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.

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.

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.

Identification and functional analysis of heterogeneous FOXP3+ Treg cell subpopulations in human pancreatic ductal adenocarcinoma.

CD4+ CD25+ regulatory T (Treg) cells express the transcription factor FOXP3 and play an essential role in preventing autoimmunity. Abundant Treg cell accumulation in tumors and tumor draining lymph nodes (TDLNs) has been reported to correlate with both poor and favorable prognosis in various cancers, which suggests that Tregs may have multiple effects on antitumor immunity. However, the heterogeneity of tumor- and TDLN-infiltrating Treg cells remains unclear. Here we provide heterogeneity analysis of tumor infiltrating human CD4+ Treg cells and their matched adjacent tissues and TDLNs. We defined three different subpopulations of tumor- and TDLN-infiltrating Treg cells by Helios and CCR8 expression in pancreatic ductal adenocarcinoma (PDAC) and confirmed their functional heterogeneity. Helios+ CCR8+ Treg cells with potent suppressor function and limited IL-2 and IFN-γ secretion were identified in tumors and TDLNs. On the contrary, Helios- CCR8- Treg cells have impaired suppressive activity, and elevated expression of pro-inflammatory cytokines. More advanced grades of PDAC have predominantly Helios+ CCR8+ Treg cells and few Helios- CCR8- Treg cells both in tumors and TDLNs that suggests poor prognosis. These data could help further define the role of Treg cells and their functional role in tumors and TDLNs.

DNMT1 cooperates with MBD4 to inhibit the expression of Glucocorticoid-induced TNFR-related protein in human T cells.

Glucocorticoid-induced TNFR-related protein (GITR) is constitutively expressed in T regulatory (Treg) cells and regulates their suppressive function. We identified two methylated CpG islands in the Gitr locus. Using a ChIP assay, we demonstrate that both DNMT1 and methyl-CpG-binding domain Protein 4 (MBD4) bind to the Gitr promoter. Moreover, knockdown of DNMT1 decreases the binding activity of MBD4. We observed much higher levels of both DNMT1 and MBD4 in human CD4+ CD25- conventional T (Tconv) cells. Moreover, co-overexpression of DNMT1 and MBD4 in Treg cells significantly inhibits GITR expression and impairs their suppressive activity. Our results reveal a novel molecular mechanism by which MBD4 inhibits GITR expression in a DNMT1-dependent manner.

MiR-125a-5p Decreases the Sensitivity of Treg cells Toward IL-6-Mediated Conversion by Inhibiting IL-6R and STAT3 Expression.

The transcription factor FOXP3 is essential for the differentiation and function of regulatory T cells (Treg). It is established that the transcription factor GATA-3 is induced in Treg cells under inflammatory conditions. GATA-3 stabilizes FOXP3 levels to avoid the differentiation of Treg cells into inflammatory-like T cells. The IL-6 signal pathway influences the sensitivity of Treg cells towards instability. The mechanism of GATA-3 in regulating FOXP3 and its relation to the IL-6 pathway remains unclear. Here we report how miR-125a-5p plays an important role in regulating the conversion of Treg cells by IL-6. miR-125a-5p expression is low in Treg cells under steady state conditions and can be induced by GATA-3 to inhibit the expression of IL-6R and STAT3. This finding reveals a GATA3/miR-125a-5p/IL-6R and STAT3/FOXP3 regulatory pathway, which determines how Treg cells respond to inflammatory IL-6-rich conditions.

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.

Kaempferol enhances the suppressive function of Treg cells by inhibiting FOXP3 phosphorylation.

Kaempferol is a natural flavonoid found in many vegetables and fruits. Epidemiologic studies have described that Kaempferol intake could reduce risk of cancer, especially lung, gastric, pancreatic and ovarian cancers. Recent studies have shown that Kaempferol could also be beneficial to the body to defend against inflammation, and infection by bacteria and viruses; however, the molecular mechanism of its immunoregulatory function remains largely unknown. Through screening a small molecule library of traditional Chinese medicine (TCM), we identified that Kaempferol could enhance the suppressive function of regulatory T cells (Tregs). Kaempferol was found to increase FOXP3 expression level in Treg cells and prevent pathological symptoms of collagen-induced arthritis in a rat animal model. Kaempferol could also reduce PIM1-mediated FOXP3 phosphorylation at S422. Our study reveals a molecular mechanism that underlies the anti-inflammatory action of Kaempferol for the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis.

FOXP3+ regulatory T cells and their functional regulation.

FOXP3(+) regulatory T (Treg) cells are critical in maintaining immune tolerance and homeostasis of the immune system. The molecular mechanisms underlying the stability, plasticity and functional activity of Treg cells have been much studied in recent years. Here, we summarize these intriguing findings, and provide insight into their potential use or manipulation during Treg cell therapy for the treatment of autoimmune diseases, graft-versus-host disease (GVHD) and cancer.

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.

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.

USP22 is a positive regulator of NFATc2 on promoting IL2 expression.

Nuclear factor of activated T cells (NFAT) is an important regulator of T cell activation. However, the molecular mechanism whereby NFATc2 regulates IL2 transcription is not fully understood. In this study, we showed that ubiquitin-specific protease 22 (USP22), known as a cancer stem cell marker, specifically interacted with and deubiquitinated NFATc2. USP22 stabilized NFATc2 protein levels, which required its deubiquitinase activity. Consistent with these observations, depletion of USP22 in T cells reduced the expression of IL2, which is a cytokine that signifies T effector cell activation. Our findings thus unveil a previously uncharacterized positive regulator of NFATc2, suggesting that targeting the deubiquitinase activity of USP22 could have therapeutic benefit to control IL2 expression and T cell function.

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.

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.

Ubiquitination signals critical to regulatory T cell development and function.

Protein ubiquitination has emerged as a crucial modulator of the immune system, participating in the control of T cell differentiation, intracellular signal transduction and the induction of immune tolerance. CD4(+)CD25(+)FOXP3(+) regulatory T cells are a unique subset of cells that mediate central and peripheral immune tolerance. In this review, we highlight our current understanding of the molecular mechanisms and signaling pathways that modulate protein ubiquitination in Treg cells, and how ubiquitination determines Treg cell development and function. Understanding how FOXP3 activity is regulated by ubiquitination and deubiquitination under molecular level will promote regulatory T cell therapy for treating inflammation in autoimmune disease, infection, transplantation and cancer.

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.

Synergy between IL-6 and TGF-ß signaling promotes FOXP3 degradation.

The forkhead family transcription factor FOXP3 is critical for the differentiation and function of CD4(+) CD25(+) regulatory T cells (Treg). How FOXP3 protein level is negatively regulated under the inflammatory microenvironment is largely unknown. Here we report that the combination of transforming growth factor-beta (TGF-ß) and IL-6 treatment (IL-6/TGF-ß) can synergistically downregulate FOXP3 at the posttranslational level by promoting FOXP3 protein degradation. In our FOXP3 overexpression model, we found that IL-6/TGF-ß treatment upregulated IL-6R expression but did not affect the stability of FOXP3 mRNA. Moreover, we found that the proteasome inhibitor MG132 could inhibit IL-6/TGF-ß-mediated downregulation of FOXP3 protein, which reveals a potential pathway for modulating Treg activity by preventing FOXP3 degradation during inflammation.

Centrosome docking at the immunological synapse is controlled by Lck signaling.

Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor-activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane.

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.

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.