs), are highly effective in ameliorating experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. ER-Tregs maintain lineage stability, possess enhanced suppressive function, and display a distinct gene expression profile, including the upregulation of Cpox, an enzyme critical for heme biosynthesis. Cpox was identified as a key regulator of ER-Treg fitness, supporting their proliferation, survival, and suppressive capacity. We hypothesize that Cpox promotes Treg expansion by supporting mitochondrial oxidative phosphorylation (OXPHOS), sequesters labile iron to prevent ferroptosis, and exports heme to enhance suppressive function. Our findings suggest that inadequate Cpox expression, due to insufficient Foxp3 and tolerogenic signaling, contributes to Treg dysfunction in autoimmunity. Enhancing Cpox- dependent heme synthesis may restore Treg function and alleviate autoimmune disease symptoms. The long- term objective is to understand the role of Cpox and heme synthesis in Treg biology and identify potential therapeutic strategies for rejuvenating Tregs in autoimmune diseases. To achieve this, we propose two specific aims: Determine how Cpox promotes the fitness and suppressive function of ER-Tregs (Aim 1). We will examine whether Cpox deficiency impairs Treg OXPHOS and causes ferroptosis, and whether the suppressive defects in Cpox-deficient Tregs are related to their inability to export heme. These studies will use CRISPR-mediated knockout and overexpression approaches. We will also investigate the regulation of Cpox expression and its role in Treg dysfunction (Aim 2). We will map the regulatory pathways controlling Cpox expression using CRISPR- based genetic and epigenetic tools and assess how Cpox impacts Treg homeostasis and function under steady- state conditions and in EAE. We will also explore whether enhancing Cpox expression via epigenetic reprogramming or overexpression can restore Treg function in EAE. These studies will provide crucial insights into the role of heme biosynthesis in regulating Treg fitness and function, potentially leading to new therapies for autoimmune diseases by restoring Treg function. Project Number: 1R01AI192616-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Xudong Li | Institution: TUFTS UNIVERSITY BOSTON, BOSTON, MA | Award Amount: $647,187 | Activity Code: R01 | Study Section: Adaptive Immunity Study Section[AI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19261601