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There has been growing interest in the role of B cells in antitumour immunity and potential use in adoptive cellular therapies. To date, the success of such therapies is limited. The intrinsic capacity of B cells to specifically activate tumour-specific CD4+ T cells in vivo via TCR-dependent interactions remains poorly defined. We have developed an in vivo tumour model that utilizes MHCII I-E restriction which limits antigen presentation to tumour-specific CD4 T cells to either tumour-specific B cells or host myeloid antigen presenting cells (APCs) in lymphopenic RAG-/-mice. We have previously shown that these naive tumour-specific CD4+ T cells can successfully eradicate established tumours in this model when activated by host APCs. When naïve tumour-specific B cells are the only source of I-E+ APC, very limited proliferation of naïve CD4+ T cells is observed, whereas host I-E+ APCs are potent T cell activators. B cells pre-activated with an anti-CD40 agonistic antibody in vivo support increased T cell proliferation, although far less than host APCs. CD4+ T cells that have already differentiated to an effector/central memory phenotype proliferate more readily in response to naïve B cells, although still 100-fold less than in response to host APCs. This study demonstrates that even in a significantly lymphopenic environment, myeloid APCs are the dominant primary activators of tumour-specific T cells, in contrast to the very limited capacity of tumour-specific B cells. This suggests that future anti-tumour therapies that incorporate activated B cells should also include mechanisms that activate host APCs., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The immunological synapse allows antigen-presenting cells (APCs) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While pathways downstream of toll-like receptors rely on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFNγ). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in murine macrophages to identify the regulators of IFNγ-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multiscreen approach enabled us to identify novel pathways that preferentially control functionally distinct proteins. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFNγ signaling pathway. We report that the IFNγ response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFNγ and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity., Competing Interests: MK, KM, DM, SH, SB, CS, AO No competing interests declared

The kinetics of antigen-presenting cells (APCs) vary depending on their resident tissues and the manner of immunization. We investigated the long-term changes in mature APC and T-cell subsets over 4 weeks in the ocular surface in murine models of corneal quiescent or potent sterile inflammation, and allosensitization using partial (PT), syngeneic (Syn), and allogeneic (Allo) corneal transplantation. In PT, CD11b int CD11c hi MHCII hi CD86 hi cells increased until 4 weeks with an increase in IFNγ hi T cells. In Syn, both CD11b int CD11c hi MHCII hi CD86 hi and CD11b hi CD11c hi MHCII hi CD86 hi APC subsets increased until 4 weeks with a brief increase in CD69 hi T cells at 2 weeks. In Allo, CD11b int CD11c hi MHCII hi CD86 hi and CD11b hi CD11c hi MHCII hi CD86 hi APC subsets increased until 4 weeks, and an early increase in CD69 hi T cells was observed at 2 weeks followed by a late increase in IFNγ hi T cells at 4 weeks. The frequency of the IFNγ hi T cell subset was positively correlated with the frequency of the CD11b int CD11c hi MHCII hi CD86 hi subset, indicating the existence of APC-T cell interaction in the ocular surface. Together, the results indicate that allosensitization in mature APCs leads to T-cell activation in the ocular surface, whereas sterile inflammation merely induces a brief and non-specific T-cell activation in the ocular surface.

The failure of M. bovis BCG to induce long-term protection has been endowed to its inability to escape the phagolysosome, leading to mild activation of CD8 + mediated T cell response. Induction of apoptosis in host cells plays an important role in potentiating dendritic cells-mediated priming of CD8 + T cells, a process defined as "cross-priming." Moreover, IL-10 secretion by infected cells has been reported to hamper BCG-induced immunity against Tuberculosis (TB). Previously, we have reported that apoptosis of BCG-infected macrophages and inhibition of IL-10 secretion is FOXO3 dependent, a transcription factor negatively regulated by the pro-survival activated threonine kinase, Akt. We speculate that FOXO3-mediated induction of apoptosis and abrogation of IL-10 secretion along with M. bovis BCG immunization might enhance the protection imparted by BCG. Here, we have assessed whether co-administration of a known anti-cancer Akt inhibitor, MK-2206, enhances the protective efficacy of M. bovis BCG in mice model of infection. We observed that in vitro MK-2206 treatment resulted in FOXO3 activation, enhanced BCG-induced apoptosis of macrophages and inhibition of IL-10 secretion. Co-administration of M. bovis BCG along with MK-2206 also increased apoptosis of antigen-presenting cells in draining lymph nodes of immunized mice. Further, MK-2206 administration improved BCG-induced CD4 + and CD8 + effector T cells responses and its ability to induce both effector and central memory T cells. Finally, we show that co-administration of MK-2206 enhanced the protection imparted by M. bovis BCG against Mtb in aerosol infected mice and guinea pigs. Taken together, we provide evidence that MK-2206-mediated activation of FOXO3 potentiates BCG-induced immunity and imparts protection against Mtb through enhanced innate immune response., 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 Bouzeyen, Chugh, Gosain, Barbouche, Haoues, Rao, Essafi and Singh.)

Mitigating the risk of drug hypersensitivity reactions is an important facet of a given pharmaceutical, with poor performance in this area of safety often leading to warnings, restrictions and withdrawals. In the last 50 years, efforts to diagnose, manage, and circumvent these obscure, iatrogenic diseases have resulted in the development of assays at all stages of a drugs lifespan. Indeed, this begins with intelligent lead compound selection/design to minimize the existence of deleterious chemical reactivity through exclusion of ominous structural moieties. Preclinical studies then investigate how compounds interact with biological systems, with emphasis placed on modeling immunological/toxicological liabilities. During clinical use, competent and accurate diagnoses are sought to effectively manage patients with such ailments, and pharmacovigilance datasets can be used for stratification of patient populations in order to optimise safety profiles. Herein, an overview of some of the in-vitro approaches to predict intrinsic immunogenicity of drugs and diagnose culprit drugs in allergic patients after exposure is detailed, with current perspectives and opportunities provided., Competing Interests: SH was employed by the company ApconiX. The remaining 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 Hammond, Thomson, Meng and Naisbitt.)

The immunological synapse (IS) is a specialized contact area formed between a T cell and an antigen presenting cell (APC). Besides molecules directly involved in antigen recognition such as the TCR/CD3 complex, ion channels important in the membrane potential and intracellular free Ca 2+ concentration control of T cells are also recruited into the IS. These are the voltage-gated Kv1.3 and Ca 2+ -activated KCa3.1 K + channels and the calcium release-activated Ca 2+ channel (CRAC). However, the consequence of this recruitment on membrane potential and Ca 2+ level control is not known. Here we demonstrate that the membrane potential (MP) of murine T cells conjugated with APCs in an IS shows characteristic oscillations. We found that depolarization of the membrane by current injection or by increased extracellular K + concentration produced membrane potential oscillations (MPO) significantly more frequently in conjugated T cells than in lone T cells. Furthermore, oscillation of the free intracellular Ca 2+ concentration could also be observed more frequently in cells forming an IS than in lone cells. We suggest that in the IS the special arrangement of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation., Competing Interests: The authors declare no conflict of interest.

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90° angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

Background: Porcine circovirus (PCV) disease caused by PCV type 2 (PCV2) is mainly attributed to immunosuppression and immune damage. PCV2 can infect vascular endothelial cells and induce high expression of endothelial IL-8. Dendritic cells (DCs), as professional antigen-presenting cells, can not only present antigens but also activate naïve T-cells, causing an immune response., Methods: To demonstrate whether endothelial IL-8 is the main factor inhibiting the maturation and related functions of dendritic cells during PCV2 infection, monocyte-derived DCs (MoDCs) and porcine iliac artery endothelial cells (PIECs) processed by different methods were co-cultured in two ways. Flow cytometry, molecular probe labeling, fluorescence quantitative PCR, and the MTS assay were used to detect the changes in related functions and molecules of MoDCs., Results: Compared to those in the PIEC-DC group, the endothelial IL-8 upregulation co-culture group showed significantly lower double-positive rates for CD80/86 and MHC-II of MoDCs and significantly increased endocytosis of MoDCs. Meanwhile, the adhesion rate and average fluorescence intensity of MoDCs were significantly downregulated in migration and adhesion experiments. Furthermore, the MHC-I and LAMP7 mRNA levels in MoDCs and the proliferation of MoDC-stimulated T-cells were markedly reduced. However, the changes in MoDCs of the endothelial IL-8 downregulation co-culture group were the opposite., Conclusions: PCV2-induced endothelial IL-8 reduces the adhesion and migration ability of MoDCs, resulting in a decreased maturation rate of MoDCs, and further inhibits antigen presentation by DCs. These results may explain the immunosuppressive mechanism of PCV2 from the perspective of the interaction between endothelial cells and DCs in vitro.

Multiple sclerosis (MS) is a chronic inflammatory disease mediated by a complex interaction between the autoreactive lymphocytes and the effector myeloid cells within the central nervous system (CNS). In a murine model of MS, experimental autoimmune encephalomyelitis (EAE), Ly6C hi monocytes migrate into the CNS and further differentiate into antigen-presenting cells (APCs) during disease progression. Currently, there is no information about gene signatures that can distinguish between monocytes and the monocyte-derived APCs. We developed a surface marker-based strategy to distinguish between these two cell types during the stage of EAE when the clinical symptoms were most severe, and performed transcriptome analysis to compare their gene expression. We report here that the inflammatory CNS environment substantially alters gene expression of monocytes, compared to the monocyte differentiation process within CNS. Monocytes in the CNS express genes that encode proinflammatory cytokines and chemokines, and their expression is mostly maintained when the cells differentiate. Moreover, monocyte-derived APCs express surface markers associated with both dendritic cells and macrophages, and have a significant up-regulation of genes that are critical for antigen presentation. Furthermore, we found that Ccl17, Ccl22 , and Ccr7 are expressed in monocyte-derived APCs but not the Ly6C hi monocytes. These findings may shed light on identifying molecular signals that control monocyte differentiation and functions during EAE., (Copyright © 2019 Monaghan, Zheng, Hu and Wan.)

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm 3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates., Competing Interests: DC, LM, ST, GB, CM, HT, LM, DA, XR, KS, MD, AH, PS, PV, RM, CW No competing interests declared

Biological vestiges are used in forensic science to resolve a large number of cases by typing the genetic profile and identifying the individual to whom it belongs. However, chimeric persons that possess cells with two or more different DNA make these types of analyses difficult. This situation can occur naturally, by errors in the fertilization or early embryogenesis, or in an artificial way, for example after hematopoietic stem cell transplantation (HSCT), when host and donor cells coexist in the patient. In this paper, we will specially focus on the latter. The vestiges from transplant patients represent a challenge from a forensic perspective since the interpretation of the genetic fingerprint can be misleading because of the presence of chimerism. Due to the high number of transplant patients (and their increase over the years) and the existence of natural chimeras (probably many of them hidden), it is necessary to consider whether we are facing a possible chimeric person or someone who has been a donor of hematopoietic stem cells in a forensic context. In this review, the presence of donor bone marrow derived cells in some tissues of forensic interest will be discussed. Finally, to emphasize the importance of chimerism after HSCT in forensic genetics, some real-life cases will be examined., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This unit describes methods for quantifying phagocytosis and imaging the immunological synapse between T cells and antigen-presenting cells (APCs), with both techniques delivering valuable information about APC function. These aspects of APC biology have traditionally been challenging to quantify, and imaging flow cytometry, which harnesses the high-throughput nature of flow cytometry combined with the capacity of microscopy to deliver spatial localization, facilitates analysis of these APC functions in a fashion that was previously not possible. Imaging flow cytometry allows large numbers of events to be captured and large amounts of fluorescence data to be quantified at the physical location of markers of interest, both on the cell surface and in intracellular compartments, combining key features of traditional flow cytometry and fluorescence microscopy. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

B cells have important functions in the pathogenesis of autoimmune diseases, including autoimmune rheumatic diseases. In addition to producing autoantibodies, B cells contribute to autoimmunity by serving as professional antigen-presenting cells (APCs), producing cytokines, and through additional mechanisms. B cell activation and effector functions are regulated by immune checkpoints, including both activating and inhibitory checkpoint receptors that contribute to the regulation of B cell tolerance, activation, antigen presentation, T cell help, class switching, antibody production and cytokine production. The various activating checkpoint receptors include B cell activating receptors that engage with cognate receptors on T cells or other cells, as well as Toll-like receptors that can provide dual stimulation to B cells via co-engagement with the B cell receptor. Furthermore, various inhibitory checkpoint receptors, including B cell inhibitory receptors, have important functions in regulating B cell development, activation and effector functions. Therapeutically targeting B cell checkpoints represents a promising strategy for the treatment of a variety of autoimmune rheumatic diseases.

The immune synapse is a complex cellular structure that enables cell-cell communications between immune cells, mainly at the interface of an effector T-cell and an antigen-presenting cell (APC) that expresses the appropriate peptide-MHC complexes. With progressive technological advances, there has been increasing interest in understanding molecular events that take place in motile T-lymphocytes at the immune synapse. Here, we provide an optimized method to induce the formation of an immune synapse between a T-cell and an APC in vitro. The experimental protocol described herein would be useful in characterizing the role of cell surface molecules and downstream signaling pathways in the context of cell-to-cell communications between T-cells and APCs.

Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, a new platform technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation is presented. Artificial antigen presenting cells (aAPCs) are successfully created using supported lipid bilayers on various cell-templated silica microparticles with defined membrane fluidity and stimulating antibody density. This panel of aAPCs is used to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations are able to stimulate T cell growth, and preferentially promote CD8 + T cell growth over CD4 + T cell growth when compared to commercially available pendant antibody-conjugated particles. T cells cultured with HeLa- and red blood cell-templated aAPCs have a less-differentiated and less-exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica-supported lipid bilayers as an aAPC platform., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the skin, the epidermis is continuously exposed to various kinds of external substances and stimuli. Therefore, epidermal barriers are crucial for providing protection, safeguarding health, and regulating water balance by maintaining skin homeostasis. Disruption of the epidermal barrier allows external substances and stimuli to invade or stimulate the epidermal cells, leading to the elicitation of skin inflammation. The major components of the epidermal barrier are the stratum corneum (SC) and tight junctions (TJs). The presence of zinc in the epidermis promotes epidermal homeostasis; hence, this study reviewed the role of zinc in the formation and function of the SC and TJs. Langerhans cells (LCs) are one of the antigen-presenting cells found in the epidermis. They form TJs with adjacent keratinocytes (KCs), capture external antigens, and induce antigen-specific immune reactions. Thus, the function of zinc in LCs was examined in this review. We also summarized the general knowledge of zinc and zinc transporters in the epidermis with updated findings.

Autoimmune regulator (AIRE), whose gene mutation is considered to be a causative factor of autoimmune polyglandular syndrome type 1 (APS1), is an important transcriptional regulator. Studies on the role of AIRE in the central immune system have demonstrated that AIRE can eliminate autoreactive T cells by regulating the expression of a series of tissue specific antigens promiscuously in medullary thymic epithelial cells (mTECs) and induce regulatory T cell (Treg) production to maintain central immune tolerance. However, the related research of AIRE in peripheral tolerance is few. In order to understand the current research progress on AIRE in peripheral tolerance, this review mainly focuses on the expression and distribution of AIRE in peripheral tissues and organs, and the role of AIRE in peripheral immune tolerance such as regulating Toll-like receptor (TLR) expression and the maturation status of antigen presenting cells (APCs), inducing T cell tolerance and differentiation. This review will show us that AIRE also plays an indispensable role in the periphery.

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R + Flt3 - antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα + DCs in the steady-state human small intestine. CSF1R + Flt3 - APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocyte-derived cells, and accumulate during inflammation. CSF1R + Flt3 - APCs show typical macrophage characteristics functionally distinct from their Flt3 + cDC counterparts: under steady-state conditions they excel at antigen uptake, have a lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.

In teleosts, as in mammals, the immune system is tightly regulated by sexual steroid hormones, such as oestrogens. We investigated the effects of 17β-oestradiol on the expression of several genes related to T cell development and resulting T cell subpopulations in sea bass, Dicentrarchus labrax, for a primary lymphoid organ, the thymus, and two secondary lymphoid organs, the head-kidney and the spleen. In parallel, the oxidative burst capacity was assessed in leucocytes of the secondary lymphoid organs. Apoptosis- and proliferation-related genes, indicative of B and T cell clonal selection and lymphoid progenitor activity, were not affected by elevated oestrogen-levels. Sex-related oestrogen-responsiveness in T cell and antigen-presenting cell markers was observed, the expression of which was differentially induced by oestrogen-exposure in the three lymphoid organs. Remarkably, in the spleen, oestrogen increased regulatory T cell-related gene expression was associated with a decrease in oxidative burst capacity. To the best of our knowledge, this study indicates for the first time that physiological levels of oestrogen are likely to promote immune tolerance by modulating thymic function (i.e., T cell development and output) and peripheral T cells in teleosts, similar to previously reported oestrogenic effects in mammals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4 + T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4 + T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.

Oligomannose-coated liposomes (OMLs), containing entrapped antigens, serve as effective antigen delivery vehicles and as a novel adjuvant to induce antigen-specific cellular immune responses. However, in vitro activation of antigen-presenting cells (APCs) by OMLs has not yet been demonstrated. In this paper, we found that OMLs can deliver the antigens and the stimulatory signals into inflammatory monocytes in vitro, leading to differentiation of the cells to mature APCs. When OMLs were co-cultured with peripheral blood mononuclear cells from C57BL/6 mice in the presence of mouse serum, OMLs were preferentially incorporated into both Ly6C high monocytes and Ly6C low monocytes, which are referred to as murine inflammatory and resident monocytes, respectively. The expression of CD11c, CD80, CD86, CCR7, and MHC class II on the Ly6C high monocytes was significantly enhanced during the 24 h after OML uptake, whereas upregulation of these molecules on the Ly6C low monocytes was limited. In addition, the antigenic peptide of OVA encased in OMLs was presented on MHC class I of only Ly6C high monocytes. Furthermore, OVA-encasing OML-ingesting monocytes can activate CD8 + T cells from OT-1 mice, suggesting that antigens encapsulated in OMLs were cross-presented in inflammatory monocytes. Adoptive transfer of the monocytes that engulf OVA-encasing OMLs led to induction of an antigen-specific Th1 immune response in mice. Taken together, mature APCs can be generated from inflammatory monocytes in peripheral blood by ex vivo treatment of the cells with OMLs without any additional stimuli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Middle East respiratory syndrome coronavirus (MERS-CoV) presents an emerging threat to public health worldwide by causing severe respiratory disease in humans with high virulence and case fatality rate (about 35%) since 2012. Little is known about the pathogenesis and innate antiviral response in primary human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) upon MERS-CoV infection. In this study, we assessed MERS-CoV replication as well as induction of inflammatory cytokines and chemokines in MDMs and immature and mature MDDCs. Immature MDDCs and MDMs were permissive for MERS-CoV infection, while mature MDDCs were not, with stimulation of proinflammatory cytokine and chemokine upregulation in MDMs, but not in MDDCs. To further evaluate the antiviral activity of well-defined drugs in primary antigen presenting cells (APCs), three compounds (chloroquine, chlorpromazine and toremifine), each with broad-spectrum antiviral activity in immortalized cell lines, were evaluated in MDMs and MDDCs to determine their antiviral effect on MERS-CoV infection. While chloroquine was not active in these primary cells, chlorpromazine showed strong anti-MERS-CoV activity, but it was associated with high cytotoxicity narrowing the potential window for drug utilization. Unlike in established cells, toremifene had marginal activity when tested in antigen presenting cells, with high apparent cytotoxicity, also limiting its potential as a therapeutic option. These results demonstrate the value of testing drugs in primary cells, in addition to established cell lines, before initiating preclinical or clinical studies for MERS treatment and the importance of carefully assessing cytotoxicity in drug screen assays. Furthermore, these studies also highlight the role of APCs in stimulating a robust protective immune response to MERS-CoV infection.

Disturbance of the epidermal barrier by UVR is associated with the release of antimicrobial peptides and inflammatory cytokines for the purpose of a danger response. On the other hand, UVR causes immunosuppression via regulatory T cells (Treg) that limit the inflammatory reaction. The concurrent induction of antimicrobial peptides and Treg by UVR may represent a counter-regulatory mechanism in response to barrier disruption, preventing microbial superinfection and sensitization to contact allergens, respectively, both of which cross impaired epidermis more easily. Thus, using a model of murine contact hypersensitivity we examined if disruption of the epidermal barrier only initiates similar counter-regulatory mechanisms via the generation of Treg. Sensitization through tape-stripped skin induced a weaker contact hypersensitivity response than in control mice. This was due to the induction of antigen-specific Treg, as demonstrated in adoptive transfer and depletion experiments utilizing DEREG mice. Treg induction by tape stripping was linked to the expression of the alarmin IL-33, as blockade of IL-33 exacerbated contact hypersensitivity, whereas injection of IL-33 inhibited contact hypersensitivity and induced Treg. These results demonstrate that epidermal barrier disruption, in addition to danger signals, induces regulatory events that prevent exaggerated skin inflammation and that IL-33 appears to be critically involved in this process., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

CD103 + dendritic cells are critical for cross-presentation of tumor antigens. Here we have shown that during immunotherapy, large numbers of cells expressing CD103 arose in murine tumors via direct differentiation of Ly6c + monocytic precursors. These Ly6c + CD103 + cells could derive from bone-marrow monocytic progenitors (cMoPs) or from peripheral cells present within the myeloid-derived suppressor cell (MDSC) population. Differentiation was controlled by inflammation-induced activation of the transcription factor p53, which drove upregulation of Batf3 and acquisition of the Ly6c + CD103 + phenotype. Mice with a targeted deletion of p53 in myeloid cells selectively lost the Ly6c + CD103 + population and became unable to respond to multiple forms of immunotherapy and immunogenic chemotherapy. Conversely, increasing p53 expression using a p53-agonist drug caused a sustained increase in Ly6c + CD103 + cells in tumors during immunotherapy, which markedly enhanced the efficacy and duration of response. Thus, p53-driven differentiation of Ly6c + CD103 + monocytic cells represents a potent and previously unrecognized target for immunotherapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system, and is characterized by inflammation and myelin damage. The immune system initiates the autoimmune response, although the mechanisms of neuronal damage have not been elucidated. The purpose of the present study was to investigate autoreactive CD4+ and CD8 + T lymphocytes, in conjunction with other inflammatory cells and cytokines in active MS lesions., Methods: EAE animal models was established by plantar injections of MBP (200 μg per rat). Purified CD4+ or CD8+ T-cells were isolated from heparinized peripheral blood (EAE animals and control animals) via negative selection. To examine effects of presence of autoreactive CD4+ and CD8+ T lymphocytes, we carried out ELISA, Western blot analysis and TUNEL. In addition, we examined the direct effects of various factors on neuronal cell death using MTT assay., Results: The data revealed that CD8+ T-cells were more toxic to neurons compared to CD4+ T-cells, in both the MBP and EAE conditions. Bax was greater increased when neurons were co-cultured with CD8+ T-cells in the MBP group. There is a significant increase in IL-17 secretion by CD4+ T-cells in both the MBP group and EAE group. Neuronal viability were affected by Perforin (1.5 μg/mL)., Conclusion: The present study extends previous research by demonstrating the role of CD8+ T-cells in MS and supports perforin secretion by CD8+ T-cells as a potential therapeutic factor. Furthermore, we determined that CD4 + T-cells can enhance CD8 + T-cell neuronal cytotoxicity via induction of intense inflammation.

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients, graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference, zinc finger nuclease, and the CRISPR/Cas9 system. To improve HLA gene editing, we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore, artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.

CD69 is a membrane-bound, type II C-lectin receptor. It is a classical early marker of lymphocyte activation due to its rapid appearance on the surface of the plasma membrane after stimulation. CD69 is expressed by several subsets of tissue resident immune cells, including resident memory T (TRM) cells and gamma delta (γδ) T cells, and is therefore considered a marker of tissue retention. Recent evidence has revealed that CD69 regulates some specific functions of selected T-cell subsets, determining the migration-retention ratio as well as the acquisition of effector or regulatory phenotypes. Specifically, CD69 regulates the differentiation of regulatory T (Treg) cells as well as the secretion of IFN-γ, IL-17, and IL-22. The identification of putative CD69 ligands, such as Galectin-1 (Gal-1), suggests that CD69-induced signaling can be regulated not only during cognate contacts between T cells and antigen-presenting cells in lymphoid organs, but also in the periphery, where cytokines and other metabolites control the final outcome of the immune response. Here, we will discuss new aspects of the molecular signaling mediated by CD69 and its involvement in the metabolic reprogramming regulating TH-effector lineages., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Immune checkpoint blockade therapy (ICBT), which blocks negative immune-activating signals and maintains the antitumor response, has elicited a remarkable clinical response in certain cancer patients. However, intrinsic resistance (i.e., insensitivity of the tumors to therapy) remains a daunting challenge. The efficacy of ICBT is tightly modulated by the function of each step in the antitumor immunity cycle. Mechanistically, the number of mutations determines tumor immunogenicity. The properties of the tumor microenvironment control T-cell infiltration, distribution, and function in tumor tissues. Low tumor immunogenicity and a strong immunosuppressive tumor microenvironment cause significant intrinsic resistance to ICBT. With our evolving understanding of intrinsic resistance, people have successfully tested, in preclinical models, treatments targeting specific resistance mechanisms to sensitize ICBT-resistant tumors. Translation of those preclinical findings to the clinical arena will help generate personalized ICBT strategies that target tumor-specific resistance mechanisms. Progress in the new personalized ICBT strategies will expand the reach of immunotherapy to more cancer types, thus enabling more patients to benefit. Cancer Res; 77(4); 817-22. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The ability to culture and expand B cells in vitro has become a useful tool for studying human immunity. A limitation of current methods for human B cell culture is the capacity to support mature B cell proliferation. We developed a culture method to support the efficient activation and proliferation of naive and memory human B cells. This culture supports extensive B cell proliferation, with ∼10 3 -fold increases following 8 d in culture and 10 6 -fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naive B cells undergo isotype switching and differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved and, when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHC class II, CD80, and CD86. CD B cells act as APCs and present alloantigens and microbial Ags to T cells. We are able to activate and expand Ag-specific memory B cells; these cultured cells are highly effective in presenting Ag to T cells. We characterized the TCR repertoire of rare Ag-specific CD4 + T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vβ usage by TT-activated CD4 + T cells differs from resting and unspecifically activated CD4 + T cells. Moreover, we found that TT-specific TCR Vβ usage by CD4 + T cells was substantially different between donors. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Certain cytotoxic chemotherapeutic drugs are immunogenic, stimulating tumor immunity through mechanisms that are not completely understood. Here we show how the DNA-damaging drug cisplatin modulates tumor immunity. At the maximum tolerated dose (MTD), cisplatin cured 50% of mice with established murine TC-1 or C3 tumors, which are preclinical models of human papillomavirus (HPV)-associated cancer. Notably, the curative benefit of cisplatin relied entirely upon induction of tumor-specific CD8 + T cells. Mechanistic investigations showed that cisplatin stimulated tumor infiltration of inflammatory antigen-presenting cells (APC) expressing relatively higher levels of the T-cell costimulatory ligands CD70, CD80, and CD86. Cell death triggered by cisplatin was associated with the release of at least 19 proteins in the tumor environment that could act as damage-associated molecular patterns and upregulate costimulatory molecules, either alone or in concert, but the responsible proteins remain unknown. Essentially, the curative effect of cisplatin was abrogated in mice lacking expression of CD80 and CD86 on APCs. Furthermore, cisplatin treatment was improved by CTLA-4 blockade, which increases the availability of CD80/86 to bind to CD28. In contrast, there was no effect of CD27 stimulation, which replaces CD70 interaction. At the cisplatin MTD, cure rates could also be increased by vaccination with synthetic long peptides, whereas cures could also be achieved at similar rates at 80% of the MTD with reduced side effects. Our findings reveal an essential basis for the immunogenic properties of cisplatin, which are mediated by the induction of costimulatory signals for CD8 + T-cell-dependent tumor destruction. Cancer Res; 76(20); 6017-29. ©2016 AACR., (©2016 American Association for Cancer Research.)

Eosinophils are a minority circulating granulocyte classically viewed as being involved in host defense against parasites and promoting allergic reactions. However, a series of new regulatory functions for these cells have been identified in the past decade. During homeostasis, eosinophils develop in the bone marrow and migrate from the blood into target tissues following an eotaxin gradient, with interleukin-5 being a key cytokine for eosinophil proliferation, survival, and priming. In multiple target tissues, eosinophils actively regulate a variety of immune functions through their vast arsenal of granule products and cytokines, as well as direct cellular interaction with cells in proximity. The immunologic regulation of eosinophils extends from innate immunity to adaptive immunity and also involves non-immune cells. Herein, we summarize recent findings regarding novel roles of murine and human eosinophils, focusing on interactions with other hematopoietic cells. We also review new experimental tools available and remaining questions to uncover a greater understanding of this enigmatic cell.

Accumulating evidence indicated that B lymphocytes exerted complex functions in tumor immunity. On the one hand, B lymphocytes can inhibit tumor development through antibody generation, antigen presentation, tumor tissue interaction, and direct killing. On the other hand, B lymphocytes have tumor-promoting functions. A typical type of B lymphocytes, termed regulatory B cells, is confirmed to attenuate immune response in a tumor environment. In this paper, we summarize the current understanding of B-cell functions in tumor immunology, which may shed light on potential therapeutic strategies against cancer.

Runt-related transcription factor 1 (Runx1), a master regulator of hematopoiesis, is expressed in preosteoclasts. Previously we evaluated the bone phenotype of CD11b-Cre Runx1(fl/fl) mice and demonstrated enhanced osteoclasts and decreased bone mass in males. However, an assessment of the effects of Runx1 deletion in female osteoclast precursors was impossible with this model. Moreover, the role of Runx1 in myeloid cell differentiation into other lineages is unknown. Therefore, we generated LysM-Cre Runx1(fl/fl) mice, which delete Runx1 equally (∼80% deletion) in myeloid precursor cells from both sexes and examined the capacity of these cells to differentiate into osteoclasts and phagocytic and antigen-presenting cells. Both female and male LysM-Cre Runx1(fl/fl) mice had decreased trabecular bone mass (72% decrease in bone volume fraction) and increased osteoclast number (2-3 times) (P < .05) without alteration of osteoblast histomorphometric indices. We also demonstrated that loss of Runx1 in pluripotential myeloid precursors with LysM-Cre did not alter the number of myeloid precursor cells in bone marrow or their ability to differentiate into phagocytizing or antigen-presenting cells. This study demonstrates that abrogation of Runx1 in multipotential myeloid precursor cells significantly and specifically enhanced the ability of receptor activator of nuclear factor-κB ligand to stimulate osteoclast formation and fusion in female and male mice without affecting other myeloid cell fates. In turn, increased osteoclast activity in LysM-Cre Runx1(fl/fl) mice likely contributed to a decrease in bone mass. These dramatic effects were not due to increased osteoclast precursors in the deleted mutants and argue that inhibition of Runx1 in multipotential myeloid precursor cells is important for osteoclast formation and function.

T cell receptor (TCR) engagement opens Ca(2+) release-activated Ca(2+) (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca(2+) influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca(2+)-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca(2+) as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca(2+) influx may modulate TCR signaling.

Endocytosis is a fundamental cell biological process, which carries out essential functions in a polarized epithelial cell such as enterocytes provided with a huge surface area of the brush border membrane. Major tasks of enterocytes, which are regulated by endocytic signals, are digestion and absorption of nutrients and drugs/pharmacological agents, barrier permeability to microorganism, toxins and antigens, and transcytotic crosstalk between intestinal lumen and lamina propria cells with access to the circulation.Investigations on inflammatory bowel diseases such as food allergy, celiac disease, Crohn's disease, and ulcerative colitis focus on immune processes originating within enterocytes as antigen presenting cells. Thus the initiation of oral tolerance, that is, the binding of food antigens to MHC class II proteins, might be localized within late endosomes of enterocytes. Furthermore, the late endosomal compartment of enterocytes seems to be involved in the processing of luminal antigens during the pathogenesis of celiac disease and inflammatory bowel diseases. Investigations of inherited diseases such as microvillus inclusion disease have revealed a pathogenetic defect in the autophagocytotic and/or recycling pathway of enterocytes.Our progress in the cell and molecular biological understanding of the endocytosis and the methodical opportunities of translational research offer now new therapeutic options for patients suffering from endocytosis-related diseases of enterocytes.

Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.

In addition to its well-documented involvement in mineral homeostasis, vitamin D seems to have broad effects on human health that go beyond the skeletal system. Prominent among these so-called nonclassical effects of vitamin D are its immunomodulatory properties. In vitro studies have shown anti-inflammatory effects of 1,25-dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin D. In addition, epidemiological analysis of patients with established inflammatory disease identified associations between vitamin D deficiency (low serum concentrations of inactive 25-hydroxyvitamin D, abbreviated to 25(OH)D) and inflammatory conditions, including rheumatoid arthritis (RA). The association of vitamin D deficiency with RA severity supports the hypothesis of a role for vitamin D in the initiation or progression of the disease, or possibly both. However, whether 25(OH)D status is a cause or consequence of RA is still incompletely understood and requires further analysis in prospective vitamin D supplementation trials. The characterization of factors that promote the transition from preclinical to clinical phases of RA has become a major focus of research, with the aim to facilitate earlier diagnosis and treatment, and improve therapeutic outcomes. In this Review, we aim to describe the current knowledge of vitamin D and the immune system specifically in RA, and discuss the potential benefits that vitamin D might have on slowing RA progression.

B cells play a central role in the immunopathogenesis of glomerulonephritides and transplant rejection. B cells secrete antibodies that contribute to tissue injury via multiple mechanisms. In addition, B cells contribute to disease pathogenesis in autoimmunity and alloimmunity by presenting antigens as well as providing costimulation and cytokines to T cells. B cells also play an immunomodulatory role in regulating the immune response by secreting cytokines that inhibit disease onset and/or progression. B cell-targeted approaches for treating immune diseases of the kidney and other organs have gained significant momentum. However, much remains to be understood about B-cell biology in order to determine the timing, duration, and context of optimal therapeutic response to B cell-targeted approaches. In this review, we discuss the multifaceted roles of B cells as enhancers and regulators of immunity with relevance to kidney disease and transplantation., (Copyright © 2016 by the American Society of Nephrology.)

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS., (© 2015 The Authors.)

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that involves the slow, progressive destruction of islet β cells and loss of insulin production, as a result of interaction with environmental factors, in genetically susceptible individuals. The gut microbiome is established very early in life. Commensal microbiota establish mutualism with the host and form an important part of the environment to which individuals are exposed in the gut, providing nutrients and shaping immune responses. In this study, we studied the impact of targeting most Gram-negative bacteria in the gut of NOD mice at different time points in their life, using a combination of three antibiotics--neomycin, polymyxin B, and streptomycin--on diabetes development. We found that the prenatal period is a critical time for shaping the immune tolerance in the progeny, influencing development of autoimmune diabetes. Prenatal neomycin, polymyxin B, and streptomycin treatment protected NOD mice from diabetes development through alterations in the gut microbiota, as well as induction of tolerogenic APCs, which led to reduced activation of diabetogenic CD8 T cells. Most importantly, we found that the protective effect was age dependent, and the most profound protection was found when the mice were treated before birth. This indicates the importance of the prenatal environment and early exposure to commensal bacteria in shaping the host immune system and health., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c(+) lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers.

Type 1 diabetes (T1D) is currently an incurable disease, characterized by a silent prodromal phase followed by an acute clinical phase, reflecting progressive autoimmune destruction of insulin-producing pancreatic β-cells. Autoreactive T cells play a major role in β-cell destruction, but innate immune cell cytokines and costimulatory molecules critically affect T-cell functional status. We show that an agonistic monoclonal antibody to TLR4/MD-2 (TLR4-Ab) reverses new-onset diabetes in a high percentage of NOD mice. TLR4-Ab induces antigen-presenting cell (APC) tolerance in vitro and in vivo, resulting in an altered cytokine profile, decreased costimulatory molecule expression, and decreased T-cell proliferation in APC:T-cell assays. TLR4-Ab treatment increases T-regulatory cell (Treg) numbers in both the periphery and the pancreatic islet, predominantly expanding the Helios(+)Nrp-1(+)Foxp3(+) Treg subset. TLR4-Ab treatment in the absence of B cells in NOD.scid mice prevents subsequent T cell-mediated disease, further suggesting a major role for APC tolerization in disease protection. Specific stimulation of the innate immune system through TLR4/MD-2, therefore, can restore tolerance in the aberrant adaptive immune system and reverse new-onset T1D, suggesting a novel immunological approach to treatment of T1D in humans., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The macrophage cell system was identified by Metchnikoff on the basis of its phagocytic ability. Later on, the reticulohistiocytic system was defined as being composed of antigen-presenting reticulum cells and macrophages. Van Furth proposed that the mononuclear phagocyte system includes all tissue macrophages as well as antigen-presenting cells and blood monocytes as their precursors. Recent findings have shown that blood monocytes are not just transient forms involved in the recruitment of macrophages but that different dendritic and monocytic subpopulations can be observed in blood. In tissue, self-renewing macrophages derived from the yolk sac as well as monocyte-derived dendritic cells and monocyte-derived macrophages can be distinguished. Due to their plasticity and polarization, under inflammatory conditions monocyte-derived macrophages may be beneficial for the reestablishment of homeostasis or may contribute to mostly chronic diseases. Because of their ubiquitous distribution, monocytes and macrophages are increasingly considered to be possible therapeutic targets.

T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/-80 Pa), dendritic cells (DCs, 440 +110/-90 Pa), and macrophages (MPHs, 900 +110/-100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young's modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)