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Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development1–4. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota5–8, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells have not been identified. Here we describe the identification of a class of RORγt+ antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I–TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pTreg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pTreg generation, including the TGF-β-activating integrin αvβ8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pTreg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pTreg generation, further implicating TC IV as the tolerogenic RORγt+ antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.

Th1.17 characteristics of CD73+ CD4+ Teff based on the expression of CXCR3, CCR6, CRTH2, the migration in response to CXCL10 or CCL20 gradient and the MDR-1-specific Rhodamine 123 exclusion assay

Effect of ERRa expression on CCL17, CCL20 TGF-b3 in human BCa cell lines.

Cytotoxic activity in the spleen and the lung and CD8+ T proliferation

ChIP-seq data analysis for ERRa binding sites

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFβ3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFβ signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth.Significance:This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.

Human and mouse primer sequences and PCR cycling and ChIP conditions used.

The production of CD73-derived adenosine (Ado) by Tregs has been proposed as a resistance mechanism to anti-PD-1 therapy in murine tumor models. We reported that human Tregs express the ectonucleotidase CD39, which generates AMP from ATP, but do not express the AMPase CD73. In contrast, CD73 defined a subset of effector CD4+ T cells (Teffs) enriched in polyfunctional Th1.17 cells characterized by expression of CXCR3, CCR6, and MDR1, and production of IL17A/IFNγ/IL22/GM-CSF. CD39+ Tregs selectively targeted CD73+ Teffs through cooperative degradation of ATP into Ado inhibiting and restricting the ability of CD73+ Teffs to secrete IL17A. CD73+ Teffs infiltrating breast and ovarian tumors were functionally blunted by Tregs expressing upregulated levels of CD39 and ATPase activity. Moreover, tumor-infiltrating CD73+ Teffs failed to express inhibitory immune checkpoints, suggesting that CD73 might be selected under pressure from immune checkpoint blockade therapy and thus may represent a nonredundant target for restoring antitumor immunity.Significance: Polyfunctional CD73+ T-cell effectors lacking other immune checkpoints are selectively targeted by CD39 overexpressing Tregs that dominate the breast tumor environment. Cancer Res; 78(13); 3604–18. ©2018 AACR.

"Minimal Network" of ESRRA, CCL17 and CCL20"

Cytokines and inflammation sign gene expression array

Cumulative data of percentages of memory T CD4+ cells, Treg and CD73+CD4+ Teff and cytokine production profile of CD73neg and CD73+ Teff in HD-Blood, and blood and tumors from breast or ovarian patients.

Innate cell homeostasis in the bone colonized by 4T1-ERRα

Extended network default node

Impact of CD39 or CD73 inhibitor (ARL67156, APCP) on ATP-mediated inhibition of CD73+ or CD73neg CD4+ Teff in the presence of Treg based on the proliferation markers profiles (Cell trace Violet or CFSE)

Sensitivity of CD73+ and CD73neg CD4+ Teff proliferation to AMP and impact of CD73 inhibitor (APCP) assessed by microscopy and flow cytometry analysis.

Impact of rh-ADA on Ado-mediated inhibition of CD73+ or CD73neg CD4+ Teff based on the proliferation marker profiles (Cell Trace Violet or CFSE)

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation (5mC) and cytosine hydroxymethylation (5hmC) are DNA modifications that identify stable cell phenotypes but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a new method to profile Th cell identity using cas9-targeted single molecule nanopore sequencing and found that 5mC and 5hmC can be used as markers of cellular identity. Targeting as few as 10 selected genomic loci, we were able to distinguish major differentiated T cell subtypes as well as intermediate phenotypes by their native DNA 5mC/5hmC patterns. Moreover, by using off-target sequences we were able to infer transcription factor activities relevant to each cell subtype. Our analysis demonstrates the importance of epigenetic regulation by 5mC and 5hmC modifications in the establishment of Th cell identity. Furthermore, our data highlight the potential to exploit this immune profiling application to elucidate the pathogenic role of Th transition states in autoimmune diseases.

SMAD4, a mediator of TGF-β signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-β receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-β signature in naive CD8+ T cells. Mechanistically, prior to TGF-β signaling, SMAD4 binds to promoters and enhancers of several TGF-β target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-β signal, SMAD4 limits the expression of TGF-β negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-β. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-β control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-β-independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFβ3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFβ signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. Significance: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.

Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (T

Despite the lack of unanimous consent on hematopoietic cell development, the current paradigm is that early hematopoietic progenitors are exclusively embryonic and short-lived. Their progeny resides in the adult and maintains its homeostasis exclusively by self-renewing without de novo generation. Here, we show that hematopoietic progenitor cells (HPC), which passed through a CD4 positive stage (HPC-CD4) give rise to a fraction of {gamma}{delta}T cells, B cells, and dendritic cells, a larger part of liver and lung macrophages, and megakaryocyte-erythroblast progenitors. These hematopoietic progenitor cells emerged during embryonic life, are observed but not generated in adults, where their graft allows the production of myeloid and lymphoid cells. Thus, we propose HPC-CD4 as a distinct embryonic HPC, which maintains a continuous generation of a fraction of hematopoietic cells through the lifespan.

SMAD4, a key mediator of TGF-β signaling, plays a crucial role in T cells to prevent chronic gut inflammation. However, the molecular mechanisms underlying this control remain elusive. Using different genetic and epigenetic approaches, we unexpectedly reveal that SMAD4 in CD8 T cells prevents chronic intestinal inflammation by a feedforward mechanism that is TGF-β-independent. Prior to any TGF-β-receptor engagement, SMAD4 acts as an active and basal repressor of epigenetic, transcriptional and functional TGF-β imprinting in CD8 T cells. Thus, in sharp opposition to total TGF-β signaling deletion, SMAD4 deletion impairs naïve CD8 T cell effector predisposition but promotes CD8 T cell accumulation and epithelial retention by promoting their response to IL-7 and their expression of integrins such as Itgae. Besides, SMAD4 deletion unleashes the induction of a wide range of TGF-β-signaling-repressors such as Smad7, Ski, Skil, and Smurf2 and hampers TGF-β-mediated CD8 T cell immunosuppression. Mechanistically, prior to any TGF-β signal, SMAD4 binds to the loci of several TGF-β-target genes, and by regulating histone acetylation, represses their expression. The massive gut epithelial colonization, associated with their escape from the immunoregulatory TGF-β effects overtakes their poor effector preconditioning and elicits microbiota-driven chronic epithelial CD8 T cell activation. Hence, in an anticipatory manner, independently of TGF-β, SMAD4 governs a feedforward regulation of TGF-β effects in CD8 T cells, preventing chronic intestinal inflammation.

Among the strategies allowing cancer cells to escape the immune system, the presence of TGF-b in the tumor micro-environment is one of the most potent. However, TGF-b is secreted in an inactive form and mechanisms responsible for its activation within the tumor remain unknown. Here, we demonstrate that regulatory T cells (Tregs) compose the main cells expressing the b8 chain of avb8 integrin (Itgb8) in the tumors and that the Itgb8pos Treg population activates TGF-b produced by the cancer cells and stored in the tumor micro-environment. Itgb8 ablation in Tregs impaired TGF-b signaling in T lymphocytes present in the tumor but not in the tumor draining lymph nodes. The cytotoxic function of CD8pos T lymphocytes infiltrating the tumors was subsequently exacerbated leading to an efficient control of the tumor growth. Similar observations were made in patient tumors after anti-Itgb8 antibody treatment. Thus, this study reveals that Tregs work in concert with cancer cells to produce bioactive-TGF-b and create a powerful-immunosuppressive micro-environment.

© The Author(s), under exclusive licence to Springer Nature America, Inc. 2020, Tissue-resident memory T (TRM) cells, functionally distinct from circulating memory T cells, have a critical role in protective immunity in tissues, are more efficacious when elicited after vaccination and yield more effective antitumor immunity, yet the signals that direct development of TRM cells are incompletely understood. Here we show that type 1 regulatory T (Treg) cells, which express the transcription factor T-bet, promote the generation of CD8+ TRM cells. The absence of T-bet-expressing type 1 Treg cells reduces the presence of TRM cells in multiple tissues and increases pathogen burden upon infectious challenge. Using infection models, we show that type 1 Treg cells are specifically recruited to local inflammatory sites via the chemokine receptor CXCR3. Close proximity with effector CD8+ T cells and Treg cell expression of integrin-β8 endows the bioavailability of transforming growth factor-β in the microenvironment, thereby promoting the generation of CD8+ TRM cells., The project leading to these results has received funding from the European Union H2020 ERA project (no. 667824, EXCELLtoINNOV), Fundo iMM-Laço and ‘la caixa’ Foundation (ID 100010434) under agreement LCF/PR/HR19/52160005 for work in the Veldhoen laboratory, with additional funding from the Fundação para a Ciência e a Tecnologia to P.F.-C. (SFRH/BD/131605/2017), to L.B. (PD/BD/138847/2018) and to A.B. (SFRH/BD/138900/2018). In addition, the work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) SFB1366 (project no. 394046768-SFB 1366; C02) and by SPP 1937 (CE 140/2-1) to A.C and SFB1292 (TP13) and TR156 (TPB02) to H.C.P. A.L. was supported by Ligue Nationale Contre le Cancer. Other grants supporting this study: Foncer Contre le Cancer (JCM) and EL-2016 LNCC Labelisation Ligue Nationale Contre Cancer.

The production of CD73-derived adenosine (Ado) by Tregs has been proposed as a resistance mechanism to anti-PD-1 therapy in murine tumor models. We reported that human Tregs express the ectonucleotidase CD39, which generates AMP from ATP, but do not express the AMPase CD73. In contrast, CD73 defined a subset of effector CD4 + T cells (Teffs) enriched in polyfunctional Th1.17 cells characterized by expression of CXCR3, CCR6, and MDR1, and production of IL17A/IFNγ/IL22/GM-CSF. CD39 + Tregs selectively targeted CD73 + Teffs through cooperative degradation of ATP into Ado inhibiting and restricting the ability of CD73 + Teffs to secrete IL17A. CD73 + Teffs infiltrating breast and ovarian tumors were functionally blunted by Tregs expressing upregulated levels of CD39 and ATPase activity. Moreover, tumor-infiltrating CD73 + Teffs failed to express inhibitory immune checkpoints, suggesting that CD73 might be selected under pressure from immune checkpoint blockade therapy and thus may represent a nonredundant target for restoring antitumor immunity.Significance: Polyfunctional CD73 + T-cell effectors lacking other immune checkpoints are selectively targeted by CD39 overexpressing Tregs that dominate the breast tumor environment. Cancer Res; 78(13); 3604-18. ©2018 AACR.

Tissue-resident memory T (T

International audience; DCC (Deleted in Colorectal Carcinoma) has been demonstrated to constrain tumor progression by inducing apoptosis unless engaged by its ligand netrin-1. This has been shown in breast and colorectal cancers; however, this tumor suppressive function in other cancers is not established. Using a transgenic mouse model, we report here that inhibition of DCC-induced apoptosis is associated with lymphomagenesis. In human diffuse large B-cell lymphoma (DLBCL), an imbalance of the netrin-1/DCC ratio suggests a loss of DCC-induced apoptosis, either via a decrease in DCC expression in germinal center subtype or by up-regulation of netrin-1 in activated B-cell (ABC) one. Such imbalance is also observed in mantle cell lymphoma (MCL). Using a netrin-1 interfering antibody, we demonstrate both in vitro and in vivo that netrin-1 acts as a survival factor for ABC-DLBCL and MCL tumor cells. Together, these data suggest that interference with the netrin-1/DCC interaction could represent a promising therapeutic strategy in netrin-1-positive DLBCL and MCL

The production of CD73-derived adenosine (Ado) by Tregs has been proposed as a resistance mechanism to anti-PD-1 therapy in murine tumor models. We reported that human Tregs express the ectonucleotidase CD39, which generates AMP from ATP, but do not express the AMPase CD73. In contrast, CD73 defined a subset of effector CD4

Summary T helper-17 (Th17) cells are associated with inflammatory disorders and cancer. We report that environmental conditions resulting in cellular stress, such as low oxygen, glucose, and isotonic stress, particularly enhance the generation of Th17 cells. Pharmacological inhibition of cell stress reduces Th17 cell differentiation while stress inducers enhance the development of Th17 cells. The cellular stress response results in Th17 cell development via sustained cytoplasmic calcium levels and, in part, XBP1 activity. Furthermore, in an inflammatory environment, conditions resulting in cell stress can bring about de novo Th17 cell differentiation, even in the absence of transforming growth factor β (TGF-β) signaling. In vivo, cell stress inhibition enhances resistance to Th17-mediated autoimmunity while stress-exposed T cells enhance disease severity. Adverse metabolic environments during inflammation provide a link between adaptive immunity and inflammation and may represent a risk factor for the development of chronic inflammatory conditions by facilitating Th17 cell differentiation., Graphical Abstract, Highlights • Limited metabolite availability results in cellular stress • Cellular stress enhances Th17 cell polarization • Cellular stress induces de novo Th17 cell differentiation • Cellular stress can substitute for TGF-β in Th17 cell differentiation, Brucklacher-Waldert et al. show how environmental conditions resulting in cellular stress, such as low metabolite levels, specifically enhance Th17 cell differentiation. Under inflammatory conditions, this stress can substitute for TGF-β signaling resulting in de novo Th17 cell differentiation.

T follicular helper (Tfh) cells contribute to the establishment of humoral immunity by controlling the delivery of helper signals to activated B cells; however, Tfh development must be restrained, as aberrant accumulation of these cells is associated with positive selection of self-reactive germinal center B cells and autoimmunity in both humans and mice. Here, we show that TGF-β signaling in T cells prevented Tfh cell accumulation, self-reactive B cell activation, and autoantibody production. Using mice with either T cell–specific loss or constitutive activation of TGF-β signaling, we demonstrated that TGF-β signaling is required for the thymic maturation of CD44+CD122+Ly49+CD8+ regulatory T cells (Tregs), which induce Tfh apoptosis and thus regulate this cell population. Moreover, peripheral Tfh cells escaping TGF-β control were resistant to apoptosis, exhibited high levels of the antiapoptotic protein BCL2, and remained refractory to regulation by CD8+ Tregs. The unrestrained accumulation of Tfh cells in the absence of TGF-β was dependent on T cell receptor engagement and required B cells. Together, these data indicate that TGF-β signaling restrains Tfh cell accumulation and B cell–associated autoimmunity and thereby controls self-tolerance.

NUPR1 (nuclear protein 1), also called P8 (molecular mass 8 kDa) or COM1 (candidate of metastasis 1), is involved in the stress response and in cancer progression. In the present study, we investigated whether human NUPR1 expression was regulated by TGFβ (transforming growth factor β), a secreted polypeptide largely involved in tumorigenesis. We demonstrate that the expression of NUPR1 was activated by TGFβ at the transcriptional level. We show that this activation is mediated by the SMAD proteins, which are transcription factors specifically involved in the signalling of TGFβ superfamily members. NUPR1 promoter analysis reveals the presence of a functional TGFβ-response element binding the SMAD proteins located in the genomic DNA region corresponding to the 5′-UTR (5′-untranslated region). Altogether, the molecular results of the present study, which demonstrate the existence of a TGFβ/SMAD/NUPR1 activation cascade, open the way to consider and investigate further a new mechanism enabling TGFβ to promote tumorigenesis by inducing stress resistance.

Invariant natural killer T (iNKT) cells have the ability to rapidly secret cytokines in response to diverse stimuli, and therefore influence numerous immune reactions. Although IFN-γ and IL-4 are thought to dominate iNKT cytokine production, a distinct subset of iNKT cells, expressing RORγt and producing IL-17, has now been identified in both mice and humans. Although a role in pathogen and allergic responses has been assigned to the RORγt+ iNKT subset, factors controlling their development and function remain illusive. Here, we demonstrate that RORγt+ iNKT-cell differentiation obeys transforming growth factor-β (TGF-β) signaling control, different from that described for conventional iNKT cells. We reveal that TGF-β signaling, and particularly its SMAD4-dependent pathway, is required for both the survival of RORγt+ iNKT cells during their development and IL-17 production at the periphery. Moreover, constitutive TGF-β signaling in RORγt+ iNKT cells drives higher peripheral numbers and increased tissue distribution. Finally, we found that SMAD4-dependent TGF-β signaling is mandatory for the peripheral expansion of the RORγt+ iNKT cells responding to inflammatory signals. Thus, this work demonstrates that both the development and responsiveness of the newly described IL-17–producing iNKT cell subset is under the control of a dedicated TGF-β signaling pathway.

We have previously generated a transgenic mouse strain (LSL-TβRICA) containing a Cre-inducible constitutively active TGFβ type I receptor (Bartholin L. et al. 2008. Generation of mice with conditionally activated transforming growth factor beta signaling through the TbetaRI/ALK5 receptor, GENESIS). Transgene expression depends on the excision of a floxed-transcriptional STOP (LSL, Lox-STOP-Lox) located upstream the TβRICA coding sequence. In order to evaluate the correct excision of the STOP signal in the presence of Cre-recombinase, we developed a rapid screening based on an original PCR genotyping strategy. More precisely, we designed a set of primers flanking the LSL containing region. The size of the amplified products will differ according to recombination status of the LSL-TβRICA allele. Indeed, the size of the STOP containing PCR product is 1.93 kb, but is reduced to 0.35 kb when the STOP signal is removed after Cre-mediated recombination. We validated excision in several compartments, including pancreas, liver, T lymphocytes and embryos using different Cre expressing transgenic mouse strains. This represents a simple and efficient way of monitoring the tissue specific recombination of the LSL-TβRICA allele.

Objective—Impairment of transforming growth factor (TGF)-β1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-β1 expression would retard atherosclerosis. The role of TGF-β1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-β1 in hyperlipidemic mice affects atherogenesis and aortic dilation.Methods and Results—We generated apolipoprotein E-null mice with transgenes that allow regulated overexpression of active TGF-β1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-β1, less aortic root atherosclerosis (P≤0.002), fewer lesions in the thoracic and abdominal aortae (P≤0.01), less aortic root dilation (PP=0.02). Mechanistic studies revealed no effect of TGF-β1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-β1-overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2.Conclusions—When overexpressed in the heart and plasma, TGF-β1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

We generated a transgenic mouse strain (LSL-TβRICA) containing a latent constitutively active TGFβ type I receptor (TβRI/ALK5) by using a knock-in strategy into the X chromosome-linked hypoxanthine phosphoribosyl-transferase (Hprt) locus. Transgene expression, under the control of the ubiquitous CAG (human cytomegalovirus enhancer and chicken β-actin) promoter, is repressed by a floxed transcriptional “Stop” (LSL, Lox-Stop-Lox). In the presence of cre-recombinase, the “Stop” is excised to allow TβRICA transgene expression. We showed that restricted expression of TβRICA in T lymphocytes efficiently activates TGFβ signaling and rescues the T-cell autoimmune disorders of TGFβRII conditional knockouts. Unexpectedly, our study reveals that TGFβ signaling upregulation controls T-cell activation but does not impair their development or their peripheral homeostasis. In addition to the information provided on TGFβ effects on T-cell biology, LSL-TβRICA mouse constitutes an attractive tool to address the effect of TGFβ signaling upregulation in any cell type expressing the cre-recombinase. genesis 46:724–731, 2008. Published 2008 Wiley-Liss, Inc.

T cell activation requires that the cell meet increased energetic and biosynthetic demands. We showed that exogenous nutrient availability regulated the differentiation of naive CD4(+) T cells into distinct subsets. Activation of naive CD4(+) T cells under conditions of glutamine deprivation resulted in their differentiation into Foxp3(+) (forkhead box P3-positive) regulatory T (Treg) cells, which had suppressor function in vivo. Moreover, glutamine-deprived CD4(+) T cells that were activated in the presence of cytokines that normally induce the generation of T helper 1 (TH1) cells instead differentiated into Foxp3(+) Treg cells. We found that alpha-ketoglutarate (alphaKG), the glutamine-derived metabolite that enters into the mitochondrial citric acid cycle, acted as a metabolic regulator of CD4(+) T cell differentiation. Activation of glutamine-deprived naive CD4(+) T cells in the presence of a cell-permeable alphaKG analog increased the expression of the gene encoding the TH1 cell-associated transcription factor Tbet and resulted in their differentiation into TH1 cells, concomitant with stimulation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Together, these data suggest that a decrease in the intracellular amount of alphaKG, caused by the limited availability of extracellular glutamine, shifts the balance between the generation of TH1 and Treg cells toward that of a Treg phenotype.

Transforming growth factor-beta (TGF-beta) has been implicated in the control of differentiation and proliferation of multiple cell types. However, a role for TGF-beta in the control of immune homeostasis is not fully understood because of its pleiotropic action. Here we report that complete ablation of the TGF-beta signaling in T cells engendered aggressive early-onset, multiorgan, autoimmune-associated lesions with 100% mortality. Peripheral CD4+ and CD8+ T cells with TGF-beta-receptor II (TGF-betaRII) deficiency activated cytolytic and T helper 1 (Th1) differentiation program in a cell-intrinsic T cell receptor (TCR)-specific fashion. Furthermore, TGF-betaRII deficiency blocked the development of canonical CD1d-restricted NKT cells. Instead, it facilitated generation of a highly pathogenic T cell subset exhibiting multiple hallmarks of NK cells and sharply elevated amounts of FasL, perforin, granzymes, and interferon-gamma. Thus, TGF-beta signaling in peripheral T cells is crucial in restraining TCR activation-dependent Th1, cytotoxic, and NK cell-like differentiation program which, when left unchecked, leads to rapidly progressing fatal autoimmunity.

Although only a few blood cells are infected during measles, this infectious disease is followed by acute immunosuppression, associated with high infant mortality. Measles virus nucleoprotein has been suggested to contribute to virus-induced inhibition of the immune response. However, it has been difficult to understand how this cytosolic viral protein could leave an infected cell and then perturb the immune response. Here we demonstrate that intracellularly synthesized nucleoprotein enters the late endocytic compartment, where it recruits its cellular ligand, the Fcγ receptor. Nucleoprotein is then expressed at the surfaces of infected leukocytes associated with the Fcγ receptor and is secreted into the extracellular compartment, allowing its interaction with uninfected cells. Finally, cell-derived nucleoprotein inhibits the secretion of interleukin-12 and the generation of the inflammatory reaction, both shown to be impaired during measles. These results reveal nucleoprotein egress from infected cells as a novel strategy in measles-induced immunosuppression.

The relationship between host organisms and their microbiota has co-evolved towards an inter-dependent network of mutualistic interactions. This interplay is particularly well studied in the gastrointestinal tract, where microbiota and host immune cells can modulate each other directly, as well as indirectly, through the production and release of chemical molecules and signals. In this review, we define the functional impact of transforming growth factor-beta (TGF-β) on this complex interplay, especially through its modulation of the activity of local regulatory T cells (Tregs), type 17 helper (Th17) cells, innate lymphoid cells (ILCs) and B cells.

Summary Regulatory T (Treg) cells play a pivotal role in suppressing self-harmful T cell responses, but how Treg cells mediate suppression to maintain immune homeostasis and limit responses during inflammation is unclear. Here we show that effector Treg cells express high amounts of the integrin αvβ8, which enables them to activate latent transforming growth factor-β (TGF-β). Treg-cell-specific deletion of integrin αvβ8 did not result in a spontaneous inflammatory phenotype, suggesting that this pathway is not important in Treg-cell-mediated maintenance of immune homeostasis. However, Treg cells lacking expression of integrin αvβ8 were unable to suppress pathogenic T cell responses during active inflammation. Thus, our results identify a mechanism by which Treg cells suppress exuberant immune responses, highlighting a key role for effector Treg-cell-mediated activation of latent TGF-β in suppression of self-harmful T cell responses during active inflammation., Graphical Abstract, Highlights • Human and mouse effector Treg cells express functional TGF-β-activating integrin αvβ8 • Treg cell integrin αvβ8-mediated TGF-β activation is not needed for T cell homeostasis • Integrin αvβ8 expression by Treg cells suppresses active inflammation • Pathway could be targeted to promote Treg-cell-mediated suppression of inflammation, Regulatory T (Treg) cells suppress harmful T cell responses, but mechanisms mediating suppressive function during homeostasis versus inflammation are poorly defined. Travis and colleagues show that activation of the cytokine TGF-β by effector Treg cells, via expression of integrin αvβ8, is crucial for Treg-cell-mediated suppression of inflammatory T cells.

Measles virus (MV) causes profound immunosuppression, resulting in high infant mortality. The mechanisms are poorly understood, largely due to the lack of a suitable animal model. Here, we report that particular MV proteins, in the absence of MV replication, could generate a systemic immunosuppression in mice through two pathways: (1) via MV-nucleoprotein and its receptor FcγR on dendritic cells; and (2) via virus envelope glycoproteins and the MV-hemagglutinin cellular receptor, CD46. The effects comprise reduced hypersensitivity responses associated with impaired function of dendritic cells, decreased production of IL-12, and the loss of antigen-specific T cell proliferation. These results introduce a novel model for testing the immunosuppressive potential of anti-measles vaccines and reveal a specific mechanism of MV-induced modulation of inflammatory reactions.

Measles virus (MV) infection causes acute childhood disease, associated in certain cases with infection of the central nervous system (CNS) and development of neurological disease. To develop a murine model of MV-induced pathology, we generated several lines of transgenic mice ubiquitously expressing as the MV receptor a human CD46 molecule with either a Cyt1 or Cyt2 cytoplasmic tail. All transgenic lines expressed CD46 protein in the brain. Newborn transgenic mice, in contrast to nontransgenic controls, were highly sensitive to intracerebral infection by the MV Edmonston strain. Signs of clinical illness (lack of mobility, tremors, and weight loss) appeared within 5 to 7 days after infection, followed by seizures, paralysis, and death of the infected animals. Virus replication was detected in neurons from infected mice, and virus was reproducibly isolated from transgenic brain tissue. MV-induced apoptosis observed in different brain regions preceded the death of infected animals. Similar results were obtained with mice expressing either a Cyt1 or Cyt2 cytoplasmic tail, demonstrating the ability of different isoforms of CD46 to function as MV receptors in vivo. In addition, maternally transferred immunity delayed death of offspring given a lethal dose of MV. These results document a novel CD46 transgenic murine model where MV neuronal infection is associated with the production of infectious virus, similarly to progressive infectious measles encephalitis seen in immunocompromised patients, and provide a new means to study pathogenesis of MV infection in the CNS.

Summary Memory CD8 + T cells induced upon immunization exhibit improved functional features that contribute to protection of immunized hosts. Although both cognate antigen recognition and inflammation are important for memory CD8 + T cell reactivation, the relative contribution of these factors and the cell types providing these signals in vivo are poorly defined. Here, we show that Ly6C + CCR2 + inflammatory monocytes, a subset of monocytes, largely orchestrate memory CD8 + T and NK lymphocytes activation by differentiating into interleukin-18 (IL-18)- and IL-15-producing cells in an inflammasome and type I interferon-IRF3-dependent manner. Memory CD8 + T cells became potent effector cells by sensing inflammation from monocytes independently of their cognate antigen. Like NK cells, they underwent rapid mobilization, upregulated intense and sustained effector functions during bacterial, viral, and parasitic infections, and contributed to innate responses and protection in vivo. Thus, inflammatory monocyte-derived IL-18 and IL-15 are critical to initiate memory CD8 + T and NK lymphocytes differentiation into antimicrobial effector cells.

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development., Author Summary Inactivation of the TGFβ tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC), a devastating malignancy. Transcriptional Intermediary Factor 1γ (TIF1γ) has recently been proposed to be involved in TGFβ signaling, a pathway inactivated in virtually all cases of this malignancy. To address the role of TIF1γ in pancreatic carcinogenesis, we used conditional Tif1γ knockout mice. In a genetic background expressing a constitutively active mutation of KRAS oncogene (KrasG12D) recurrently found in patients with PDAC, Tif1γ inactivation induces pancreatic precancerous lesions resembling those observed in the absence of Smad4, a key player involved TGFβ signal transduction. This observation strengthens the notion that TIF1γ plays an active role in TGFβ signaling. Interestingly, we also found that TIF1γ expression was markedly down-regulated in human pancreatic tumors supporting the relevance of our findings to human malignancy. Characterization of new players involved in the outbreak of early pancreatic lesions that will eventually evolve into invasive pancreatic cancer is crucial to detect the disease earlier and eventually develop new therapeutic drugs.

iNKT cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although different steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling finely and specifically orchestrates several steps of iNKT cell development. In vivo this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors allowing TGF-β to finely tune the iNKT cell-differentiation program.

Measles is a highly contagious childhood disease associated with an immunological paradox: although a strong virus-specific immune response results in virus clearance and the establishment of a life-long immunity, measles infection is followed by an acute and profound immunosuppression leading to an increased susceptibility to secondary infections and high infant mortality. In certain cases, measles is followed by fatal neurological complications. To elucidate measles immunopathology, we have analyzed the immune response to measles virus in mice transgenic for the measles virus receptor, human CD150. These animals are highly susceptible to intranasal infection with wild-type measles strains. Similarly to what has been observed in children with measles, infection of suckling transgenic mice leads to a robust activation of both T and B lymphocytes, generation of virus-specific cytotoxic T cells and antibody responses. Interestingly, Foxp3(+)CD25(+)CD4(+) regulatory T cells are highly enriched following infection, both in the periphery and in the brain, where the virus intensively replicates. Although specific anti-viral responses develop in spite of increased frequency of regulatory T cells, the capability of T lymphocytes to respond to virus-unrelated antigens was strongly suppressed. Infected adult CD150 transgenic mice crossed in an interferon receptor type I-deficient background develop generalized immunosuppression with an increased frequency of CD4(+)CD25(+)Foxp3(+) T cells and strong reduction of the hypersensitivity response. These results show that measles virus affects regulatory T-cell homeostasis and suggest that an interplay between virus-specific effector responses and regulatory T cells plays an important role in measles immunopathogenesis. A better understanding of the balance between measles-induced effector and regulatory T cells, both in the periphery and in the brain, may be of critical importance in the design of novel approaches for the prevention and treatment of measles pathology.