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The thermal resistance of a wall can be readily measured in steady-state. However, such a state is seldomly achieved in a building because of the variation of outdoor conditions as well as the high thermal inertia of building materials. This paper introduces a novel active (dynamic) method to measure the thermal resistance of a building wall. Not only are active approaches less sensitive to external temperature variations, they also enable to perform measurements within only a few hours. In the proposed methodology, an artificial thermal load is applied to a wall (heating of the indoor air) and its thermal response is monitored. Inverse techniques are used with a reduced model to estimate the value of the thermal resistance of a wall from the measured temperatures and heat fluxes. The methodology was validated on a known load-bearing wall built inside a climate chamber. The results were in good agreement with reference values derived from a steady-state characterization of the wall. The method also demonstrated a good reproducibility.

The nervous and immune systems are the primary sensory interfaces of the body, allowing it to recognize, process, and respond to various stimuli from both the external and internal environment. These systems work in concert through various mechanisms of neuro-immune crosstalk to detect threats, provide defense against pathogens, and maintain or restore homeostasis, but can also contribute to the development of diseases. Among peripheral sensory neurons (PSNs), nociceptive PSNs are of particular interest. They possess a remarkable capability to detect noxious stimuli in the periphery and transmit this information to the brain, resulting in the perception of pain and the activation of adaptive responses. Pain is an early symptom of cancer, often leading to its diagnosis, but it is also a major source of distress for patients as the disease progresses. In this review, we aim to provide an overview of the mechanisms within tumors that are likely to induce cancer pain, exploring a range of factors from etiological elements to cellular and molecular mediators. In addition to transmitting sensory information to the central nervous system, PSNs are also capable, when activated, to produce and release neuropeptides (e.g., CGRP and SP) from their peripheral terminals. These neuropeptides have been shown to modulate immunity in cases of inflammation, infection, and cancer. PSNs, often found within solid tumors, are likely to play a significant role in the tumor microenvironment, potentially influencing both tumor growth and anti-tumor immune responses. In this review, we discuss the current state of knowledge about the degree of sensory innervation in tumors. We also seek to understand whether and how PSNs may influence the tumor growth and associated anti-tumor immunity in different mouse models of cancer. Finally, we discuss the extent to which the tumor is able to influence the development and functions of the PSNs that innervate it., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mardelle, Bretaud, Daher and Feuillet.)

The nervous and immune systems have classically been studied as separate entities, but there is now mounting evidence for bidirectional communication between them in various organs, including the skin. The skin is an epithelial tissue with important sensory and immune functions. The skin is highly innervated with specialized subclasses of primary sensory neurons (PSNs) that can be in contact with skin-resident innate and adaptive immune cells. Neuroimmune crosstalk in the skin, through interactions of PSNs with the immune system, has been shown to regulate host cutaneous defense, inflammation, and tissue repair. Here, we review current knowledge about the cellular and molecular mechanisms involved in this crosstalk, as depicted via mouse model studies. We highlight the ways in which different immune challenges engage specialized subsets of PSNs to produce mediators acting on immune cell subsets and modulating their function., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The long lapse between the presumptive origin of schizophrenia (SCZ) during early development and its diagnosis in late adolescence has hindered the study of crucial neurodevelopmental processes directly in living patients. Dopamine, a neurotransmitter consistently associated with the pathophysiology of SCZ, participates in several aspects of brain development including pruning of neuronal extensions. Excessive pruning is considered the cause of the most consistent finding in SCZ, namely decreased brain volume. It is therefore possible that patients with SCZ carry an increased susceptibility to dopamine's pruning effects and that this susceptibility would be more obvious in the early stages of neuronal development when dopamine pruning effects appear to be more prominent. Obtaining developing neurons from living patients is not feasible. Instead, we used Monocyte-Derived-Neuronal-like Cells (MDNCs) as these cells can be generated in only 20 days and deliver reproducible results. In this study, we expanded the number of individuals in whom we tested the reproducibility of MDNCs. We also deepened the characterization of MDNCs by comparing its neurostructure to that of human developing neurons. Moreover, we studied MDNCs from 12 controls and 13 patients with SCZ. Patients' cells differentiate more efficiently, extend longer secondary neurites and grow more primary neurites. In addition, MDNCs from medicated patients expresses less D1R and prune more primary neurites when exposed to dopamine. Haloperidol did not influence our results but the role of other antipsychotics was not examined and thus, needs to be considered as a confounder., (© 2022. The Author(s).)

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFβ-dependent and mediated by SMAD7, a TGFβ- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFβ superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

T lymphocyte migration is an essential step to mounting an efficient immune response. The rapid and random motility of these cells which favors their sentinel role is conditioned by chemokines as well as by the physical environment. Morphological changes, underlaid by dynamic actin cytoskeleton remodeling, are observed throughout migration but especially when the cell modifies its trajectory. However, the signaling cascade regulating the directional changes remains largely unknown. Using dynamic cell imaging, we investigated in this paper the signaling pathways involved in T cell directionality. We monitored cyclic adenosine 3'-5' monosphosphate (cAMP) variation concomitantly with actomyosin distribution upon T lymphocyte migration and highlighted the fact that spontaneous bursts in cAMP starting from the leading edge, are sufficient to promote actomyosin redistribution triggering trajectory modification. Although cAMP is commonly considered as an immunosuppressive factor, our results suggest that, when transient, it rather favors the exploratory behavior of T cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Simao, Régnier, Taheraly, Fraisse, Tacine, Fraudeau, Benabid, Feuillet, Lambert, Delon and Randriamampita.)

The majority of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals remain paucisymptomatic, contrasting with a minority of infected individuals in danger of death. Here, we speculate that the robust disease resistance of most individuals is due to a swift production of type I interferon (IFNα/β), presumably sufficient to lower the viremia. A minority of infected individuals with a preexisting chronic inflammatory state fail to mount this early efficient response, leading to a delayed harmful inflammatory response. To improve the epidemiological scenario, we propose combining: (i) the development of efficient antivirals administered early enough to assist in the production of endogenous IFNα/β; (ii) potentiating early IFN responses; (iii) administering anti-inflammatory treatments when needed, but not too early to interfere with endogenous antiviral responses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

β-Adrenergic receptor (β-AR) signaling exerts protumoral effects by acting directly on tumor cells and angiogenesis. In addition, β-AR expression on immune cells affects their ability to mount antitumor immune responses. However, how β-AR signaling impinges antitumor immune responses is still unclear. Using a mouse model of vaccine-based immunotherapy, we showed that propranolol, a nonselective β-blocker, strongly improved the efficacy of an antitumor STxBE7 vaccine by enhancing the frequency of CD8 + T lymphocytes infiltrating the tumor (TIL). However, propranolol had no effect on the reactivity of CD8 + TILs, a result further strengthened by ex vivo experiments showing that these cells were insensitive to adrenaline- or noradrenaline-induced AR signaling. In contrast, naïve CD8 + T-cell activation was strongly inhibited by β-AR signaling, and the beneficial effect of propranolol mainly occurred during CD8 + T-cell priming in the tumor-draining lymph node. We also demonstrated that the differential sensitivity of naïve CD8 + T cells and CD8 + TILs to β-AR signaling was linked to a strong downregulation of β 2 -AR expression related to their activation status, since in vitro -activated CD8 + T cells behaved similarly to CD8 + TILs. These results revealed that β-AR signaling suppresses the initial priming phase of antitumor CD8 + T-cell responses, providing a rationale to use clinically available β-blockers in patients to improve cancer immunotherapies., (©2019 American Association for Cancer Research.)

Type I interferons (IFN) are being rediscovered as potent anti-tumoral agents. Activation of the STimulator of INterferon Genes (STING) by DMXAA (5,6-dimethylxanthenone-4-acetic acid) can induce strong production of IFNα/β and rejection of transplanted primary tumors. In the present study, we address whether targeting STING with DMXAA also leads to the regression of spontaneous MMTV-PyMT mammary tumors. We show that these tumors are refractory to DMXAA-induced regression. This is due to a blockade in the phosphorylation of IRF3 and the ensuing IFNα/β production. Mechanistically, we identify TGFβ, which is abundant in spontaneous tumors, as a key molecule limiting this IFN-induced tumor regression by DMXAA. Finally, blocking TGFβ restores the production of IFNα by activated MHCII + tumor-associated macrophages, and enables tumor regression induced by STING activation. On the basis of these findings, we propose that type I IFN-dependent cancer therapies could be greatly improved by combinations including the blockade of TGFβ.

Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ + CD39 + CD73 + Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Regressing tumors are usually associated with a large immune infiltrate, but the molecular and cellular interactions that govern a successful anti-tumor immunity remain elusive. Here, we have triggered type I Interferon (IFN) signaling in a breast tumor model (MMTV-PyMT) using 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a ligand of the STimulator of Interferon Genes, STING. The 2 main events rapidly triggered by DMXAA in transplanted PyMT tumors are 1) the disruption of the tumor vasculature, followed by hypoxia and cell death; 2) the release of chemokines. Both events converged to trigger the recruitment of 2 waves of immune cells: a swift, massive recruitment of neutrophils, followed by a delayed rise in monocytes and CD8 T cells in the tumor mass. Depletion experiments in vivo revealed that myeloid cell subsets and T cells need to cooperate to achieve full-blown recruitment and activation at the tumor site and to induce effective secondary cell death leading to tumor regression (Illustration 1). Altogether, our study highlights that the tumor regression induced by the STING agonist DMXAA results from a cascade of events, with an initial vessel destruction followed by several infiltration waves of immune cells which have to cooperate to amplify and sustain the initial effect. We thus provide the first global and detailed kinetic analysis of the anti-tumoral effect of DMXAA and of its different articulated steps.

Apoptotic cells represent an important source of self-antigens and their engulfment by dendritic cells (DCs) is usually considered to be related to tolerance induction. We report here an unexpectedly high level of human CD4(+) T-cell proliferation induced by autologous DCs loaded with autologous apoptotic cells, due to the activation of more than 10% of naive CD4(+) T cells. This proliferation is not due to an increase in the costimulatory capacity of DCs, but is dependent on apoptotic cell-associated material processed through an endo-lysosomal pathway and presented on DC MHC class II molecules. Autologous CD4(+) T cells stimulated with apoptotic cell-loaded DCs exhibit suppressive capacities. However, in the presence of bacterial lipopolysaccharide, apoptotic cell-loaded DCs induce the generation of IL-17-producing cells. Thus, apoptotic cell engulfment by DCs may lead to increased autologous responses, initially generating CD4(+) T cells with suppressive capacities able to differentiate into Th17 cells in the presence of a bacterial danger signal such as LPS., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In mice, CD8α(+) myeloid dendritic cells (mDC) optimally cross-present Ags to CD8(+) T cells and respond strongly to TLR3 ligands. Although equivalent DC have been identified by comparative genomic analysis and functional studies in humans as XCR1(+)CD141 (BDCA-3)(+)Clec9A(+)cell adhesion molecule 1(+) mDC, and in sheep as CD26(+) mDC, these cells remained elusive in nonhuman primates. To remedy this situation, we delineated precisely DC and monocyte populations by 12-color flow cytometry and transcriptomic analyses in healthy rhesus macaques. We identified a new mDC population, with strong phenotypic and transcriptional homology to human CD141(+) and murine CD8α(+) mDC, including XCR1 membrane expression as a conserved specific marker. In contrast, high CD11c expression was not characteristic of mDC in macaques, but of CD16(+) monocytes. Like their human and murine homologs, simian XCR1(+) mDC had much stronger responses to TLR3 stimulation than other myeloid cells. The importance of this new mDC population was tested in SIV(mac251) infection, the most relevant animal model for pathogenic HIV-1 infection and vaccination. This population increased sharply and transiently during acute infection, but was reduced in blood and spleen during advanced disease. The identification of XCR1(+) mDC in rhesus macaques opens new avenues for future preclinical vaccinal studies and highlights XCR1 as a prime candidate for targeted vaccine delivery.

HIV infects activated CD4⁺ T cells and induces their depletion. Progressive HIV infection leading to AIDS is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFα. This has been related to intestinal epithelial damage and microbial LPS translocation into the circulation. Using 11-color flow cytometry, cell sorting, and cell culture, we investigated the numbers and TNFα production of fully defined circulating dendritic cell and monocyte populations during HIV-1 infection. In 15 viremic, untreated patients, compared with 8 treated, virologically suppressed patients or to 13 healthy blood donors, circulating CD141 (BDCA-3)⁺ and CD1c (BDCA-1)⁺ dendritic cell counts were reduced. Conversely, CD14⁺ CD16⁺⁺ monocyte counts were increased, particularly those expressing M-DC8, while classical CD14⁺⁺ CD16⁻ M-DC8⁻ monocyte numbers were unchanged. Blood mononuclear cells from viremic patients produced more TNFα in response to LPS than those from virologically suppressed patients. M-DC8⁺ monocytes were mostly responsible for this overproduction. Moreover, M-DC8⁺ monocytes differentiated in vitro from classical monocytes using M-CSF and GM-CSF, which is increased in viremic patient's plasma. This M-DC8⁺ monocyte population, which is involved in the pathogenesis of chronic inflammatory diseases like Crohn disease, might thus be considered as a major actor in the immune hyperactivation fueling HIV infection progression.

The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1(-/-) HSC are impaired, suggesting that the ERK1(-/-)-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1(-/-) defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments.

Dendritic cells (DC) are able to elicit anti-tumoral CD8(+) T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8(+) T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8(+) T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy.

Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8alpha+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8alpha+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8alpha+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1-/- mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8alpha+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria.

Cross-presentation is an essential mechanism that allows dendritic cells (DCs) to efficiently present exogenous antigens to CD8(+) T cells. Among cellular antigen sources, apoptotic cells are commonly considered as the best for cross-presentation by DCs. However, the potential of live cells as a source of antigen has been overlooked. Here we explored whether DCs were able to capture and cross-present antigens from live cells. DCs internalized cytosolic and membrane material into vesicles from metabolically labeled live cells. Using time-lapse confocal microscopy in whole spleens, we showed that DCs internalized material from live cells in vivo. After ovalbumin uptake from live cells, DCs cross-primed ovalbumin-specific naive OT-I CD8(+) T cells in vitro. Injected into mice previously transferred with naive OT-I T cells, they also cross-primed in vivo, even in the absence of endogenous DCs able to present the epitope in the recipient mice. Interestingly, DCs induced stronger natural CD8(+) T-cell responses and protection against a lethal tumor challenge after capture of antigens from live melanoma cells than from apoptotic melanoma cells. The potential for cross-presentation from live cells uncovers a new type of cellular intercommunication and must be taken into account for induction of tolerance or immunity against self, tumors, grafts, or pathogens.

Toll-like receptors (TLRs) are key components of the immune system that detect microbial infection and trigger antimicrobial host defense responses. TLR5 is a sensor for monomeric flagellin, which is a component of bacterial flagella known to be a virulence factor. In this study we generated TLR5-deficient mice and investigated the role of TLR5 signaling in the detection of flagellin and antibacterial immune responses to Salmonella typhimurium and Pseudomonas aeruginosa. We found that TLR5 is essential for the recognition of bacterial flagellin both in vivo and ex vivo. TLR5 contribution to antibacterial host response to i.p. infection with S. typhimurium or intranasal administration of P. aeruginosa may be masked by TLR4 or other sensing mechanisms. By using radiation bone marrow chimera, we showed that upon i.p. injection of flagellin immune responses are mediated by lymphoid cells, whereas resident cells are required for the initiation of response upon intranasal flagellin administration. These results suggest that flagellin recognition in different organs is mediated by distinct TLR5-expressing cells and provide insights into the cooperation of the TLR5 and TLR4 signaling pathways used by the innate immune system in the recognition of bacterial pathogens.

The molecular mechanisms by which dendritic cells (DC) favor naive T cell survival in mice have been examined in co-cultures of DC and naive CD4+ T cells. Naive T cells can survive in the presence of IL-4 or IL-7, but DC-induced T cell survival requires direct cell-cell interactions and does not seem to be mediated by these or other soluble factors. Classical MHC II molecules on DC are not necessary for T cell survival as long as hybrid AalphaEbeta MHC class II molecules are present. In the total absence of MHC II molecules on DC, T cell survival is reduced by half, and CD3zeta phosphorylation fully disappears. These results contrast with the classical view that naive T cell survival is associated with CD3zeta phosphorylation and depends mostly on IL-7 and MHC-TCR interactions. We demonstrate that DC-induced T cell survival is a multi-factorial process that also involves CD28, LFA-1 and another (as yet undefined) surface molecule that requires the activity of src (but not phosphatidylinositol-3-) kinase.

The structure of immunological synapses formed between murine naive T cells and mature dendritic cells has been subjected to a quantitative analysis. Immunofluorescence images of synapses formed in the absence of antigen show a diffuse synaptic accumulation of CD3 and LFA-1. In electron microscopy, these antigen-free synapses present a number of tight appositions (cleft size approximately 15 nm), all along the synapse. These tight appositions cover a significantly larger surface fraction of antigen-dependent synapses. In immunofluorescence, antigen-dependent synapses show multiple patches of CD3 and LFA-1 with a variable overlap. A similar distribution is observed for PKCtheta and talin. A concentric organization characteristic of prototypical synapses is rarely observed, even when dendritic cells are paralyzed by cytoskeletal poisons. In T-DC synapses, the interaction surface is composed of several tens of submicronic contact spots, with no large-scale segregation of CD3 and LFA-1. As a comparison, in T-B synapses, a central cluster of CD3 is frequently observed by immunofluorescence, and electron microscopy reveals a central tight apposition. Our data show that it is inappropriate to consider the concentric structure as a "mature synapse" and multifocal structures as immature.

Sam68 phosphorylation correlates with Fyn but not Lck expression in T cells. This substrate has been used here to explore the possible basis of the specificity of Fyn versus Lck. We show that this specificity is not based on a spatial segregation of the two kinases, since a chimeric Lck molecule containing the membrane anchoring domain of Fyn does not phosphorylate Sam68. Moreover, a Sam68 molecule targeted to the plasma membrane by the farnesylation signal of c-Ha-Ras remains poorly phosphorylated by Lck. In T cells, Fyn appears to be the active Src kinase in rafts, but Sam68 is not expressed in rafts, and its distinct phosphorylation by Fyn and Lck is not affected by raft dispersion. The Fyn/Lck specificity does not reflect a higher kinase activity of Fyn in general, as both Fyn and Lck are similarly recognized by an anti-active Src antibody. Both also strongly phosphorylate another Src substrate in vivo. Mainly, Lck phosphorylates Sam68 when the interaction between the SH3 domain and the SH2-catalytic domain linker is altered in heterologous Src molecules or after mutating key residues in the linker that increase the accessibility of the SH3 domain. Thus, the distinct potential of Fyn and Lck to phosphorylate Sam68 is likely controlled by the interaction of the kinase SH3 domain with the linker and Sam68, possibly on a competitive binding basis.