Montauti, E. | bioRxiv Science

bioRxiv2023

Lymphocyte activation gene 3 (Lag3) supports Foxp3+ Treg cell function by restraining c-Myc-dependent aerobic glycolysis

Lymphocyte activation gene 3 (Lag3) has emerged as the next-generation immune checkpoint molecule due to its ability to inhibit effector T cell activity. Foxp3+ regulatory T (Treg) cells, a master regulator of immunity and tolerance, also highly express Lag3. While Lag3 is thought to be necessary for Treg cell-mediated regulation of immunity, the precise roles and underlying mechanisms remain largely elusive. In this study, we report that Lag3 is indispensable for Treg cells to control autoimmune inflammation. Utilizing a newly generated Treg cell specific Lag3 mutant mouse model, we found that these animals are highly susceptible to autoimmune diseases, suggesting defective Treg cell function. Genome wide transcriptome analysis further uncovered that Lag3 mutant Treg cells upregulated genes involved in metabolic processes. Mechanistically, we found that Lag3 limits Treg cell expression of Myc, a key regulator of aerobic glycolysis. We further found that Lag3-dependent Myc expression determines Treg cells metabolic programming as well as the in vivo function to suppress autoimmune inflammation. Taken together, our results uncovered a novel function of Lag3 in supporting Treg cell suppressive function by regulating Myc-dependent metabolic programming.

immunology↗

bioRxiv2020

CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

Regulatory T cells (Tregs) are required to control immune responses and maintain homeostasis but are a significant barrier to anti-tumor immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of pro-inflammatory properties2, can promote autoimmunity and/or facilitate more effective tumor immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. Despite improved functional genetic tools that now allow for systematic interrogation, dissection of the gene regulatory programs that modulate Foxp3 expression has not yet been reported. In this study, we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregs and applied this technology to perform a targeted loss-of-function screen of [~]490 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We discovered several novel modulators including ubiquitin-specific peptidase 22 (Usp22), Ataxin 7 like 3 (Atxn7l3) and ring finger protein 20 (Rnf20). Members of the deubiquitination module of the SAGA chromatin modifying complex, Usp22 and Atxn7l3, were discovered to be positive regulators that stabilized Foxp3 expression; whereas the screen suggested Rnf20, an E3 ubiquitin ligase, is a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein and created defects in their suppressive function that led to spontaneous autoimmunity but protected against tumor growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregs could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregs. These results reveal novel modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

immunology↗