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Naïve T cells and regulatory T cells, when purified, do not proliferate to the γc-cytokines IL-2, IL-7, or IL-15, despite their expression of cognate cytokine receptors. Dendritic cells (DCs) enabled the T cell proliferation to these cytokines, through cell-to-cell contact, but independent of T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in DC-depleted hosts. We propose calling this a "preconditioning effect". Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2 target genes. "Preconditioning" was necessary to activate these two pathways and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation, and prolonged S6 phosphorylation occurred. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells. [ABSTRACT FROM AUTHOR]

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer’s patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells. [ABSTRACT FROM AUTHOR]

Antigen-specific therapies hold promise for treating autoimmune diseases such as multiple sclerosis while avoiding the deleterious side effects of systemic immune suppression due to delivering the disease-specific antigen as part of the treatment. In this study, an antigen-specific dual-sized microparticle (dMP) treatment reversed hind limb paralysis when administered in mice with advanced experimental autoimmune encephalomyelitis (EAE). Treatment reduced central nervous system (CNS) immune cell infiltration, demyelination, and inflammatory cytokine levels. Mechanistic insights using single-cell RNA sequencing showed that treatment impacted the MHC II antigen presentation pathway in dendritic cells, macrophages, B cells, and microglia, not only in the draining lymph nodes but also strikingly in the spinal cord. CD74 and cathepsin S were among the common genes down-regulated in most antigen presenting cell (APC) clusters, with B cells also having numerous MHC II genes reduced. Efficacy of the treatment diminished when B cells were absent, suggesting their impact in this therapy, in concert with other immune populations. Activation and inflammation were reduced in both APCs and T cells. This promising antigen-specific therapeutic approach advantageously engaged essential components of both innate and adaptive autoimmune responses and capably reversed paralysis in advanced EAE without the use of a broad immunosuppressant. [ABSTRACT FROM AUTHOR]

Thyroid-associated ophthalmopathy (TAO) represents a disfiguring and potentially blinding autoimmune component of Graves' disease. It appears to be driven, at least in part, by autoantibodies targeting the thyrotropin receptor (TSHR)/insulin-like growth factor I receptor (IGF-IR) complex. Actions mediated through either TSHR or IGF-IR are dependent on IGF-IR activity. CD34+ fibrocytes, monocyte lineage cells, reside uniquely in the TAO orbit, where they masquerade as CD34+ orbital fibroblasts. Fibrocytes present antigens to T cells through their display of the major histocompatibility complex class II (MHC II) while providing costimulation through B7 proteins (CD80, CD86, and programmed death-ligand 1 [PD-L1]). Here, we demonstrate that teprotumumab, an anti-IGF-IR inhibitor, attenuates constitutive expression and induction by the thyroid-stimulating hormone of MHC II and these B7 members in CD34+ fibrocytes. These actions are mediated through reduction of respective gene transcriptional activity. Other IGF-IR inhibitors (1H7 and linsitinib) and knocking down IGF-IR gene expression had similar effects. Interrogation of circulating fibrocytes collected from patients with TAO, prior to and following teprotumumab treatment in vivo during a phase 2 clinical trial, demonstrated reductions in cell-surface MHC II and B7 proteins similar to those found following IGF-IR inhibitor treatment in vitro. Teprotumumab therapy reduces levels of interferon-1 and IL-17A expression in circulating CD4+ T cells, effects that may be indirect and mediated through actions of the drug on fibrocytes. Teprotumumab was approved by the US Food and Drug Administration for TAO. Our current findings identify potential mechanisms through which teprotumumab might be eliciting its clinical response systemically in patients with TAO, potentially by restoring immune tolerance. [ABSTRACT FROM AUTHOR]

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membraneanchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy. [ABSTRACT FROM AUTHOR]

Intracellular delivery of messenger RNA (mRNA)-based cancer vaccine has shown great potential to elicit antitumor immunity. To achieve robust antitumor efficacy, mRNA encoding tumor antigens needs to be efficiently delivered and translated in dendritic cells with concurrent innate immune stimulation to promote antigen presentation. Here, by screening a group of cationic lipid-like materials, we developed a minimalist nanovaccine with C1 lipid nanoparticle (LNP) that could efficiently deliver mRNA in antigen presenting cells with simultaneous Toll-like receptor 4 (TLR4) activation and induced robust T cell activation. The C1 nanovaccine entered cells via phagocytosis and showed efficient mRNAencoded antigen expression and presentation. Furthermore, the C1 lipid nanoparticle itself induced the expression of inflammatory cytokines such as IL-12 via stimulating TLR4 signal pathway in dendritic cells. Importantly, the C1 mRNA nanovaccine exhibited significant antitumor efficacy in both tumor prevention and therapeutic vaccine settings. Overall, our work presents a C1 LNPbased mRNA cancer nanovaccine with efficient antigen expression as well as self-adjuvant property, which may provide a platform for developing cancer immunotherapy for a wide range of tumor types. [ABSTRACT FROM AUTHOR]

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5'-3' direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a singlemolecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) claims 1.5 million lives per year. This situation is largely due to the low efficacy of the only licensed TB vaccine, Bacillus Calmette–Guérin (BCG) against pulmonary TB. The metabolic disease type 2 diabetes (T2D) is a risk factor for TB and the mechanisms underlying increased TB susceptibility in T2D are not well understood. Furthermore, it is unknown if new TB vaccines will provide protection in the context of T2D. Here we used a diet-induced murine model of T2D to investigate the underlying mechanisms of TB/T2D comorbidity and to evaluate the protective capacity of two experimental TB vaccines in comparison to conventional BCG. Our data reveal a distinct immune dysfunction that is associated with diminished recognition of mycobacterial antigens in T2D. More importantly, we provide compelling evidence that mucosal delivery of recombinant BCG strains expressing the Mycobacterium tuberculosis (Mtb) ESX-1 secretion system (BCG::RD1 and BCG::RD1 ESAT-6 ∆92–95) are safe and confer superior immunity against aerosol Mtb infection in the context of T2D. Our findings suggest that the remarkable anti-TB immunity by these recombinant BCG strains is achieved via augmenting the numbers and functional capacity of antigen presenting cells in the lungs of diabetic mice. [ABSTRACT FROM AUTHOR]

Double fertilization is a key innovation for the evolutionary success of angiosperms by which the two fertilized female gametes, the egg cell and central cell, generate the embryo and endosperm, respectively. The female gametophyte (embryo sac) enclosed in the sporophyte is derived from a one-celled haploid cell lineage. It undergoes successive events of mitotic divisions, cellularization, and cell specification to give rise to the mature embryo sac, which contains the two female gametes accompanied by two types of accessory cells, namely synergids and antipodals. How the cell fate of the central cell is specified has long been equivocal and is further complicated by the structural diversity of female gametophyte across plant taxa. Here, MADS-box protein AGL80 was verified as a transcriptional repressor that directly suppresses the expression of accessory cell-specific genes to specify the central cell. Further genetic rescue and phylogenetic assay of the AGL80 orthologs revealed a possible conservedmechanism in the Brassicaceae family. Results from this study provide insight into the molecular determination of the second female gamete cell in Brassicaceae. [ABSTRACT FROM AUTHOR]

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recentwork suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes. [ABSTRACT FROM AUTHOR]

An essential feature of the adaptive immune system is the proliferation of antigen-specific lymphocytes during an immune reaction to form a large pool of effector cells. This proliferation must be regulated to ensure an effective response to infection while avoiding immunopathology. Recent experiments in mice have demonstrated that the expansion of a specific clone of T cells in response to cognate antigen obeys a striking inverse power law with respect to the initial number of T cells. Here, we show that such a relationship arises naturally from a model in which T cell expansion is limited by decaying levels of presented antigen. The same model also accounts for the observed dependence of T cell expansion on affinity for antigen and on the kinetics of antigen administration. Extending the model to address expansion of multiple T cell clones competing for antigen, we find that higher-affinity clones can suppress the proliferation of lower-affinity clones, thereby promoting the specificity of the response. Using the model to derive optimal vaccination protocols, we find that exponentially increasing antigen doses can achieve a nearly optimized response. We thus conclude that the dynamics of presented antigen is a key regulator of both the size and specificity of the adaptive immune response. [ABSTRACT FROM AUTHOR]

T follicular helper (Tfh) and regulatory (Tfr) cells are terminally differentiated cells found in germinal centers (GCs), specialized secondary lymphoid organ structures dedicated to antibody production. As such, follicular T (Tfol) cells are supposed to be specific for immunizing antigens, which has been reported for Tfh cells but is debated for Tfr cells. Here, we used high-throughput T cell receptor (TCR) sequencing to analyze the repertoires of Tfh and Tfr cells, at homeostasis and after immunization with self- or foreign antigens. We observed that, whatever the conditions, Tfh and Tfr cell repertoires are less diverse than those of effector T cells and Treg cells of the same tissues; surprisingly, these repertoires still represent thousands of different sequences, even after immunization with a single antigen that induces a 10-fold increase in Tfol cell numbers. Thorough analysis of the sharing and network of TCR sequences revealed that a specific response to the immunizing antigen can only, but hardly, be detected in Tfh cells immunized with a foreign antigen and Tfr cells immunized with a self-antigen. These antigen-specific responses are obscured by a global stimulation of Tfh and Tfr cells that appears to be antigen-independent. Altogether, our results suggest a major bystander Tfol cell activation during the immune response in the GCs. [ABSTRACT FROM AUTHOR]

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell-cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation. [ABSTRACT FROM AUTHOR]

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3' regulatory region (3'RR) in a stimulus-dependent manner. Why the 3'RR-mediated up- regulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3'RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3'RR in developing B cells. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR) triggering and subsequent T-cell activation are essential for the adaptive immune response. Recently, multiple lines of evidence have shown that force transduction across the TCR complex is involved during TCR triggering, and that the T cell might use its force-generation machinery to probe the mechanical properties of the opposing antigen-presenting cell, giving rise to different signaling and physiological responses. Mechanistically, actin polymerization and turnover have been shown to be essential for force generation by T cells, but how these actin dynamics are regulated spatiotemporally remains poorly understood. Here, we report that traction forces generated by T cells are regulated by dynamic microtubules (MTs) at the interface. These MTs suppress Rho activation, nonmuscle myosin II bipolar filament assembly, and actin retrograde flow at the T-cell-substrate interface. Our results suggest a novel role of the MT cytoskeleton in regulating force generation during T-cell activation. [ABSTRACT FROM AUTHOR]

Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II-expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide-MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow-resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box-binding protein associated with epithelial-mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6Chi monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals. [ABSTRACT FROM AUTHOR]

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that theMHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8+ cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8+ T cells and play critical roles in cytotoxic T-cell-mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8+ T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4+ T-cell activation by CD11b+ DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103hi) DCs, which immigrate from the skin, and resident (CD8αhi) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8+ T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8+ T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8+ T-cell activation. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function. [ABSTRACT FROM AUTHOR]

The differentiation of naive CD4+ T cells into distinct lineages plays critical roles in mediating adaptive immunity or maintaining immune tolerance. In addition to being a first line of defense, the innate immune system also actively instructs adaptive immunity through antigen presentation and immunoregulatory cytokine production. Here we found that sirtuin 1 (SIRT1), a type III histone deacetylase, plays an essential role in mediating proinflammatory signaling in dendritic cells (DCs), consequentially modulating the balance of proinflammatory T helper type 1 (TH1) cells and antiinflammatory Foxp3+ regulatory T cells (Treg cells). Genetic deletion of SIRT1 in DCs restrained the generation of Treg cells while driving TH1 development, resulting in an enhanced T-cell-mediated inflammation against microbial responses. Beyond this finding, SIRT1 signaled through a hypoxia-inducible factor-1 alpha (HIF1α)- dependent pathway, orchestrating the reciprocal TH1 and Treg lineage commitment through DC-derived IL-12 and TGF-β1. Our studies implicates a DC-based SIRT1-HIF1α metabolic checkpoint in controlling T-cell lineage specification. [ABSTRACT FROM AUTHOR]

γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ -deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance. [ABSTRACT FROM AUTHOR]

The reciprocal activation of dendritic cells (DCs) and natural killer cells (NKs) plays a key role in both innate and adaptive immunity. The effect of aging on this cross-talk, a critical step in virus disease control and tumor immunology, has not been reported. Splenic DCs and NKs were purified from both young and old C57BL/6 mice and cocultured in the presence of polyinosinicpolycytidylic acid (poly l:C). The resulting activation of NKs was measured as expression of CD69 and secretion of IFN-γ. However, DCs from old mice could not activate NKs from either young or old mice in vitro or in vivo. In contrast, DCs from young mice efficiently activated NKs from both young and old mice. DCs from old mice were deficient in poly I: C-stimulated secretion of IL-15, IL-18, and IFN-γ. Gene expression analysis revealed many other differences between DCs of old and young mice. Young mice strongly eradicated MHC class l-negative NK-sensitive RMA-S lymphoma mutant tumor cells, but old mice did not, in concert with the previous report that mousepox kills aged, but not young, C57BL/6 mice. Furthermore, a similar dysfunction of DC and its key role in NK activation was found in 27 out of 55 healthy human donors. [ABSTRACT FROM AUTHOR]

In primary humoral responses, B-cell lymphoma 6 (Bcl6) is a master regulator of follicular helper T (TFH) cell differentiation; however, its activation mechanisms and role in memory responses remain unclear. Here we demonstrate that survival of CXCR5+ TFH memory cells, and thus subsequent recall antibody response, require Bcl6 expression. Furthermore, we show that, upon rechallenge with soluble antigen Bcl6 in memory TFH cells is rapidly induced in a dendritic cell-independent manner and that peptide:class II complexes (pMHC) on cognate memory B cells significantly contribute to this induction. Given the previous evidence that antigen-specific B cells residing in the follicles acquire antigens within minutes of injection, our results suggest that memory B cells present antigens to the cognate TFH memory cells, thereby contributing to rapid Bcl6 reexpression and differentiation of the TFH memory cells during humoral memory responses. [ABSTRACT FROM AUTHOR]

Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8+ T cells. During early-islet infiltration, T-cell interactions with CD11c+ antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell–APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling in vitro. Using timed introduction of effector T cells, we demonstrate that elements of the autoimmune-tissue microenvironment control the dynamics of autoantigen recognition by T cells and their resulting pathogenic effector functions. [ABSTRACT FROM AUTHOR]

As sentinels of the immune system, dendritic cells (DCs) continuously generate and turnover antigenic peptide–MHC class II complexes (pMHC-II). pMHC-II generation is a complex process that involves many well-characterized MHC-II biosynthetic intermediates; however, the mechanisms leading to MHC-II turnover/degradation are poorly understood. We now show that pMHC-II complexes undergoing clathrin-independent endocytosis from the DC surface are efficiently ubiquitinated by the E3 ubiquitin ligase March-I in early endosomes, whereas biosynthetically immature MHC-II– Invariant chain (Ii) complexes are not. The inability of MHC-II–Ii to serve as a March-I substrate is a consequence of Ii sorting motifs that divert the MHC-II-Ii complex away from March-I+ early endosomes. When these sorting motifs are mutated, or when clathrin-mediated endocytosis is inhibited, MHC-II–Ii complexes internalize by using a clathrin-independent endocytosis pathway and are now ubiquitinated as efficiently as pMHC-II complexes. These data show that the selective ubiquitination of internalizing surface pMHC-II in March-I+ early endosomes promotes degradation of "old" pMHC-II and spares forms of MHC-II that have not yet loaded antigenic peptides or have not yet reached the DC surface. [ABSTRACT FROM AUTHOR]

Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during differentiation of the myelomonoblast PLB-985 cell line into "neutrophil-like" cells. Functional assays indicated that human leukocyte antigen G, the preferred ligand of ILT4, inhibited the phagocytic function of neutrophils. ILT4 engagement also impaired reactive oxygen species production induced through CD32a and both receptors were found colocalized into neutrophil lipid rafts. Moreover, neutrophil degranulation induced through inflammatory stimuli increased ILT4 expression as a result of the rapid translocation of an intracellular pool to the cell surface. Consequently to this ILT4 up-regulation, the human leukocyte antigen G-mediated inhibition of neutrophil phagocytic function was enhanced. Finally, we found that ILT4 up-regulation induced on healthy donor neutrophils following stimulation was impaired in presence of plasma from patients with sepsis. Similarly, ILT4 up-regulation was inhibited in neutrophils from septic patients. Altogether, our results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders. [ABSTRACT FROM AUTHOR]

The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FC∊RI on mast cells and basophils, and CD23 (Fc∊RII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cr3 and G 4 domains (Fa-3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for crosslinking membrane-bound trim∊RIc CD23 by IgE, or membrane IgE by soluble trim∊RIc forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single Fc∊RI binding site, which lies at the opposite ends of the CE3 domains. Structural comparisons with both free IgE-Fc and its Fc∊RI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with Fc∊RI binding, but also that the converse is true. The two binding sites are allost∊RIcally linked. We demonstrate exp∊RImentally the reciprocal inhibition of CD23 and Fc∊RI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors. [ABSTRACT FROM AUTHOR]

A coordinated innate and adaptive immune response, orchestrated by antigen presenting cells (APCs), is required for effective clearance of influenza A virus (IAV). Although IAV primarily infects epithelial cells of the upper respiratory tract, APCs are also susceptible. To determine if virus transcription in these cells is required to generate protective innate and adaptive immune responses, we engineered IAV to be selectively attenuated in cells of hematopoietic origin. Incorporation of hematopoietic-specific miR-142 target sites into the nucleoprotein of IAV effectively silenced virus transcription in APCs, but had no significant impact in lung epithelial cells. Here we demonstrate that inhibiting IAV replication in APCs in vivo did not alter clearance, or the generation of IAV-specific CD8 T cells, suggesting that cross-presentation is sufficient for cytotoxic T lymphocyte activation. In contrast, loss of in vivo virus infection, selectively in APCs, resulted in a significant reduction of retinoic acid-inducible gene l-dependent type I IFN (IFN-I). These data implicate the formation of virus replication intermediates in APCs as the predominant trigger of IFN-I in vivo. Taking these data together, this research describes a unique platform to study the host response to IAV and provides insights into the mechanism of antigen presentation and the induction of IFN-I. [ABSTRACT FROM AUTHOR]

Τ cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients ("go" signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition ("stop" signal). However, the mechanisms by which Τ cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell Chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4+ Τ cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows Τ cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals. [ABSTRACT FROM AUTHOR]

lnterleukin-4 is a cytokine widely known for its role in CD4+ T cell polarization and its ability to alternatively activate macrophage populations. In contrast, the impact of IL-4 on the activation and function of dendritic cells (DCs) is poorly understood. We report here that DCs respond to IL-4 both in vitro and in vivo by expression of multiple alternative activation markers with a different expression pattern to that of macrophages. We further demonstrate a central role for DC IL-4Rα expression in the optimal induction of IFNy responses in vivo in both Th1 and Th2 settings, through a feedback loop in which IL-4 promotes DC secretion of IL-12. Finally, we reveal a central role for RELMa during T-cell priming, establishing that its expression by DCs is critical for optimal IL-10 and IL-13 promotion in vitro and in vivo. Together, these data highlight the significant impact that IL-4 and RELMa can have on DC activation and function in the context of either bacterial or helminth pathogens. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) are antigen-presenting cells found in both lymphoid and nonlymphoid organs, including the brain (bDC) of Cd11c/eyfp transgenic C57BL/6 mice. Using an intranasal vesicular stomatitis virus infection, we demonstrated that EYFP+ cells amass in areas associated with viral antigens, take on an activated morphology, and project their processes into infected neuronal tissue within the olfactory bulb. These bDC separated into three EYFP+ CD45+ CD11b+ populations, all but one being able to functionally promote both T lymphocyte proliferation and TH1 cytokine production. One population was shown to emanate from the brain and a second population was peripherally derived. The third population was of indeterminate origin, being both radiosensitive and not replenished by donor bone marrow. Finally, each EYFP+ population contained CD11b+ CD103+ subpopulations and could be distinguished in terms of CD115, Gr-1, and Ly-6C expression, highlighting mucosal and monocyte-derived DC lineages. [ABSTRACT FROM AUTHOR]

an antigenic peptide bound to a major histocompatibility complex (pMHC) molecule on an antigen-presenting cell (APC). In addition, T-cell activation generally requires binding of this same pMHC to a CD4 or CD8 coreceptor. Here, we report the structure of a complete TCR-pMHC-CD4 ternary complex involving a human autoimmune TCR, a myelin-derived self-peptide bound to HLA-DR4, and CD4. The complex resembles a pointed arch in which TCR and CD4 are each tilted ∼65° relative to the T-cell membrane. By precluding direct contacts between TCR and CD4, the structure explains how TCR and CD4 on the T cell can simultaneously, yet independently, engage the same pMHC on the APC. The structure, in conjunction with previous mutagenesis data, places TCR-associated CD3εγ and CD3εδ subunits, which transmit activation signals to the T cell, inside the TCR-pMHC-CD4 arch, facing CD4. By establishing anchor points for TCR and CD4 on the T-cell membrane, the complex provides a basis for understanding how the CD4 coreceptor focuses TCR on MHC to guide TCR docking on pMHC during thymic T-cell selection. [ABSTRACT FROM AUTHOR]

Acute graft-versus-host disease (GvHD) is a major complication that prevents successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies demonstrate that donor T cells and host antigen-presenting cells along with several proinflammatory cytokines are required for the induction of GvHD and contribute to its severity. Increasing evidence demonstrates that human serumderived αalpha-1- anti-trypsin (AAT) reduces production of promflammatory cytokines, induces anti-inflammatory cytokines, and interferes with maturation of dendritic cells. Using well-characterized mouse models of BMT, we have studied the effects of AAT on GvHD severity. Administration of AAT early after BMT decreased mortality in three models of GvHD and reduced serum levels of proinflammatory cytokines in the allogeneic recipients compared with vehicle (albumin) treated animals. AAT treatment reduced the expansion of alloreactive T effector cells but enhanced the recovery of T regulatory T cells, (Tregs) thus altering the ratio of donor T effector to T regulatory cells in favor of reducing the pathological process. However, despite altering the ratio in vivo. AAT had no direct effects on either the donor Teffector cells or T regulatory cells Tregs in vitro. In contrast. AAT suppressed LPSinduced in vitro secretion of proinflammatory cytokines such as TNF-α and IL-1β enhanced the production of the anti-inflammatory cytokine IL-10, and impaired NF-KB translocation in the host dendritic cells. In light of its long history of safety in humans, these findings suggest that administration of AAT represents a novel unique and viable strategy to mitigate clinical GvHD. [ABSTRACT FROM AUTHOR]

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b+Gr-1+ cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b+Gr-1+ cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis. [ABSTRACT FROM AUTHOR]

Successful priming of adaptive immune responses is crucially dependent on innate activation signals that convert resting antigenpresenting cells (APCs) into immunogenic ones. APC5 expressing the relevant innate pattern recognition receptors can be directly activated by pathogen-associated molecular patterns (PAMPs) to become competent to prime T-cell responses. Alternatively, it has been suggested that APCs could be activated indirectly by promflammatory mediators synthesized by PAMP-exposed cells. However, data obtained with CD4+ T cells suggest that inflammatory signals often cannot substitute for direct pattern recognition in APC activation for the priming of T helper responses. To test whether the same is true for CD8+ T cells, we studied cytotoxic T lymphocyte development in vitro and in mixed chimeric mice in which coexisting APCs can either present a preprocessed model antigen or directly recognize a given PAMP, but not both. We show that indirectly activated APCs promote antigen-specific proliferation of naïve CD8+ T cells but fail to support their survival and cytotoxic T lymphocyte differentiation. Furthermore, CD8+ T cells primed by indirectly activated APCs are unable to reject tumors. Thus, inflammation cannot substitute for direct recognition of single PAMPs in CD8+ T-cell priming. These findings have important practical implications for vaccine design, indicating that adjuvants must be judiciously chosen to trigger the relevant pattern recognition receptors in APCs. [ABSTRACT FROM AUTHOR]

Extrafollicular (EF) B-cell responses are increasingly being recognized as an alternative pathway of B-cell activation, particularly in autoimmunity. Critical cellular interactions required for the EF B-cell response are unclear. A key question in autoimmunity, in which Toll-like receptor (TLR) signals are costimulatory and could be sufficient for B-cell activation, is whether T cells are required for the response. This is pivotal, because autoreactive B cells are considered antigen-presenting cells for autoreactive T cells, but where such interactions occur has not been identified. Here, using AM14 site-directed transgenic rheumatoid factor (RF) mice, we report that B cells can be activated, differentiate, and isotype-switch independent of antigen-specific T-cell help, αβ T cells, CD40L signaling, and IL-21 signaling to B cells. However, T cells do dramatically enhance the response, and this occurs via CD40L and IL-21 signals. Surprisingly, the response is completely inducible T-cell costimulator ligand independent. These results establish that, although not required, T cells substantially amplify EF autoantibody production and thereby implicate T-independent autoreactive B cells as a potential vector for breaking T-cell tolerance. We suggest that these findings explain why autoreactivity first focuses on self-components for which B cells carry TLR ligands, because these will uniquely be able to activate B cells independently of T cells, with subsequent T-B interactions activating autoreactive T cells, resulting in chronic autoimmunity. [ABSTRACT FROM AUTHOR]

Arenaviruses are a major cause of hemorrhagic fevers endemic to Sub-Saharan Africa and South America, and thus a major public health and medical concern. The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is widely used as a model system for studying persistent and acute infections, as well as for gaining an understanding of mammalian immune function. When originally characterized three decades ago, the LCMV isolate, Armstrong, which causes an acute infection in adult mice, was found to differ from the LCMV Clone 13 strain that causes a persistent infection by two amino acid changes, one within the virus surface glycoprotein (GP1: F260L) and the other within the virus L polymerase (K1076Q). Mutation F260L was considered solely responsible for the exceptionally strong binding affinity of Clone 13 (L at GP1 260) to its cellular receptor, a-dystroglycan, which among cells of the immune system is preferentially expressed on dendritic cells, and consequently, alters dendritic cell function leading to viral persistence. Recently, we noted a previously overlooked nucleotide difference between these two strains that results in an additional amino acid change in GP1, N176D. To investigate the potential contribution of this newly identified mutation to the Clone 13 phenotype, we used reverse-genetics approaches to generate recombinant LCM viruses with each of these individual mutations. Phenotypic characterization of these rLCMV showed that mutation F260L, but not N176D, in the GP1 of LCMV is essential for mediating the long-term persistence of Clone 13 infections. This work emphasizes the importance of subtle differences in viral strains that determine disease outcomes. [ABSTRACT FROM AUTHOR]

Herein we investigated a new strategy for the modulation of cardiac macrophages to a reparative state, at a predetermined time after myocardial infarction (MI), in aim to promote resolution of inflammation and elicit infarct repair. The strategy employed intravenous injections of phosphatidylserine (PS)-presenting liposomes, mimicking the anti-inflammatory effects of apoptotic cells. Following PS-liposome uptake by macrophages in vitro and in vivo, the cells secreted high levels of anti-inflammatory cytokines [transforming growth factor β (TGFβ) and interleukin 10 (IL-10)] and upregulated the expression of the mannose receptor—CD206, concomitant with downregulation of proinflammatory markers, such as tumor necrosis factor α (TNFα) and the surface marker CD86. In a rat model of acute MI, targeting of PS-presenting liposomes to infarct macrophages after injection via the femoral vein was demonstrated by magnetic resonance imaging (MRI). The treatment promoted angiogenesis, the preservation of small scars, and prevented ventricular dilatation and remodeling. This strategy represents a unique and accessible approach for myocardial infarct repair. [ABSTRACT FROM AUTHOR]

B1 cells represent a distinct subset of B cells that produce most of the natural serum lgM and much of the gut lgA and function as an important component of early immune responses to pathogens. The development of B1 cells depends on the nuclear factor of activated T cells c1 (NFAT1), a transcription factor abundantly expressed by B1 cells but not by conventional B2 cells. However, the factors that regulate the expression of NFATc1 in B1 cells remain unknown. Here we show that a vitamin A-deficient diet results in reduction of NFATc1 expression in B1 cells and almost complete loss of the B1 cell compartment. As a consequence, vitamin A-deficient mice have reduced serum lgM and are unable to mount T cell-independent antibody responses against bacterial antigens. We demonstrate that injection of all-trans retinoic acid induces the expression of NFATc1, particularly from the constitutive P2 promoter, and leads to the increase of the B1 cells. Thus, the retinoic acid-dependent pathway is critical for regulating NFATc1 expression and for maintenance of the natural memory B cell compartment. [ABSTRACT FROM AUTHOR]

Memory T cells of the effector type (TEM) account for the characteristic rapidity of memory T-cell responses, whereas memory T cells of the central type (TCM) account for long-lasting, vigorously proliferating memory T-cell responses. How antigen-stimulated (primed) T cells develop into different memory T-cell subsets with diverse tissue distributions is largely unknown. Here we show that after respiratory tract infection of mice with influenza virus, viral antigen associated with dendritic cells (DCs) was abundant in lungdraining lymph nodes (DLN) and the spleen for more than a week but was scant and transient in nondraining lymph nodes (NDLN). Correspondingly, activated CD8 T cells proliferated extensively in DLN and the spleen but minimally in NDLN. Strikingly, however, although most persisting CD8 T cells in DLN and spleen exhibited the TEM phenotype, those persisting in NDLN exhibited the TCM phenotype. Reducing antigen exposure by depleting DCs at the peak of primary T-cell responses enhanced the development of TCM, whereas subjecting primed CD8 T cells from NDLN to additional antigen stimulation inhibited TCM development. These findings demonstrate that differences in persistence of antigen bearing DCs in various tissues regulate the tissue-specific pattern of memory CD8 T-cell development. The findings have significant implications for design of vaccines and immunization strategies. [ABSTRACT FROM AUTHOR]

Dendritic spikes appear to be a ubiquitous feature of dendritic excitability. In cortical pyramidal neurons, dendritic spikes increase the efficacy of distal synapses, providing additional inward current to enhance axonal action potential (AP) output, thus increasing synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger synaptic plasticity, but their influence on axonal output is not well understood. We have used simultaneous somatic and dendritic patch-clamp recordings to directly assess the impact of dendritic calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, followed by pauses in spiking, which cancelled out the extra spikes in the burst. As a result, average output firing rates during trains of input remained independent of the input strength, thus flattening synaptic gain. We demonstrate that this "clamping" of AP output by the pause following dendritic spikes is due to activation of high conductance calcium-dependent potassium channels by dendritic spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, thus have differential effects on temporally coded and rate coded information: increasing the impact of transient parallel fiber input. while depressing synaptic gain for sustained parallel fiber inputs. [ABSTRACT FROM AUTHOR]

The cross-talk among cells of the innate immunity can greatly affect both innate and adaptive responses. Here we analyzed the molecular interactions between human natural killer (NK) cells and autologous macrophages. Activated NK cells killed MO and M2. whereas M1 macrophages were more resistant to lysis because of their higher expression of HLA class I molecules. Following exposure to LPS or bacillus Calmette-Guérin, MO and M2. but not polarized (endotoxin tolerant) M1 macrophages. induced strong activation of resting NK cells. The expression of CD69 and CD25 activation markers and the acquisition of cytotoxicity against tumor cells and immature dendritic cells required soluble factors being mostly contact independent. On the contrary, IFN-γ production was contact dependent and required the interaction of DNAM- 1 and 2B4 (on NK) with their ligands on macrophages as well as IL- 18. 11-18 was involved also in the acquisition of CCR7 by NK cells. Interestingly, MO and M2 cells expressed a membrane-bound form of IL-18, which was released in small amounts after LPS treatment. Our data indicate that, upon interaction with MO macrophages exposed to microbial products. NK cells may amplify classical type 1 immune responses. In addition, M1-polarizing stimuli can rescue M2 macrophages from their immunomodulatory state and shape their functional behavior toward NK stimulatory capability. [ABSTRACT FROM AUTHOR]

There has beenconsiderable interest in virulence genes in the plasticity region of Helicobacter pylon, but little is known about many of these genes. JHP94O, one of the virulence factors encoded by the plasticity region of H. pylon strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-ºB in cultured macrophages. Here we have characterized the structure and function of JHP94O to provide the framework for better understanding its role in inflammation by H. pylon. Our work demonstrates that JHP94O is the first example of a eukaryotic-type Ser/Thr kinase from H. pylon. We show that JHP94O is catalytically active as a protein kinase and translocates into cultured human cells. Further- more, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP94O in inflammation and subsequent pathogenesis caused by H. pylon. We propose to rename the jhp940 gene as ctkA (cell translocating kinase A). [ABSTRACT FROM AUTHOR]

Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts. [ABSTRACT FROM AUTHOR]

Lymphoid organs are characterized by a complex network of phenotypically distinct dendritic cells (DC) with potentially unique roles in pathogen recognition and immunostimulation. Classical DC (cDC) include two major subsets distinguished in the mouse by the expression of CD8a. Here we describe a subset of CD8a DC in lymphoid organs of naïve mice characterized by expression of the CX3CR1 chemokine receptor. CX3CR1 CD8L DC lack hallmarks of classical CD8a DC, including IL-12 secretion, the capacity to cross- present antigen, and their developmental dependence on the transcriptional factor BatF3. Gene-expression profiling showed that CX3CR1 CD8a DC resemble CD8a cDC. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX3CR1 CD8u DC. A PDC relationship of the cells is supported further by the fact that they harbor characteristic D-J Ig gene rearrangements and that development of CX3CR1 CD8u DC re- quires E2-2, the critical transcriptional regulator of PDC. Thus, CX3CR1 CD8u DC represent a unique DC subset, related to but distinct from PDC. Collectively, the expression-profiling data of this study refine the resolution of previous DC definitions, sharpen the border of classical CD8a and CD8C DC, and should assist the identification of human counterparts of murine DC subsets. [ABSTRACT FROM AUTHOR]

T helper type 17 (TH17) cells are highly proinflammatory effector T cells that are characterized by the production of high amounts of 1L-17A, IL-17F, IL-21, and IL-22. Furthermore, TH17 cells have been associated with a number of autoimmune diseases. However, it is not clear whether TH17 cells can also serve as effective helper cells. Here we show that TH17 cells can function as B-cell helpers in that they not only induce a strong proliferative response of B cells in vitro but also trigger antibody production with class switch recombination in vivo. Transfer of TH17 cells into WT or T-cell receptor α-deficient mice, which lack endogenous T cells, induces a pronounced antibody response with preferential isotype class switching to IgG1, IgG2a, IgG2b, and IgG3. as well as the formation of germinal centers. Conversely, blockade of IL-17 signaling results in a significant reduction in both number and size of germinal centers. Whereas IL-21 is known to help B cells. IL-17 on its own drives B cells to undergo preferential isotype class switching to IgG2a and IgG3 subtypes. These observations provide insights into the unappreciated role of TH17 cells and their signature cytokines in mediating B-cell differentiation and class switch recombination. [ABSTRACT FROM AUTHOR]

MHC class I plays a critical role in the immune defense against viruses and tumors by presenting antigens to CD8 T cells. An NLR protein, class II transactivator (CIITA), is a key regulator of MHC class II gene expression that associates and cooperates with transcription factors in the MHC class II promoter. Although CIITA also transactivates MHC class! gene promoters, loss of CIITA in humans and mice results in the severe reduction of only MHC class II expression, suggesting that additional mechanisms regulate the expression of MHC class I. Here, we identify another member of the NLR protein family, NLRCS, as a transcriptional regulator of MHC class I genes. Similar to CIITA, NLRC5 is an IFN-γ-inducible nuclear protein, and the expression of NLRC5 resulted in enhanced MHC class I expression in lymphoid as well as epithelial cell lines. Using chromatin immunoprecipitation and reporter gene assays, we show that NLRC5 associates with and activates the promoters of MHC class I genes. Furthermore, we show that the IFN-i-induced up-regulation of MHC class I requires NLRC5, because knockdown of NLRC5specifically impaired the expression of MHC class I. In addition to MHC class I genes, NLRC5 also induced the expression of β2-microglobulin, transporter associated with antigen processing, and large multifunctional protease, which are essential for MHC class I antigen presentation. Our results suggest that NLRC5 is a transcriptional regulator, orchestrating the concerted expression of critical components in the MHC class I pathway. [ABSTRACT FROM AUTHOR]

TIM-4, a member of the TIM family expressed on antigen-presenting cells, binds to phosphatidylserine exposed on the surface of apoptotic bodies. However, the significance of this interaction in vivo remains unknown because other receptors have been implicated in the clearance of apoptotic bodies and could compensate for the TIM-4 deficiency in vivo. In this study, we describe the generation of TIM-4-deficient mice and address whether TIM-4 serves a unique function in vivo. We show that TlM-4-/- peritoneal macrophages and B-1 cells do not efficiently engulf apoptotic bodies in vitro, or clear apoptotic bodies in vivo. TIM-4-deficient mice have hyperactive T and B cells, elevated levels of serum lg. and develop antibodies to double-stranded DNA. Taken together, we show that TIM-4 is critical for the clearance of apoptotic bodies in vivo, and that lack of TIM-4 results in aberrant persistence of apoptotic bodies leading to dysregulated lymphocyte activation and signs of systemic autoimmunity. [ABSTRACT FROM AUTHOR]

The functional capacities of CD8+ T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4+ T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8+ T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-γ, TNF-α, MIP1β, and CD107a, showed that aAPC-generated CD8+ T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8+ T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8+ T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8+ T cell function. [ABSTRACT FROM AUTHOR]