Insulin signaling establishes a developmental trajectory of adipose regulatory T cells.

Systematic characterizations of adipose regulatory T (Treg) cell subsets and their phenotypes remain uncommon. Using single-cell ATAC-sequencing and paired single-cell RNA and T cell receptor (TCR) sequencing to map mouse adipose Treg cells, we identified CD73hiST2lo and CD73loST2hi subsets with distinct clonal expansion patterns. Analysis of TCR-sharing data implied a state transition between CD73hiST2lo and CD73loST2hi subsets. Mechanistically, we revealed that insulin signaling occurs through a HIF-1α-Med23-PPAR-γ axis to drive the transition of CD73hiST2lo into a CD73loST2hi adipose Treg cell subset. Treg cells deficient in insulin receptor, HIF-1α or Med23 have decreased PPAR-γ expression that in turn promotes accumulation of CD73hiST2lo adipose Treg cells and physiological adenosine production to activate beige fat biogenesis. We therefore unveiled a developmental trajectory of adipose Treg cells and its dependence on insulin signaling. Our findings have implications for understanding the dynamics of adipose Treg cell subsets in aged and obese contexts.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

The ubiquitin ligase Stub1 negatively modulates regulatory T cell suppressive activity by promoting degradation of the transcription factor Foxp3.

Regulatory T (Treg) cells suppress inflammatory immune responses and autoimmunity caused by self-reactive T cells. The key Treg cell transcription factor Foxp3 is downregulated during inflammation to allow for the acquisition of effector T cell-like functions. Here, we demonstrate that stress signals elicited by proinflammatory cytokines and lipopolysaccharides lead to the degradation of Foxp3 through the action of the E3 ubiquitin ligase Stub1. Stub1 interacted with Foxp3 to promote its K48-linked polyubiquitination in an Hsp70-dependent manner. Knockdown of endogenous Stub1 or Hsp70 prevented Foxp3 degradation. Furthermore, the overexpression of Stub1 in Treg cells abrogated their ability to suppress inflammatory immune responses in vitro and in vivo and conferred a T-helper-1-cell-like phenotype. Our results demonstrate the critical role of the stress-activated Stub1-Hsp70 complex in promoting Treg cell inactivation, thus providing a potential therapeutic target for the intervention against autoimmune disease, infection, and cancer.

The deubiquitinase USP44 promotes Treg function during inflammation by preventing FOXP3 degradation.

The transcription factor forkhead box P3 (FOXP3) is essential for the development of regulatory T cells (Tregs) and their function in immune homeostasis. Previous studies have shown that in natural Tregs (nTregs), FOXP3 can be regulated by polyubiquitination and deubiquitination. However, the molecular players active in this pathway, especially those modulating FOXP3 by deubiquitination in the distinct induced Treg (iTreg) lineage, remain unclear. Here, we identify the ubiquitin-specific peptidase 44 (USP44) as a novel deubiquitinase for FOXP3. USP44 interacts with and stabilizes FOXP3 by removing K48-linked ubiquitin modifications. Notably, TGF-ß induces USP44 expression during iTreg differentiation. USP44 co-operates with USP7 to stabilize and deubiquitinate FOXP3. Tregs genetically lacking USP44 are less effective than their wild-type counterparts, both in vitro and in multiple in vivo models of inflammatory disease and cancer. These findings suggest that USP44 plays an important role in the post-translational regulation of Treg function and is thus a potential therapeutic target for tolerance-breaking anti-cancer immunotherapy.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

The histone demethylase LSD1 has been known as a key transcriptional coactivator for DNA viruses such as herpes virus. Inhibition of LSD1 was found to block viral genome transcription and lytic replication of DNA viruses. However, RNA virus genomes do not rely on chromatin structure and histone association, and the role of demethylase activity of LSD1 in RNA virus infections is not anticipated. Here, we identify that, contrary to its role in enhancing DNA virus replication, LSD1 limits RNA virus replication by demethylating and activating IFITM3 which is a host restriction factor for many RNA viruses. We have found that LSD1 is recruited to demethylate IFITM3 at position K88 under IFNα treatment. However, infection by either Vesicular Stomatitis Virus (VSV) or Influenza A Virus (IAV) triggers methylation of IFITM3 by promoting its disassociation from LSD1. Accordingly, inhibition of the enzymatic activity of LSD1 by Trans-2-phenylcyclopropylamine hydrochloride (TCP) increases IFITM3 monomethylation which leads to more severe disease outcomes in IAV-infected mice. In summary, our findings highlight the opposite role of LSD1 in fighting RNA viruses comparing to DNA viruses infection. Our data suggest that the demethylation of IFITM3 by LSD1 is beneficial for the host to fight against RNA virus infection.

The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.

Killing by cytotoxic T lymphocytes (CTLs) is mediated by the secretion of lytic granules. The centrosome plays a key role in granule delivery, polarizing to the central supramolecular activation complex (cSMAC) within the immunological synapse upon T cell receptor (TCR) activation. Although stronger TCR signals lead to increased target cell death than do weaker signals, it is not known how the strength of TCR signal controls polarization of the centrosome and lytic granules. By using TCR transgenic OT-I CTLs, we showed that both high- and low-avidity interactions led to centrosome polarization to the cSMAC. However, only high-avidity interactions, which induced a higher threshold of intracellular signaling, gave rise to granule recruitment to the polarized centrosome at the synapse. By controlling centrosome and granule polarization independently, the centrosome is able to respond rapidly to weak signals so that CTLs are poised and ready for the trigger for granule delivery.

Inflammation negatively regulates FOXP3 and regulatory T-cell function via DBC1.

Forkhead box P3 (FOXP3)-positive Treg cells are crucial for maintaining immune homeostasis. FOXP3 cooperates with its binding partners to elicit Treg cells' signature and function, but the molecular mechanisms underlying the modulation of the FOXP3 complex remain unclear. Here we report that Deleted in breast cancer 1 (DBC1) is a key subunit of the FOXP3 complex. We found that DBC1 interacts physically with FOXP3, and depletion of DBC1 attenuates FOXP3 degradation in inflammatory conditions. Treg cells from Dbc1-deficient mice were more resistant to inflammation-mediated abrogation of Foxp3 expression and function and delayed the onset and severity of experimental autoimmune encephalomyelitis and colitis in mice. These findings establish a previously unidentified mechanism regulating FOXP3 stability during inflammation and reveal a pathway for potential therapeutic modulation and intervention in inflammatory diseases.

Cutting edge: Ubiquitin-specific protease 4 promotes Th17 cell function under inflammation by deubiquitinating and stabilizing RORγt.

RORγt is a key transcription factor that controls the development and function of inflammatory Th17. The mechanisms that regulate RORγt stability remain unclear. We report that Th17 cells highly express the deubiquitinase ubiquitin-specific protease (USP)4, which is essential for maintaining RORγt and Th17 cell function. Inhibition of the catalytic activity of USP4 with vialinin A, a compound derived from Chinese traditional medicine, dampened Th17 differentiation. USP4 interacted and deubiquitinated K48-linked polyubiquitination of RORγt, thereby promoting RORγt function and IL-17A transcription. Interestingly, TGF-ß plus IL-6 enhanced USP4-mediated deubiquitination of RORγt. Moreover, USP4 and IL-17 mRNA, but not RORγt mRNA, were significantly elevated in CD4(+) T cells from patients with rheumatic heart disease. Thus, USP4 could be a novel therapeutic target for the treatment of Th17-modulated autoimmune diseases.

PIM1 kinase phosphorylates the human transcription factor FOXP3 at serine 422 to negatively regulate its activity under inflammation.

Previous reports have suggested that human CD4(+) CD25(hi)FOXP3(+) T regulatory cells (Tregs) have functional plasticity and may differentiate into effector T cells under inflammation. The molecular mechanisms underlying these findings remain unclear. Here we identified the residue serine 422 of human FOXP3 as a phosphorylation site that regulates its function, which is not present in murine Foxp3. PIM1 kinase, which is highly expressed in human Tregs, was found to be able to interact with and to phosphorylate human FOXP3 at serine 422. T cell receptor (TCR) signaling inhibits PIM1 induction, whereas IL-6 promotes PIM1 expression in in vitro expanded human Tregs. PIM1 negatively regulates FOXP3 chromatin binding activity by specifically phosphorylating FOXP3 at Ser(422). Our data also suggest that phosphorylation of FOXP3 at the Ser(418) site could prevent FOXP3 phosphorylation at Ser(422) mediated by PIM1. Knockdown of PIM1 in in vitro expanded human Tregs promoted FOXP3-induced target gene expression, including CD25, CTLA4, and glucocorticoid-induced tumor necrosis factor receptor (GITR), or weakened FOXP3-suppressed IL-2 gene expression and enhanced the immunosuppressive activity of Tregs. Furthermore, PIM1-specific inhibitor boosted FOXP3 DNA binding activity in in vitro expanded primary Tregs and also enhanced their suppressive activity toward the proliferation of T effector cells. Taken together, our findings suggest that PIM1 could be a new potential therapeutic target in the prevention and treatment of human-specific autoimmune diseases because of its ability to modulate the immunosuppressive activity of human Tregs.

The E3 deubiquitinase USP17 is a positive regulator of retinoic acid-related orphan nuclear receptor γt (RORγt) in Th17 cells.

Stable retinoic acid-related orphan nuclear receptor γt (RORγt) expression is pivotal for the development and function of Th17 cells. Here we demonstrate that expression of the transcription factor RORγt can be regulated through deubiquitination, which prevents proteasome-mediated degradation. We establish that USP17 stabilizes RORγt protein expression by reducing RORγt polyubiquitination at its Lys-360 residue. In contrast, knockdown of endogenous USP17 in Th17 cells resulted in decreased RORγt protein levels and down-regulation of Th17-related genes. Furthermore, USP17 expression was up-regulated in CD4(+) T cells from systemic lupus erythematosus patients. Our data reveal a molecular mechanism in which RORγt expression in Th17 cells can be positively regulated by USP17, thereby modulating Th17 cell functions.

60-kDa Tat-interactive protein (TIP60) positively regulates Th-inducing POK (ThPOK)-mediated repression of eomesodermin in human CD4+ T cells.

The abundant expression of IFNγ in Th-inducing POK (ThPOK)-deficient CD4(+) T cells requires the activation of Eomesodermin (Eomes); however, the underlying mechanism of this phenomenon remains unclear. Here we report that ThPOK binds directly to the promoter region of the Eomes gene to repress its expression in CD4(+) T cells. We identified the histone acetyltransferase TIP60 as a co-repressor of ThPOK-target genes, where ectopically expressed TIP60 increased ThPOK protein stability by promoting its acetylation at its Lys(360) residue to then augment the transcriptional repression of Eomes. Moreover, knockdown of endogenous TIP60 abolished the stabilization of ThPOK in CD4(+) T cells, which led to the transcriptional activation of Eomes and increased production of IFNγ. Our results reveal a novel pathway by which TIP60 and ThPOK synergistically suppresses Eomes function and IFNγ production, which could contribute to the regulation of inflammation.

Negative regulation of interferon-induced transmembrane protein 3 by SET7-mediated lysine monomethylation.

Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases.

Identification of the E3 deubiquitinase ubiquitin-specific peptidase 21 (USP21) as a positive regulator of the transcription factor GATA3.

The expression of the transcription factor GATA3 in FOXP3(+) regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

Regulatory T-cell differentiation and their function in immune regulation.

Regulatory T-cells (Treg) represent a subset of CD4+ T-cells characterized by high suppressive capacity, which can be generated in the thymus or induced in the periphery. The deleterious phenotype of the Scurfy mouse, which develops an X-linked lymphoproliferative disease resulting from defective T-cell tolerance, clearly demonstrates the importance of Treg cells for the maintenance of immune homeostasis. Although significant progress has been achieved, much information regarding the development, characteristics and function of Treg cells remain lacking. This chapter highlights the most recent discoveries in the field of Treg biology, focusing on the development and role of this cell subset in the maintenance of immune balance.

Preoperative risk factors influencing the incidence of postoperative sepsis in human immunodeficiency virus-infected patients: a retrospective cohort study.

BACKGROUND: Compared to noninfected patients, human immunodeficiency virus (HIV)-infected patients undergoing surgery have an increased postoperative risk of developing sepsis. We aimed to investigate the preoperative risk factors that affect the incidence of sepsis after surgery in HIV-infected patients. METHODS: Clinical parameters of 215 patients with HIV/acquired immunodeficiency syndrome (AIDS) who had undergone surgery between January 2011 and February 2012 were examined retrospectively for the effect of HIV/AIDS on the incidence of postoperative sepsis. RESULTS: Logistic regression analysis identified four independent risk factors of postoperative sepsis in HIV-infected patients: CD4 counts [B = -0.007, odds ratio (OR) 0.993]; blood albumin levels (B = -0.077, OR 0.926); surgical infection (B = 1.887, OR 6.598); major surgery (B = 1.013, OR 2.754). The incidence of postoperative sepsis was high with CD4 counts ≤ 100 cells/µl, albumin levels <35 g/L, the presence of surgical infection, the patient had undergone major surgery--81.25%, 39/48; 76.47%, 26/34; 70.73%, 29/41; and 54.76%, 46/84, respectively, compared to that of the total cohort (40.93%, 88/215). When CD4 counts were >350 cells/µl, the incidence of postoperative sepsis was significantly lower (16.36%, 9/55). CONCLUSIONS: Low CD4 cell counts, hypoalbuminemia, surgical infection, and major surgery are independent risk factors for the development of postoperative sepsis among HIV-infected patients. CD4 cell numbers and albumin levels negatively correlated with the incidence of postoperative sepsis, whereas surgical infections and major surgical procedures positively correlated with the incidence of postoperative sepsis.

Two distinct cytoplasmic regions of the beta2 integrin chain regulate RhoA function during phagocytosis.

AlphaMbeta2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. AlphaMbeta2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during alphaMbeta2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to alphaMbeta2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of beta2, but not of alphaM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the beta2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for beta2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.

Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer.

BACKGROUND: The discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%-30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003). METHODS: To determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further. RESULTS: PM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo. CONCLUSIONS: A unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

Single-cell RNA-seq unveils critical regulators of human FOXP3+ regulatory T cell stability.

The heterogeneity and plasticity of T lymphocytes is critical for determining immune response outcomes. Functional regulatory T (Treg) cells are commonly characterized by stable FOXP3 expression and have reported to exhibit heterogeneous phenotypes under inflammatory conditions. However, the interplay between inflammation and Treg cell suppressive activity still remains elusive. Here, we utilized single-cell RNA sequencing to investigate how human Treg cells respond to the pro-inflammatory cytokine interleukin-6 (IL-6). We observed that Treg cells divided into two subpopulations after IL-6 stimulation. TIGIT- unstable Treg cells lost FOXP3 expression and gained an effector-like T cell phenotype, whereas TIGIT+ Treg cells retained robust suppressive function. Single cell transcriptome analysis revealed a spectrum of cellular states of IL-6-stimulated Treg cells and how cytochrome P450 family 1 subfamily A member 1 (CYP1A1) is a crucial regulator of Treg cell suppressive capability and stability. CYP1A1-deficient human Treg cells developed a Th17-like phenotype after IL-6 stimulation. Our findings implicate CYP1A1 as a previously unidentified regulator of Treg cells that may have target potential for clinical application for biotherapies.

A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy.

In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provide a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain of the viral Spike (S) glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic viral infection in a standard 96-h co-incubation assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.