CD4+ T cells in antitumor immunity.

Advances in cancer immunotherapy have transformed cancer care and realized unprecedented responses in many patients. The growing arsenal of novel therapeutics - including immune checkpoint inhibition (ICI), adoptive T cell therapies (ACTs), and cancer vaccines - reflects the success of cancer immunotherapy. The therapeutic benefits of these treatment modalities are generally attributed to the enhanced quantity and quality of antitumor CD8+ T cell responses. Nevertheless, CD4+ T cells are now recognized to play key roles in both the priming and effector phases of the antitumor immune response. In addition to providing T cell help through co-stimulation and cytokine production, CD4+ T cells can also possess cytotoxicity either directly on MHC class II-expressing tumor cells or to other cells within the tumor microenvironment (TME). The presence of specific populations of CD4+ T cells, and their intrinsic plasticity, within the TME can represent an important determinant of clinical response to immune checkpoint inhibitors, vaccines, and chimeric antigen receptor (CAR) T cell therapies. Understanding how the antitumor functions of specific CD4+ T cell types are induced while limiting their protumorigenic attributes will enable more successful immunotherapies.

Inhibitory co-receptor Lag3 supports Foxp3+ regulatory T cell function by restraining Myc-dependent metabolic programming.

Lymphocyte activation gene 3 (Lag3) is an inhibitory co-receptor expressed on activated T cells and has been proposed to regulate regulatory T (Treg) cell function. However, its precise modality and mechanisms remain elusive. We generated Treg cell-specific Lag3-mutant mouse models and found that Lag3 was essential for Treg cell control of autoimmunity. RNA sequencing analysis revealed that Lag3 mutation altered genes associated with metabolic processes, especially Myc target genes. Myc expression in Lag3-mutant Treg cells was increased to the level seen in conventional T helper (Th)1-type effector cells and directly correlated with their metabolic profiles and in vivo suppressive functions. The phosphatidylinositol 3-kinase (PI3K)-Akt-Rictor pathway was activated in Lag3-mutant Treg cells, and inhibiting PI3K, Rictor, or lactate dehydrogenase A (Ldha), a key Myc target enzyme converting pyruvate to lactate, was sufficient to restore normal metabolism and suppressive function in Lag3-mutant Treg cells. These findings indicate that Lag3 supports Treg cell suppression partly by tuning Myc-dependent metabolic programming.

Acidity induces durable enhancement of Treg cell suppressive functions for tumor immune evasion.

The microenvironment within solid tumors often becomes acidic due to various factors associated with abnormal metabolism and cellular activities, including increased lactate production as a result of dysregulated tumor glycolysis. Recently, we have identified multiple tumor microenvironment (TME) factors that potentiate regulatory T (Treg) cell function in evading anti-tumor immunosurveillance. Despite the strong correlation between lactate and acidity, the potential roles of acidity in intratumoral Treg cell adaptation and underlying molecular mechanisms have gone largely unstudied. In this study, we demonstrate that acidity significantly enhances immunosuppressive functions of nTreg cells, but not iTreg cells, without altering the expression of either FoxP3 or the cell surface receptors CD25, CTLA4, or GITR in these cells. Surprisingly, the addition of lactate, often considered a major contributor to increased acidity of the TME, completely abolished the acidity-induced enhancement of nTreg suppressive functions. Consistently, metabolic flux analyses showed elevated basal mitochondrial respiratory capacity and ATP-coupled respiration in acidity-treated nTreg cells without altering glycolytic capacity. Genome-wide transcriptome and metabolomics analyses revealed alterations in multiple metabolic pathways, particularly the one-carbon folate metabolism pathway, with reduced SAM, folate, and glutathione, in nTreg cells exposed to low pH conditions. Addition of a one-carbon metabolic contributor, formate, diminished the acidity-induced enhancement in nTreg cell suppressive functions, but neither SAM nor glutathione could reverse the phenotype. Remarkably, in vitro transient treatment of nTreg cells resulted in sustained enhancement of their functions, as evidenced by more vigorous tumor growth observed in mice adoptively receiving acidity-treated nTreg cells. Further analysis of intratumoral infiltrated T cells confirmed a significant reduction in CD8+ T cell frequency and their granzyme B production. In summary, our study elucidates how acidity-mediated metabolic reprogramming leads to sustained Treg-mediated tumor immune evasion.

Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation.

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

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.

A deubiquitination module essential for Treg fitness in the tumor microenvironment.

The tumor microenvironment (TME) enhances regulatory T (Treg) cell stability and immunosuppressive functions through up-regulation of lineage transcription factor Foxp3, a phenomenon known as Treg fitness or adaptation. Here, we characterize previously unknown TME-specific cellular and molecular mechanisms underlying Treg fitness. We demonstrate that TME-specific stressors including transforming growth factor-ß (TGF-ß), hypoxia, and nutrient deprivation selectively induce two Foxp3-specific deubiquitinases, ubiquitin-specific peptidase 22 (Usp22) and Usp21, by regulating TGF-ß, HIF, and mTOR signaling, respectively, to maintain Treg fitness. Simultaneous deletion of both USPs in Treg cells largely diminishes TME-induced Foxp3 up-regulation, alters Treg metabolic signatures, impairs Treg-suppressive function, and alleviates Treg suppression on cytotoxic CD8+ T cells. Furthermore, we developed the first Usp22-specific small-molecule inhibitor, which dramatically reduced intratumoral Treg Foxp3 expression and consequently enhanced antitumor immunity. Our findings unveil previously unappreciated mechanisms underlying Treg fitness and identify Usp22 as an antitumor therapeutic target that inhibits Treg adaptability in the TME.

HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N6-methyladenosine (m6A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with liver-specific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m6A modification pathways, termed the ERm6A pathway, for ER stress adaptation to proteotoxicity.

Regulation of Treg Functions by the Ubiquitin Pathway.

Regulatory T (Tregs) cells, required to maintain immune homeostasis, have significant power in disease outcomes. Treg dysfunction, predominantly characterized by the loss of the master transcription factor FoxP3 and the acquisition of Teff-like phenotypes, can promote autoimmunity as well as enhance anti-tumor immunity. As FoxP3 expression and stability are pinnacle for Treg suppressive functions, understanding the pathways that regulate FoxP3 is crucial to ascertain Treg-mediated therapies for autoimmune diseases and cancer. Mechanisms controlling FoxP3 expression and stability range from transcriptional to posttranslational, revealing multiple therapeutic opportunities. While many of the transcriptional pathways have been explored in detail, a recent surge in interest on the posttranslational mechanisms regulating FoxP3 has arisen. Particularly, the role of ubiquitination on Tregs both directly and indirectly involving FoxP3 has gained interest. Here, we summarize the current knowledge on ubiquitin-dependent, FoxP3-mediated control of Treg function as it pertains to human diseases.

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown 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.

The E3 ligase Hrd1 stabilizes Tregs by antagonizing inflammatory cytokine-induced ER stress response.

Treg differentiation, maintenance, and function are controlled by the transcription factor FoxP3, which can be destabilized under inflammatory or other pathological conditions. Tregs can be destabilized under inflammatory or other pathological conditions, but the underlying mechanisms are not fully defined. Herein, we show that inflammatory cytokines induce ER stress response, which destabilizes Tregs by suppressing FoxP3 expression, suggesting a critical role of the ER stress response in maintaining Treg stability. Indeed, genetic deletion of Hrd1, an E3 ligase critical in suppressing the ER stress response, leads to elevated expression of ER stress-responsive genes in Treg and largely diminishes Treg suppressive functions under inflammatory condition. Mice with Treg-specific ablation of Hrd1 displayed massive multiorgan lymphocyte infiltration, body weight loss, and the development of severe small intestine inflammation with aging. At the molecular level, the deletion of Hrd1 led to the activation of both the ER stress sensor IRE1α and its downstream MAPK p38. Pharmacological suppression of IRE1α kinase, but not its endoribonuclease activity, diminished the elevated p38 activation and fully rescued the stability of Hrd1-null Tregs. Taken together, our studies reveal ER stress response as a previously unappreciated mechanism underlying Treg instability and that Hrd1 is crucial for maintaining Treg stability and functions through suppressing the IRE1α-mediated ER stress response.

USP22 deficiency leads to myeloid leukemia upon oncogenic Kras activation through a PU.1-dependent mechanism.

Ras mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic KrasG12D/+ USP22 deficiency in KrasG12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient KrasG12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient KrasG12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient KrasG12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis.

Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa.

The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process.