TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

Tissue-restricted control of established central nervous system autoimmunity by TNF receptor 2-expressing Treg cells.

CD4+Foxp3+ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell-mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.

Phylogenomics illuminates the phylogeny of flower weevils (Curculioninae) and reveals ten independent origins of brood-site pollination mutualism in true weevils.

Weevils are an unusually species-rich group of phytophagous insects for which there is increasing evidence of frequent involvement in brood-site pollination. This study examines phylogenetic patterns in the emergence of brood-site pollination mutualism among one of the most speciose beetle groups, the flower weevils (subfamily Curculioninae). We analysed a novel phylogenomic dataset consisting of 214 nuclear loci for 202 weevil species, with a sampling that mainly includes flower weevils as well as representatives of all major lineages of true weevils (Curculionidae). Our phylogenomic analyses establish a uniquely comprehensive phylogenetic framework for Curculioninae and provide new insights into the relationships among lineages of true weevils. Based on this phylogeny, statistical reconstruction of ancestral character states revealed at least 10 independent origins of brood-site pollination in higher weevils through transitions from ancestral associations with reproductive structures in the larval stage. Broadly, our results illuminate the unexpected frequency with which true weevils-typically specialized phytophages and hence antagonists of plants-have evolved mutualistic interactions of ecological significance that are key to both weevil and plant evolutionary fitness and thus a component of their deeply intertwined macroevolutionary success.

Regulatory T Cell Stability and Migration Are Dependent on mTOR.

CD4+ Foxp3+ regulatory T cells (Treg) are essential to maintain immune tolerance, as their loss leads to a fatal autoimmune syndrome in mice and humans. Conflicting findings have been reported concerning their metabolism. Some reports found that Treg have low mechanistic target of rapamycin (mTOR) activity and would be less dependent on this kinase compared with conventional T cells, whereas other reports suggest quite the opposite. In this study, we revisited this question by using mice that have a specific deletion of mTOR in Treg. These mice spontaneously develop a severe and systemic inflammation. We show that mTOR expression by Treg is critical for their differentiation into effector Treg and their migration into nonlymphoid tissues. We also reveal that mTOR-deficient Treg have reduced stability. This loss of Foxp3 expression is associated with partial Foxp3 DNA remethylation, which may be due to an increased activity of the glutaminolysis pathway. Thus, our work shows that mTOR is crucial for Treg differentiation, migration, and identity and that drugs targeting this metabolism pathway will impact on their biology.

Circulating Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Contribute to Sepsis-Induced Immunosuppression in Patients During Septic Shock.

BACKGROUND: Septic shock remains a major cause of death that can be complicated by long-term impairment in immune function. Among regulatory T (Treg) cells, the tumor necrosis factor receptor 2 positive (TNFR2pos) Treg-cell subset endorses significant immunosuppressive functions in human tumors and a sepsis mouse model but has not been investigated during septic shock in humans. METHODS: We prospectively enrolled patients with septic shock hospitalized in intensive care units (ICU). We performed immunophenotyping and functional tests of CD4+ T cells, Treg cells, and TNFR2pos Treg cells on blood samples collected 1, 4, and 7 days after admission to ICU. RESULTS: We investigated 10 patients with septic shock compared to 10 healthy controls. Although the proportions of circulating Treg cells and TNFR2pos Treg-cell subsets were not increased, their CTLA4 expression and suppressive functions in vitro were increased at 4 days of septic shock. Peripheral blood mononuclear cells from healthy donors cultured with serum from septic shock patients had increased CTLA4 expression in TNFR2pos Treg cells compared to TNFR2neg Treg cells. CONCLUSIONS: In patients with septic shock, CTLA4 expression and suppressive function were increased in circulating TNFR2pos Treg cells. We identify TNFR2pos Treg cells as a potential attractive target for therapeutic intervention.

Tumor necrosis factor receptor family costimulation increases regulatory T-cell activation and function via NF-κB.

Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3+ regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.

Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Play a Major Role in CD4+ T-Cell Impairment During Sepsis.

Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.

The TNF/TNFR2 signaling pathway is a key regulatory factor in endothelial progenitor cell immunosuppressive effect.

BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).

Diagnostic value of frozen section examination of sentinel lymph nodes in early-stage cervical cancer at the time of ultrastaging.

OBJECTIVES: We aimed to assess the diagnostic value of frozen-section pathologic examination (FSE) of sentinel lymph nodes (SLN) in patients with early-stage cervical cancer. METHODS: Two French prospective multicentric database on SLN biopsy for cervical cancer (SENTICOL I and II) were analysed. Patients with IA to IIA1 2018 FIGO stage, who underwent SLN biopsy with both FSE and ultrastaging examination were included. RESULTS AND DISCUSSION: Between 2005 and 2012, 313 patients from 25 centers fulfilled the inclusion criteria. Metastatic involvement of SLN was diagnosed in 52 patients (16.6%). Macrometastases, micrometastases and isolated tumor cells (ITCs) were found in 27, 12 and 13 patients respectively. Among the 928 SLNs analysed, FSE identified 23 SLNs with macrometastases in 20 patients and 5 SLNs with micrometastases in 2 patients whereas no ITCs were identified. Ultrastaging of negative SLNs by FSE found macrometastases, micrometastases and ITCs in additional 7, 11 and 17 SLNs. Ultrastaging increased significantly the rate of patients with positive SLN from 7% to 16.6% (p < 0.0001). The sensitivity and the negative predictive value of FSE were 42.3% and 89.7% respectively or 56.4% and 94.1% if ITCs were excluded. False-negative cases were more frequent with tumor size ≥ 20 mm (OR = 4.46, 95%IC = [1.45-13.66], p = 0.01) and preoperative brachytherapy (OR = 4.47, 95%IC = [1.37-14.63], p = 0.01) and less frequent with patients included in higher volume center (>5 patients/year) (OR = 0.09, 95%IC = [0.02-0.51], p = 0.01). CONCLUSIONS: FSE of SLN had a low sensitivity for detecting micrometastases and ITCs and a high negative predictive value for SLN status. Clinical impact of false-negative cases has to be assessed by further studies.

Surface currents in the Alderney Race from high-frequency radar measurements and three-dimensional modelling.

Two weeks of high-frequency radar measurements collected at the Alderney Race are compared with the results of a three-dimensional fully coupled wave-current model. Spatial current measurements are rare in this site, otherwise well investigated through modelling. Thus, the radar measurements offer a unique opportunity to examine the spatial reliability of numerical results, and can help to improve our understanding of the complex currents in the area. Comparison of observed and modelled surface current velocities showed a good agreement between the methods, represented by root mean squared errors ranging from 14 to 40 cm s-1 and from 18 to 60 cm s-1 during neap and spring tides, respectively. Maximum errors were found in shallow regions with consistently high current velocities, represented by mean neap and spring magnitudes of 1.25 m s-1 and 2.7 m s-1, respectively. Part of the differences between modelled and observed surface currents in these areas are thought to derive from limitations in the k-epsilon turbulence model used to simulate vertical mixing, when the horizontal turbulent transport is high. In addition, radar radial currents showed increased variance over the same regions, and might also be contributing to the discrepancies found. Correlation analyses yielded magnitudes above 0.95 over the entire study area, with better agreement during spring than during neap tides, probably because of an increase in the phase lag between radar and model velocities during the latter. This article is part of the theme issue 'New insights on tidal dynamics and tidal energy harvesting in the Alderney Race'.

Patterns and drivers of genetic diversity and structure in the biological control parasitoid Habrobracon hebetor in Niger.

When a promising natural enemy of a key pest exists locally, it is a common practice in biological control (BC) to rear and release it for supplementary control in the targeted agroecosystem even though significant knowledge gaps concerning pre/post release may still exist. Incorporating genetic information into BC research fills some of these gaps. Habrobracon hebetor, a parasitoid of many economically important moths that infest stored and field crops worldwide is commonly used, particularly against the millet head miner (MHM), a key pest of millet in Sahelian countries. To advance our knowledge on how H. hebetor that occurs naturally in open-field cropping systems and grain stores as well as being mass-produced and released for MHM control, performs in millet agroecosystems in Niger we evaluated its population genetics using two mitochondrial and 21 microsatellite markers. The field samples were genetically more diverse and displayed heterozygote excess. Very few field samples had faced significant recent demographic bottlenecks. The mating system (i.e. nonrandom mating with complementary sex determination) of this species may be the major driver of these findings rather than bottlenecks caused by the small number of individuals released and the scarcity of hosts during the longlasting dry season in Niger. H. hebetor population structure was represented by several small patches and genetically distinct individuals. Gene flow occurred at local and regional scales through human-mediated and natural short-distance dispersal. These findings highlight the importance of the mating system in the genetic diversity and structure of H. hebetor populations, and contribute to our understanding of its reported efficacy against MHM in pearl millet fields.

Control of GVHD by regulatory T cells depends on TNF produced by T cells and TNFR2 expressed by regulatory T cells.

Therapeutic CD4(+)Foxp3(+) natural regulatory T cells (Tregs) can control experimental graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) by suppressing conventional T cells (Tconvs). Treg-based therapies are currently tested in clinical trials with promising preliminary results in allo-HCT. Here, we hypothesized that as Tregs are capable of modulating Tconv response, it is likely that the inflammatory environment and particularly donor T cells are also capable of influencing Treg function. Indeed, previous findings in autoimmune diabetes revealed a feedback mechanism that renders Tconvs able to stimulate Tregs by a mechanism that was partially dependent on tumor necrosis factor (TNF). We tested this phenomenon during alloimmune response in our previously described model of GVHD protection using antigen specific Tregs. Using different experimental approaches, we observed that control of GVHD by Tregs was fully abolished by blocking TNF receptor type 2 (TNFR2) or by using TNF-deficient donor T cells or TNFR2-deficient Tregs. Thus, our results show that Tconvs exert a powerful modulatory activity on therapeutic Tregs and clearly demonstrate that the sole defect of TNF production by donor T cells was sufficient to completely abolish the Treg suppressive effect in GVHD. Importantly, our findings expand the understanding of one of the central components of Treg action, the inflammatory context, and support that targeting TNF/TNFR2 interaction represents an opportunity to efficiently modulate alloreactivity in allo-HCT to either exacerbate it for a powerful antileukemic effect or reduce it to control GVHD.

Treatment of Uveitis by In Situ Administration of Ex Vivo-Activated Polyclonal Regulatory T Cells.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell therapy is a promising approach for the treatment of autoimmune diseases. To be effective, Treg cells should be in an activated state in the target tissue. This can be achieved by systemic administration of Ag-specific Treg cells, which are difficult to produce in conditions that can be translated to the clinic. In this paper, we propose an alternative approach consisting of in situ injection of preactivated polyclonal Treg cells that would exert bystander suppression in the target tissue. We show that polyclonal Treg cells suppressed uveitis in mice as efficiently as Ag-specific Treg cells but only when preactivated and administered in the vitreous. Uveitis control was correlated with an increase of IL-10 and a decrease of reactive oxygen species produced by immune cell infiltrates in the eye. Thus, our results reveal a new mechanism of Treg cell-mediated suppression and a new Treg cell therapy approach.

Bank vole immunoheterogeneity may limit Nephropatia Epidemica emergence in a French non-endemic region.

Ecoevolutionary processes affecting hosts, vectors and pathogens are important drivers of zoonotic disease emergence. In this study, we focused on nephropathia epidemica (NE), which is caused by Puumala hantavirus (PUUV) whose natural reservoir is the bank vole, Myodes glareolus. We questioned the possibility of NE emergence in a French region that is considered to be NE-free but that is adjacent to a NE-endemic region. We first confirmed the epidemiology of these two regions and we demonstrated the absence of spatial barriers that could have limited dispersal, and consequently, the spread of PUUV into the NE-free region. We next tested whether regional immunoheterogeneity could impact PUUV chances to circulate and persist in the NE-free region. We showed that bank voles from the NE-free region were sensitive to experimental PUUV infection. We observed high levels of immunoheterogeneity between individuals and also between regions. Antiviral gene expression (Tnf and Mx2) reached higher levels in bank voles from the NE-free region. During experimental infections, anti-PUUV antibody production was higher in bank voles from the NE-endemic region. These results indicated a lower susceptibility to PUUV for bank voles from this NE-free region, which might limit PUUV persistence and therefore, the risk of NE.

Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction.

The dynamics of CD4(+) effector T cells (Teff cells) and CD4(+)Foxp3(+) regulatory T cells (Treg cells) during diabetes progression in nonobese diabetic mice was investigated to determine whether an imbalance of Treg cells and Teff cells contributes to the development of type 1 diabetes. Our results demonstrated a progressive decrease in the Treg cell:Teff cell ratio in inflamed islets but not in pancreatic lymph nodes. Intra-islet Treg cells expressed reduced amounts of CD25 and Bcl-2, suggesting that their decline was due to increased apoptosis. Additionally, administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabetes. Together, these results suggest intra-islet Treg cell dysfunction secondary to defective IL-2 production is a root cause of the progressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice.

Effector T cells boost regulatory T cell expansion by IL-2, TNF, OX40, and plasmacytoid dendritic cells depending on the immune context.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a major role in peripheral tolerance. Multiple environmental factors and cell types affect their biology. Among them, activated effector CD4(+) T cells can boost Treg cell expansion through TNF or IL-2. In this study, we further characterized this effector T (Teff) cell-dependent Treg cell boost in vivo in mice. This phenomenon was observed when both Treg and Teff cells were activated by their cognate Ag, with the latter being the same or different. Also, when Treg cells highly proliferated on their own, there was no additional Treg cell boost by Teff cells. In a condition of low inflammation, the Teff cell-mediated Treg cell boost involved TNF, OX40L, and plasmacytoid dendritic cells, whereas in a condition of high inflammation, it involved TNF and IL-2. Thus, this feedback mechanism in which Treg cells are highly activated by their Teff cell counterparts depends on the immune context for its effectiveness and mechanism. This Teff cell-dependent Treg cell boost may be crucial to limit inflammatory and autoimmune responses.

How the truffle got its mate: insights from genetic structure in spontaneous and planted Mediterranean populations of Tuber melanosporum.

The life cycles and dispersal of edible fungi are still poorly known, thus limiting our understanding of their evolution and domestication. The prized Tuber melanosporum produces fruitbodies (fleshy organs where meiospores mature) gathered in natural, spontaneously inoculated forests or harvested in plantations of nursery-inoculated trees. Yet, how fruitbodies are formed remains unclear, thus limiting yields, and how current domestication attempts affect population genetic structure is overlooked. Fruitbodies result from mating between two haploid individuals: the maternal parent forms the flesh and the meiospores, while the paternal parent only contributes to the meiospores. We analyzed the genetic diversity of T. melanosporum comparatively in spontaneous forests vs. plantations, using SSR polymorphism of 950 samples from South-East France. All populations displayed strong genetic isolation by distance at the metric scale, possibly due to animal dispersal, meiospore persistence in soil, and/or exclusion of unrelated individuals by vegetative incompatibility. High inbreeding was consistently found, suggesting that parents often develop from meiospores produced by the same fruitbody. Unlike maternal genotypes, paternal mycelia contributed to few fruitbodies each, did not persist over years, and were undetectable on tree mycorrhizae. Thus, we postulate that germlings from the soil spore bank act as paternal partners. Paternal genetic diversity and outbreeding were higher in plantations than in spontaneous truffle-grounds, perhaps because truffle growers disperse fruitbodies to maintain inoculation in plantations. However, planted and spontaneous populations were not genetically isolated, so that T. melanosporum illustrates an early step of domestication where genetic structure remains little affected.

In vivo activation of transferred regulatory T cells specific for third-party exogenous antigen controls GVH disease in mice.

Treg cells hold enormous promise for therapeutic application in GVH disease, a lethal complication of allogeneic HSC transplantation. Mouse studies showed that donor-derived recipient-specific Treg (rsTreg) cells are far more efficient than polyclonal Treg cells in suppressing GVH disease. However, clinical grade preparations of rsTreg cells carries the risk of containing significant numbers of highly pathogenic recipient-specific effector T cells. We hypothesized that an alternative approach using Treg cells specific for an exogenous (i.e. nondonor, nonrecipient) Ag (exoTreg cells) can overcome this risk by taking advantage of the bystander suppressive effect of Treg cells. For this, we used a murine model for aggressive GVH disease. We expanded ex vivo exoTreg cells that are primed against the HY Ag, which is only expressed in males. ExoTreg cells supressed GVH disease as efficiently as rsTreg cells in recipient male mice. We also applied this strategy in female mice that do not express this Ag. While exoTreg cells were not effective in female recipients when applied alone, providing the cognate HY Ag in vivo along side effectively activated exoTreg cells and completely abrogated GVH disease, establishing a targeted on/off system to provide a suppressive effect on alloreactive effector T cells.

Evolutionary history of almond tree domestication in the Mediterranean basin.

Genetic diversity of contemporary domesticated species is shaped by both natural and human-driven processes. However, until now, little is known about how domestication has imprinted the variation of fruit tree species. In this study, we reconstruct the recent evolutionary history of the domesticated almond tree, Prunus dulcis, around the Mediterranean basin, using a combination of nuclear and chloroplast microsatellites [i.e. simple sequence repeat (SSRs)] to investigate patterns of genetic diversity. Whereas conservative chloroplast SSRs show a widespread haplotype and rare locally distributed variants, nuclear SSRs show a pattern of isolation by distance with clines of diversity from the East to the West of the Mediterranean basin, while Bayesian genetic clustering reveals a substantial longitudinal genetic structure. Both kinds of markers thus support a single domestication event, in the eastern side of the Mediterranean basin. In addition, model-based estimation of the timing of genetic divergence among those clusters is estimated sometime during the Holocene, a result that is compatible with human-mediated dispersal of almond tree out of its centre of origin. Still, the detection of region-specific alleles suggests that gene flow from relictual wild preglacial populations (in North Africa) or from wild counterparts (in the Near East) could account for a fraction of the diversity observed.