IL-18 is required for the TH1-adaptation of TREG cells and the selective suppression of TH17 responses in acute and chronic infections.

Interleukin (IL)-18, a member of the IL-1 family of alarmins, is abundantly released in the lungs following influenza A (IAV) infections yet its role in orchestrating the local adaptive immune response remains ill defined. Through genetic disruption of the IL-18 receptor, we demonstrate that IL-18 not only promotes pulmonary TH1 responses but also influences regulatory T cells (TREG) function in the infected lungs. As the response unfolds, TREG cells accumulating in the lungs express Helios, T-bet, CXCR3, and IL-18R1 and produce interferon γ in the presence of IL-12. During IAV, IL-18R1 is required for TREG cells to control TH17, but not TH1, responses and promote a return to lung homeostasis, revealing a novel mechanism of selective suppression. Moreover, this observation was not limited to the lungs, as skin-localized TREG cells require an IL-18 signal to specifically suppress IL-17A production by TH17 and γδ T cells in a model of chronic cutaneous Leishmania major infection. Overall, these results uncover how IL-18 orchestrates the tissue adaptation of TREG cells to selectively favor TH1 over TH17 responses during TH1-driven immune responses and provide a novel perspective into how IL-18 dictates the immune response during viral and parasitic infections.

The eIF4EBP-eIF4E axis regulates CD4+ T cell differentiation through modulation of T cell activation and metabolism.

CD4+ T cells are critical for adaptive immunity, differentiating into distinct effector and regulatory subsets. Although the transcriptional programs underlying their differentiation are known, recent research has highlighted the importance of mRNA translation in determining protein abundance. We previously conducted genome-wide analysis of translation in CD4+ T cells revealing distinct translational signatures distinguishing these subsets, identifying eIF4E as a central differentially translated transcript. As eIF4E is vital for eukaryotic translation, we examined how altered eIF4E activity affected T cell function using mice lacking eIF4E-binding proteins (BP-/-). BP-/- effector T cells showed elevated Th1 responses ex vivo and upon viral challenge with enhanced Th1 differentiation observed in vitro. This was accompanied by increased TCR activation and elevated glycolytic activity. This study highlights how regulating T cell-intrinsic eIF4E activity can influence T cell activation and differentiation, suggesting the eIF4EBP-eIF4E axis as a potential therapeutic target for controlling aberrant T cell responses.

A Hemagglutinin 1 Carrying Plant-Based Virus-like Particle Vaccine Generates an Efficacious Cellular Response by Exploiting IL-1 Signaling in Both Adult and Aged Mice.

Inactivated influenza vaccines have struggled to provide consistent protection in older individuals. Circumventing immune senescence, an aging of the immune response characterized by weak humoral responses to vaccines, and unchecked inflammation during infection require novel immunization strategies. Plant-based virus-like particles (VLPs) bearing recombinant hemagglutinin proteins have been shown to provide protection in older animals in preclinical challenge studies, despite eliciting relatively low or absent humoral responses. The nature of the cellular response induced by these vaccines and its evolution during infection have not yet been fully characterized, however. Using a murine model that recapitulates features of human immune senescence, we assessed T cell responses to vaccination with a VLP bearing the hemagglutinin of H1N1/California 07/2009 (H1-VLP) before and after challenge in young and aged BALB/c mice (2 and 18 mo old, respectively). We report that two i.m. doses of H1-VLP (3 µg) vaccine 21 d apart generated H1-specific Th1 and Th2 cells associated with the prevention of prolonged pulmonary inflammation and mortality in both adult and aged mice. While investigating the regulation of cellular immunity, we identified a unique IL-1R1+ tissue-adapted regulatory T cell population in the lungs of both H1-VLP-vaccinated adult and aged mice, suggesting a novel regulatory T cell population associated with vaccine-mediated protection. Collectively, this study provides preclinical evidence that the plant-based H1-VLP vaccine may act, in part, by preventing exacerbated immune responses against influenza A.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3+ Regulatory T Cells.

Regulatory T (Treg) cells integrate diverse environmental signals to modulate their function for optimal suppression. Translational regulation represents a favorable mechanism for Treg cell environmental sensing and adaptation. In this study, we carry out an unbiased screen of the Treg cell translatome and identify serum/glucocorticoid-regulated kinase 1 (SGK1), a known salt sensor in T cells, as being preferentially translated in activated Treg cells. We show that high salt (HS) drives thymic Treg cells to adopt a T helper type 17 (Th17)-like phenotype and enhances generation of Th17-like induced Treg cells in a SGK1-dependent manner, all the while maintaining suppressive function. Salt-mediated Th17-like differentiation of Treg cells was evident in mice fed with HS diet or injected with HS-preconditioned T cells. Overall, SGK1 enables Treg cells to adapt their function in response to environmental cues. By understanding these environmental-sensing mechanisms, we envision targeted approaches to fine-tune Treg cell function for better control of inflammation.

Rare Genetic Variants of Large Effect Influence Risk of Type 1 Diabetes.

Most replicated genetic determinants for type 1 diabetes are common (minor allele frequency [MAF] >5%). We aimed to identify novel rare or low-frequency (MAF <5%) single nucleotide polymorphisms with large effects on risk of type 1 diabetes. We undertook deep imputation of genotyped data followed by genome-wide association testing and meta-analysis of 9,358 type 1 diabetes case and 15,705 control subjects from 12 European cohorts. Candidate variants were replicated in a separate cohort of 4,329 case and 9,543 control subjects. Our meta-analysis identified 27 independent variants outside the MHC, among which 3 were novel and had MAF <5%. Three of these variants replicated with P replication < 0.05 and P combined < P discovery In silico analysis prioritized a rare variant at 2q24.3 (rs60587303 [C], MAF 0.5%) within the first intron of STK39, with an effect size comparable with those of common variants in the INS and PTPN22 loci (combined [from the discovery and replication cohorts] estimate of odds ratio [ORcombined] 1.97, 95% CI 1.58-2.47, P combined = 2.9 × 10-9). Pharmacological inhibition of Stk39 activity in primary murine T cells augmented effector responses through enhancement of interleukin 2 signaling. These findings provide insight into the genetic architecture of type 1 diabetes and have identified rare variants having a large effect on disease risk.

Sexual dimorphism and the role of estrogen in the immune microenvironment of liver metastases.

Liver metastases (LM) remain a major cause of cancer-associated death and a clinical challenge. Here we explore a sexual dimorphism observed in the regulation of the tumor immune microenvironment (TIME) of LM, wherein the accumulation of myeloid-derived suppressor cells (MDSC) and regulatory T cells in colon and lung carcinoma LM is TNFR2-dependent in female, but not in male mice. In ovariectomized mice, a marked reduction is observed in colorectal, lung and pancreatic carcinoma LM that is reversible by estradiol reconstitution. This is associated with reduced liver MDSC accumulation, increased interferon-gamma (IFN-γ) and granzyme B production in CD8+ T cells and reduced TNFR2, IDO2, TDO and Serpin B9 expression levels. Treatment with tamoxifen increases liver cytotoxic T cell accumulation and reduces colon cancer LM. The results identify estrogen as a regulator of a pro-metastatic immune microenvironment in the liver and a potential target in the management of liver metastatic disease.

The Deubiquitinating Enzyme Ubiquitin-Specific Peptidase 11 Potentiates TGF-ß Signaling in CD4+ T Cells to Facilitate Foxp3+ Regulatory T and TH17 Cell Differentiation.

Foxp3+ regulatory T (TREG) cells are central mediators in the control of peripheral immune responses. Genome-wide transcriptional profiles show canonical signatures for Foxp3+ TREG cells, distinguishing them from Foxp3- effector T (TEFF) cells. We previously uncovered distinct mRNA translational signatures differentiating CD4+ TEFF and TREG cells through parallel measurements of cytosolic (global) and polysome-associated (translationally enhanced) mRNA levels in both subsets. We show that the mRNA encoding for the ubiquitin-specific peptidase 11 (USP11), a known modulator of TGF-ß signaling, was preferentially translated in TCR-activated TREG cells compared with conventional, murine CD4+ T cells. TGF-ß is a key cytokine driving the induction and maintenance of Foxp3 expression in T cells. We hypothesized that differential translation of USP11 mRNA endows TREG cells with an advantage to respond to TGF-ß signals. In an in vivo mouse model promoting TREG cells plasticity, we found that USP11 protein was expressed at elevated levels in stable TREG cells, whereas ectopic USP11 expression enhanced the suppressive capacity and lineage commitment of these cells in vitro and in vivo. USP11 overexpression in TEFF cells enhanced the activation of the TGF-ß pathway and promoted TREG or TH17, but not Th1, cell differentiation in vitro and in vivo, an effect abrogated by USP11 gene silencing or the inhibition of enzymatic activity. Thus, USP11 potentiates TGF-ß signaling in both TREG and TEFF cells, in turn driving increased suppressive function and lineage commitment in thymic-derived TREG cells and potentiating the TGF-ß-dependent differentiation of TEFF cells to peripherally induced TREG and TH17 cells.

The alarmins IL-1 and IL-33 differentially regulate the functional specialisation of Foxp3+ regulatory T cells during mucosal inflammation.

CD4+Foxp3+ regulatory T (TREG) cells are critical mediators of peripheral tolerance and modulators of immune responses. Functional adaptation of TREG cells, through acquisition of secondary transcription factors is critical for their effector differentiation towards local inflammatory stimuli including infections. The drivers and consequences of this adaptation of TREG cell function remain largely unknown. Using an unbiased screen, we identified receptors of the IL-1 family controlling the adaptation of TREG cells. Through respiratory infection models, we show that the IL-33 receptor (ST2) and the IL-1 receptor (IL1R1) selectively identify stable and unstable TREG cells at mucosal surfaces, respectively. IL-33, not IL-1, is specifically required for maintaining the suppressive function of TREG cells. In the absence of ST2, TREG cells are prone to lose Foxp3 expression and acquire RORγT and IL1R1, while, in the absence of IL-1R1, they maintain Foxp3 expression and resist the acquisition of a Th17 phenotype. Finally, lack of IL-1 signalling enhances the accumulation of ST2+ TREG over pro-inflammatory TREG cells in a Cryptococcus neoformans infection. These observations show that IL-1 and IL-33 exert opposing functions in controlling the functional adaptation of TREG cells, ultimately dictating the dynamics of adaptive immunity to pathogens.

CD4+ Regulatory T Lymphocytes Prevent Impaired Cerebral Blood Flow in Angiotensin II-Induced Hypertension.

Background Immune cells are key regulators of the vascular inflammatory response characteristic of hypertension. In hypertensive rodents, regulatory T lymphocytes (Treg, CD 4+ CD 25+) prevented vascular injury, cardiac damage, and endothelial dysfunction of mesenteric arteries. Whether Treg modulate the cerebrovascular damage induced by hypertension is unknown. Methods and Results C57 BL /6 mice were perfused with angiotensin II (Ang II ; 1000 ng/kg per minute) for 14 days and adoptive transfer of 3×105 CD 4+ CD 25+ T cells was performed via 2 intravenous injections. Control mice received a sham surgery and PBS . Treg prevented Ang II -induced neurovascular uncoupling ( P<0.05) and endothelial impairment ( P<0.05), evaluated by laser Doppler flowmetry in the somatosensory cortex. The neuroprotective effect of Treg was abolished when they were isolated from mice deficient in interleukin-10. Administration of interleukin-10 (60 ng/d) to hypertensive mice prevented Ang II -induced neurovascular uncoupling ( P<0.05). Treg adoptive transfer also diminished systemic inflammation induced by Ang II ( P<0.05), examined with a peripheral blood cytokine array. Mice receiving Ang II + Treg exhibited reduced numbers of Iba-1+ cells in the brain cortex ( P<0.05) and hippocampus ( P<0.001) compared with mice infused only with Ang II. Treg prevented the increase in cerebral superoxide radicals. Overall, these effects did not appear to be directly modulated by Treg accumulating in the brain parenchyma, because only a nonsignificant number of Treg were detected in brain. Instead, Treg penetrated peripheral tissues such as the kidney, inguinal lymph nodes, and the spleen. Conclusions Treg prevent impaired cerebrovascular responses in Ang II -induced hypertension. The neuroprotective effects of Treg involve the modulation of inflammation in the brain and periphery.

Translational control in the tumor microenvironment promotes lung metastasis: Phosphorylation of eIF4E in neutrophils.

The translation of mRNAs into proteins serves as a critical regulatory event in gene expression. In the context of cancer, deregulated translation is a hallmark of transformation, promoting the proliferation, survival, and metastatic capabilities of cancer cells. The best-studied factor involved in the translational control of cancer is the eukaryotic translation initiation factor 4E (eIF4E). We and others have shown that eIF4E availability and phosphorylation promote metastasis in mouse models of breast cancer by selectively augmenting the translation of mRNAs involved in invasion and metastasis. However, the impact of translational control in cell types within the tumor microenvironment (TME) is unknown. Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression. Mice bearing a mutation in the phosphorylation site of eIF4E (S209A) in cells comprising the TME are resistant to the formation of lung metastases in a syngeneic mammary tumor model. This is associated with reduced survival of prometastatic neutrophils due to decreased expression of the antiapoptotic proteins BCL2 and MCL1. Furthermore, we demonstrate that pharmacological inhibition of eIF4E phosphorylation prevents metastatic progression in vivo, supporting the development of phosphorylation inhibitors for clinical use.

Suppression by human FOXP3+ regulatory T cells requires FOXP3-TIP60 interactions.

CD4+FOXP3+ regulatory T (Treg) cells are critical mediators of immune tolerance, and their deficiency owing to FOXP3 mutations in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) patients results in severe autoimmunity. Different FOXP3 mutations result in a wide range of disease severity, reflecting the relative importance of the affected residues in the integrity of the FOXP3 protein and its various molecular interactions. We characterized the cellular and molecular impact of the most common IPEX mutation, p.A384T, on patient-derived Treg cells. We found that the p.A384T mutation abrogated the suppressive capacity of Treg cells while preserving FOXP3's ability to repress inflammatory cytokine production. This selective functional impairment is partly due to a specific disruption of FOXP3A384T binding to the histone acetyltransferase Tat-interacting protein 60 (TIP60) (KAT5) and can be corrected using allosteric modifiers that enhance FOXP3-TIP60 interaction. These findings reveal the functional impact of TIP60 in FOXP3-driven Treg biology and provide a potential target for therapeutic manipulation of Treg activity.

KLRG1 expression identifies short-lived Foxp3+ Treg effector cells with functional plasticity in islets of NOD mice.

A progressive waning in Foxp3+ regulatory T (Treg) cell function provokes autoimmunity in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), a cellular defect rescued by prophylactic IL-2 therapy. We showed that most islet-infiltrating Treg cells express inducible T-cell co-stimulator (ICOS) in pre-diabetic NOD mice, and that ICOS+ Treg cells display enhanced fitness and suppressive function in situ. Moreover, T1D progression is associated with decreased expansion and suppressive activity of ICOS+Foxp3+ Treg cells, in islets, an observation consistent with the exacerbated T1D seen in NOD.BDC2.5 mice in which the ICOS pathway is abrogated. Here, we show that a large proportion of islet-resident Treg cells express the KLRG1 marker of terminally differentiation, in contrast to islet-infiltrating ICOS- Treg or Teff cells. We hypothesized that KLRG1 expression designates a subpopulation of ICOS+ Treg cells in islets that progressively loses function, and contributes to the immune dysregulation observed at T1D onset. Indeed, KLRG1-expressing ICOS+ Treg cells are prone to apoptosis, and have an impaired proliferative capacity and suppressive function in vitro and in vivo. T1D protective low-dose IL-2 treatment in vivo could not rescue the loss of KLRG1-expressing Treg cells in situ. While the global pool of Foxp3+ Treg cells displays some degree of functional plasticity in vivo, the KLRG1+ ICOS+ Treg cell subset is particularly susceptible to lose Foxp3 expression and reprogram into Th1- or Th17-like effector T (Teff) cells in the pancreas microenvironment. Overall, KLRG1 expression delineates a subpopulation of dysfunctional Treg cells during T1D progression in autoantigen-specific TCR transgenic NOD mice.

Posttranscriptional and Translational Control of Gene Regulation in CD4+ T Cell Subsets.

The immune system is under strict regulatory control to ensure homeostasis of inflammatory responses, lying dormant when not needed but quick to act when called upon. Small changes in gene expression can lead to drastic changes in lineage commitment, cellular function, and immunity. Conventional assessment of these changes centered on the analysis of mRNA levels through a variety of methodologies, including microarrays. However, mRNA synthesis does not always correlate directly to protein synthesis and downstream functional activity. Work conducted in recent years has begun to shed light on the various posttranscriptional changes that occur in response to a dynamic external environment that a given cell type encounters. We provide a critical review of key posttranscriptional mechanisms (i.e., microRNA) and translational mechanisms of regulation of gene expression in the immune system, with a particular emphasis on these regulatory processes in various CD4(+) T cell subsets.