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Smooth muscle antibodies (SMA) with anti-microfilament actin (MF-SMA) specificity are regarded as highly specific markers of type 1 autoimmune hepatitis (AIH-1) but their recognition relying on immunofluorescence of vessel, glomeruli, and tubules (SMA-VGT pattern) in rodent kidney tissue, is restricted by operator-dependent interpretation. A gold standard method for their identification is not available. We assessed and compared the diagnostic accuracy for AIH-1 of an embryonal aorta vascular smooth muscle (VSM) cell line-based assay with those of the rodent tissue-based assay for the detection of MF-SMA pattern in AIH-1 patients and controls. Sera from 138 AIH-1 patients and 295 controls (105 primary biliary cholangitis, 40 primary sclerosing cholangitis, 50 chronic viral hepatitis, 20 alcohol-related liver disease, 40 steatotic liver disease, and 40 healthy controls) were assayed for MF-SMA and SMA-VGT using VSM-based and rodent tissue-based assays, respectively. MF-SMA and SMA-VGT were found in 96 (70%) and 87 (63%) AIH-1 patients, and 2 controls (P < 0.0001). Compared with SMA-VGT, MF-SMA showed similar specificity (99%), higher sensitivity (70% vs 63%, P = ns) and likelihood ratio for a positive test (70 vs 65). Nine (7%) AIH-1 patients were MF-SMA positive despite being SMA-VGT negative. Overall agreement between SMA-VGT and MF-SMA was 87% (kappa coefficient 0.870, [0.789-0.952]). MF-SMA were associated with higher serum γ-globulin [26 (12-55) vs 20 g/l (13-34), P < 0.005] and immunoglobulin G (IgG) levels [3155 (1296-7344) vs 2050 mg/dl (1377-3357), P < 0.002]. The easily recognizable IFL MF-SMA pattern on VSM cells strongly correlated with SMA-VGT and has an equally high specificity for AIH-1. Confirmation of these results in other laboratories would support the clinical application of the VSM cell-based assay for reliable detection of AIH-specific SMA., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Antigen processing and antigen-specific CD8 T cell activation form part and parcel of cell-mediated immunity to infections. Yet, several lacunae remain in our understanding of how antigen processing and CD8 T cell response are coordinated. In this study, using mouse bone marrow-derived dendritic cells (BMDC) as antigen-presenting cells and Ovalbumin (OVA)/DQ-Ovalbumin (DQ-OVA) as model antigen we demonstrated that Wnt5A signaling in BMDC supports antigen processing/presentation and concomitant CD8 T cell activation through regulation of actin and proteasome dynamics. Recombinant Wnt5A conditioning of BMDC and associated actin assembly facilitated DQ-OVA processing, which was inhibited by the proteasome inhibitor MG132. Moreover, Wnt5A depletion led to a significant reduction in OVA processing and presentation. Impaired DQ-OVA processing in Wnt5A depleted BMDC correlated with altered dynamics of both actin and the proteasome regulator PA28α-PA28β, and reduced association of DQ-OVA with actin and proteasome subunits. Inhibited OVA processing/presentation in the Wnt5A depleted BMDC also resulted in subdued activation of OVA-sensitized CD8 T cells in co-culture with the BMDC. In concurrence with these findings, we demonstrated reduced OVA processing and impaired CD8 T cell response to OVA immunization in Wnt5A heterozygous mice lacking a copy of the Wnt5A gene in comparison to the wild-type cohorts. Taken together, our results reveal a crucial requirement of Wnt5A signaling in antigen processing/presentation and CD8 T cell activation, thus unveiling a vital regulatory node of cell-mediated immunity, unidentified thus far., 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 © 2022 Sarraf and Sen.)

Naïve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions, T cell activation, and T cell expansion in vivo . Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Finally, we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions, which promotes the initiation of stable T cell conjugates during APC scanning. Therefore, Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo ., 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 © 2022 Waldman, Rahkola, Sigler, Chung, Willett, Kedl, Friedman and Jacobelli.)

Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity., (© 2022. The Author(s).)

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca 2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions., 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 © 2022 Kim, Yi, Kwon, Park, Kwon and Kim.)

Beta-actin (ACTB), a highly conserved cytoskeleton structural protein, has been regarded as a common housekeep gene and used as a reference gene for years. However, accumulating evidence indicates that ACTB is abnormally expressed in multiple cancers and hence changes the cytoskeleton to affect the invasiveness and metastasis of tumors. This study aimed to investigate the function and clinical significance of ACTB in pan-cancer. The role of ACTB for prognosis and immune regulation across 33 tumors was explored based on the datasets of gene expression omnibus and the cancer genome atlas. Differential expression of ACTB was found between cancer and adjacent normal tissues, and significant associations was found between ACTB expression and prognosis of tumor patients. In most cancers, ACTB expression was associated with immune cells infiltration, immune checkpoints and other immune modulators. Relevance between ACTB and metastasis and invasion was identified in various types of cancers by CancerSEA. Moreover, focal adhesion and actin regulation-associated pathways were included in the functional mechanisms of ACTB. The expression of ACTB was verified by quantitative real-time polymerase chain reaction. Knockdown of ACTB inhibited head and neck squamous carcinoma cell migration and invasion by NF-κB and Wnt/β-catenin pathways. Our first pan-cancer study of ACTB offers insight into the prognostic and immunological roles of ACTB across different tumors, indicating ACTB may be a potential biomarker for poor prognosis and immune infiltration in cancers, and the role of ACTB as a reference gene in cancers was challenged.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI ( capri kd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3β, and cofilin; and excessive actin polymerization. capri kd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients., Competing Interests: The authors declare no competing interest.

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 membrane-anchored 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., Competing Interests: The authors declare no competing interest.

Monogenic diseases of the immune system, also known as inborn errors of immunity (IEIs), are caused by single-gene mutations and result in immune deficiency and dysregulation. More than 400 monogenic diseases have been described to date, and this number is rapidly expanding. The increasing availability of next-generation sequencing is now facilitating the diagnosis of IEIs. It is known that IEIs can predispose a person to not only infectious diseases but also cancer and immune disorders, such as inflammatory, autoimmune, and atopic diseases. IEIs with eosinophilia and atopic diseases can occur in several disorders. IEIs with eosinophilia have provided insights into human immunity and the pathogenesis of allergic diseases. Eosinophilia is not a rare finding in clinical practice, and it often poses problems in terms of etiologic research and differential diagnoses. Secondary eosinophilia is the most common form. The main underlying conditions are infectious diseases such as parasitic infections, allergic disorders, drug reactions, and of course IEIs. In clinical settings, the recognition of IEIs in the context of an allergic phenotype with eosinophilia is critical for prompt diagnosis and appropriate treatment aimed at modulating pathophysiological mechanisms and improving clinical symptoms., (Copyright © 2021 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Objective: The parasite Trichomonas vaginalis (T. vaginalis) causes one of the most common non-viral sexually transmitted infections in humans. T. vaginalis is notorious for its inconspicuous appearance in vaginal smears. It can be missed under the microscope., Method: In the present study, we investigate the immunoreactivity of T. vaginalis to smooth muscle actin (SMA) in the vaginal smear., Result: T. vaginalis trophozoite and pseduocyst are immunoreactive for SMA in all of the study group cases (n = 21) and in none of the control group cases (n = 21). Thus, SMA immunostain is a sensitive method for the demonstration of T. vaginalis. Moreover, the protozoan attains a conspicuous and unique appearance. By SMA immunohistochemical stain, the apperance of T. vaginalis floated freely or located in the cytoplasm of the epithelial cells is easily identified., Conclusion: We recommend performing SMA immunostain in every vaginal smear with clinical or pathologic suspicion of trichomoniasis., (© 2021 Wiley Periodicals LLC.)

The tunicate, Styela plicata (Lesueur, 1823) present an open circulator system with a tubular heart and blood flowing in lacunae among organs, bathing the tissues directly. Blood vascular lacunae are present in the tunica that is situated outside the epidermis and present a fibrous structure. The cells of the tunic are in straight contact with the blood vessels or are highly mobile. Ascidians are considered model organisms in comparative immunology of the chordate, and hold an important phylogenetic position as sister group of vertebrates. In recent years, numerous studies have reported the presence of Toll-like receptors (TLRs) in the genome of non-mammalian organisms including invertebrates. Two TLRs, designated Ci-TLR1 and Ci-TLR2 were expressed in the stomach, intestine and in numerous hemocytes of Ciona intestinalis, demonstrating that these key transmembrane proteins are evolutionarily conserved in ascidians. In this study for the first time, hemocytes aggregates were identified by confocal immunofluorescence techniques, using TLR2 antibody in the tunica of Styela plicata; furthermore, α-Smooth Muscle Actin (α-SMA) expression has been shown in the cells lining the vessels of the tunic. Our results support the view that the TLR-mediated innate immune functions are conserved in ascidian tissues., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Immune cells identify and destroy tumors by recognizing cellular traits indicative of oncogenic transformation. In this study, we found that myocardin-related transcription factors (MRTFs), which promote migration and metastatic invasion, also sensitize cancer cells to the immune system. Melanoma and breast cancer cells with high MRTF expression were selectively eliminated by cytotoxic lymphocytes in mouse models of metastasis. This immunosurveillance phenotype was further enhanced by treatment with immune checkpoint blockade (ICB) antibodies. We also observed that high MRTF signaling in human melanoma is associated with ICB efficacy in patients. Using biophysical and functional assays, we showed that MRTF overexpression rigidified the filamentous actin cytoskeleton and that this mechanical change rendered mouse and human cancer cells more vulnerable to cytotoxic T lymphocytes and natural killer cells. Collectively, these results suggest that immunosurveillance has a mechanical dimension, which we call mechanosurveillance, that is particularly relevant for the targeting of metastatic disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background Information: We have previously observed that in response to antigenic activation, T cells produce actin-rich protrusions that generate forces involved in T cell activation. These forces are influenced by the mechanical properties of antigen-presenting cells (APCs). However, how external forces, which can be produced by APCs, influence the dynamic of the actin protrusion remains unknown. In this study, we quantitatively characterised the effects of external forces in the dynamic of the protrusion grown by activated T cells., Results: Using a micropipette force probe, we applied controlled compressive or pulling forces on primary T lymphocytes activated by an antibody-covered microbead, and measured the effects of these forces on the protrusion generated by T lymphocytes. We found that the application of compressive forces slightly decreased the length, the time at which the protrusion stops growing and retracts and the velocity of the protrusion formation, whereas pulling forces strongly increased these parameters. In both cases, the applied forces did not alter the time required for the T cells to start growing the protrusion (delay). Exploring the molecular events controlling the dynamic of the protrusion, we showed that inhibition of the Arp2/3 complex impaired the dynamic of the protrusion by reducing both its maximum length and its growth speed and increasing the delay to start growing. Finally, T cells developed similar protrusions in more physiological conditions, that is, when activated by an APC instead of an activating microbead., Conclusions: Our results suggest that the formation of the force-generating protrusion by T cells is set by an intracellular constant time and that its dynamic is sensitive to external forces. They also show that actin assembly mediated by actin-related protein Arp2/3 complex is involved in the formation and dynamic of the protrusion., Significance: Actin-rich protrusions developed by T cells are sensory organelles that serve as actuators of immune surveillance. Our study shows that forces experienced by this organelle modify their dynamic suggesting that they might modify immune responses. Moreover, the quantitative aspects of our analysis should help to get insight into the molecular mechanisms involved in the formation of the protrusion., (© 2021 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Dendritic cells (DCs) are crucial for the priming of naive T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell-cell contact, the immunological synapse (IS). While it is established that F-actin dynamics regulates signaling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. Here, we show that the DC actin cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. DC actin at the IS appears in transient foci that are dynamized by the WAVE regulatory complex (WRC). The absence of the WRC in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin-mediated cell-cell adhesion. This results in lower numbers of activated and proliferating T cells, demonstrating an important role for DC actin in the regulation of immune synapse functionality., (© 2021 Leithner et al.)

Although immunotherapy holds promising cytotoxic activity against lymphoma or leukemia, the immunosuppressive mechanisms of solid tumors remain challenging. In this study, we developed and applied a hypergravity exposure system as a novel strategy to improve the responsiveness of breast cancer cells to natural killer (NK) cells for efficient immunotherapy. Following exposure to hypergravity, either in the presence or absence of NK cells, we investigated for changes in the cell cytoskeletal structure, which is related to the F-actin mediated immune evasion mechanism (referred to as "actin response") of cancer cells. Breast cancer cell line MDA-MB-231 cells were exposed thrice to a 20 min hypergravitational condition (10 × g), with a 20 min rest period between each exposure. The applied hypergravity induces changes in the intracellular cytoskeleton structure without decreasing the cell viability but increasing the cytotoxicity of MDA-MB-231 from 4 to 18% (4.5-fold) at a 3:1 ratio (NK-to-target). Analyses related to F-actin further demonstrate that the applied hypergravity results in rearrangement of the cytoskeleton, leading to inhibition of the actin response of MDA-MB-231. Taken together, our results suggest that the mechanical load increases through application of hypergravity, which potentially improves efficiency of cell-based immunotherapies by sensitizing tumors to immune cell-mediated cytotoxicity.

Cellular function is reliant on the dynamic interplay between the plasma membrane and the actin cytoskeleton. This critical relationship is of particular importance in immune cells, where both the cytoskeleton and the plasma membrane work in concert to organize and potentiate immune signaling events. Despite their importance, there remains a critical gap in understanding how these respective dynamics are coupled, and how this coupling in turn may influence immune cell function from the bottom up. In this review, we highlight recent optical technologies that could provide strategies to investigate the simultaneous dynamics of both the cytoskeleton and membrane as well as their interplay, focusing on current and future applications in immune cells. We provide a guide of the spatio-temporal scale of each technique as well as highlighting novel probes and labels that have the potential to provide insights into membrane and cytoskeletal dynamics. The quantitative biophysical tools presented here provide a new and exciting route to uncover the relationship between plasma membrane and cytoskeletal dynamics that underlies immune cell function., 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 Schneider, Colin-York and Fritzsche.)

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1 -/- ) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1 -/- mice. Inhibition of macrophage IL-1β production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO 3 ) 2 NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO 3 ) 2 NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO 3 ) 2 NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB ( NF-κB ) and selected cytokines, such as tumor necrosis factor alpha ( TNFα ), transforming growth factor beta 1 ( TGFβ1 ), interleukin 6( IL-6 ), IL-1α and IL-1β , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO 3 ) 2 NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.

Skeletal muscle myopathies represent a common non-pulmonary manifestation of influenza infection, leading to reduced physical function and hospitalization in older adults. However, underlying mechanisms remain poorly understood. Our study examined the effects of influenza virus A pulmonary infection on contractile function at the cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) levels in soleus and extensor digitorum longus muscles of aged (20 months) C57BL/6 male mice that were healthy or flu-infected for 7 (7-days post-infection; 7-DPI) or 12 days (12-DPI). Cross-sectional area (CSA) of myosin heavy chain (MHC) IIA and IIB fibers was reduced at 12-DPI relative to 7-DPI and healthy. Maximal isometric force in MHC IIA fibers was also reduced at 12-DPI relative to 7-DPI and healthy, resulting in no change in specific force (maximal isometric force divided by CSA). In contrast, MHC IIB fibers produced greater isometric force and specific force at 7-DPI compared to 12-DPI or healthy. The increased specific force in MHC IIB fibers was likely due to greater myofilament lattice stiffness and/or an increased number or stiffness of strongly bound myosin-actin cross-bridges. At the molecular level, cross-bridge kinetics were slower in MHC IIA fibers with infection, while changes in MHC IIB fibers were largely absent. In both fiber types, greater myofilament lattice stiffness was positively related to specific force. This study provides novel evidence that cellular and molecular contractile function is impacted by influenza infection in a fiber type-specific manner, suggesting potential molecular mechanisms to help explain the impact of flu-induced myopathies., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Peritoneal metastasis (PM) in gastric cancer (GC) is characterized by diffusely infiltrating and proliferating cancer cells accompanied by extensive stromal fibrosis in the peritoneal space. The prognosis of GC with PM is still poor regardless of the various current treatments. In order to elucidate the cause of difficulties in PM treatment, we compared the tumor immune microenvironment (TME) in primary and PM lesions in GC. In addition, a PM model with fibrous stroma was constructed using immunocompetent mice to determine whether its TME was similar to that in patients., Methods: Immuno-histochemical analyses of infiltrating immune cells were performed in paired primary and PM lesions from 28 patients with GC. A C57BL/6 J mouse model with PM was established using the mouse GC cell line YTN16 either with or without co-inoculation of mouse myofibroblast cell line LmcMF with α-SMA expression. The resected PM from each mouse model was analyzed the immunocompetent cells using immunohistochemistry., Results: The number of CD8 + cells was significantly lower in PM lesions than in primary lesions (P < 0.01). Conversely, the number of CD163 + cells (M2 macrophages) was significantly higher in PM lesions than in primary lesions (P = 0.016). Azan staining revealed that YTN16 and LmcMF co-inoculated tumors were more fibrous than tumor with YTN16 alone (P < 0.05). Co-inoculated fibrous tumor also showed an invasive growth pattern and higher progression than tumor with YTN16 alone (P = 0.045). Additionally, YTN16 and LmcMF co-inoculated tumors showed lower infiltration of CD8 + cells and higher infiltration of M2 macrophages than tumors with YTN16 alone (P < 0.05, P < 0.05). These results indicate that LmcMF plays as cancer-associated fibroblasts (CAFs) by crosstalk with YTN16 and CAFs contribute tumor progression, invasion, fibrosis, and immune suppression., Conclusions: This model is the first immunocompetent mouse model similar to TME of human clinical PM with fibrosis. By using this model, new treatment strategies for PM, such as anti-CAFs therapies, may be developed.

T cell responses to antigen are initiated by engagement of the T cell receptor (TCR) 1 , leading to activation of diverse signaling cascades, including an incompletely defined pathway that triggers rapid remodeling of the actin cytoskeleton. Defects in the control of actin dynamics and organization are associated with several human immunodeficiency diseases, emphasizing the importance of cytoskeletal remodeling in the functioning of the adaptive immune system. Here, we investigate the role of the adaptor protein Bcl10 2 in the control of actin dynamics. Although Bcl10 is primarily known as a component of the pathway connecting the TCR to activation of the NF-κB 3 transcription factor, a few studies have implicated Bcl10 in antigen receptor-dependent control of actin polymerization and F-actin-dependent functional responses. However, the role of Bcl10 in the regulation of cytoskeletal dynamics remains largely undefined. To investigate the contribution of Bcl10 in the regulation of TCR-dependent cytoskeletal dynamics, we monitored actin dynamics at the immune synapse of primary murine CD8 effector T cells. Quantification of these dynamics reveals two distinct temporal phases distinguished by differences in speed and directionality. Our results indicate that effector CD8 T cells lacking Bcl10 display faster actin flows and more dynamic lamellipodia, compared to wild-type cells. These studies define a role for Bcl10 in TCR-dependent actin dynamics, emphasizing that Bcl10 has important cytoskeleton-directed functions that are likely independent of its role in transmission of NF-κB -activating signals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aliphatic di- and polyisocyanates are crucial chemical ingredients in many industrial processes and are a well-recognized cause of occupational asthma. Serologic detection of "chemical epitopes" in biological samples could serve as an exposure surveillance approach toward disease prevention, and thus we sought to generate aliphatic isocyanate-specific monoclonal antibodies (mAbs). Three hybridomas were generated from Balb/c mice immunized with a commercial product containing a combination of uretdione, homopolymer, and monomeric forms of hexamethylene diisocyanate (HDI). Three stable hybridomas were subcloned by limiting dilution, two secreting IgG1κ and one secreting IgMκ mAb that bind aliphatic di- and polyisocyanates (conjugated to albumin), but not aromatic toluene or methylene diphenyl diisocyanate (TDI or MDI). Each mAb demonstrates slight differences in epitope specificity, for example, recognition of hydrogenated MDI (HMDI) or different carrier proteins (transferrin, actin) reacted with vapor phase HDI, and is encoded by unique recombination of different germline antibody genes, with distinct complementary determining regions. By western blot, all three mAbs detect a molecule with characteristics of an albumin adduct uniquely in urine from mice skin exposed to a mixture of aliphatic di- and polyisocyanate. Together, the data define molecular determinants of humoral immune recognition of aliphatic di- and polyisocyanates through new mAbs, which will serve as useful research reagents and may be applicable to future exposure surveillance efforts.

CXCL8 is the principal human neutrophil-attracting chemokine and a major mediator of inflammation. The chemokine exerts its neutrophil-chemotactic and neutrophil-activating activities via interaction with glycosaminoglycans (GAGs) and activation of the G protein-coupled receptors (GPCRs) CXCR1 and CXCR2. Natural CXCL8 displays an exceptional degree of amino (NH 2 )-terminal heterogeneity. Most CXCL8 forms result from proteolytic processing of authentic CXCL8(1-77). Here, we compared the potencies to activate and recruit neutrophils of the 3 most abundant natural CXCL8 forms: full-length 77 amino acid CXCL8 and the 2 major natural truncated forms lacking 5 or 8 NH 2 -terminal amino acids. NH 2 -terminal truncation hardly affected the capacity of CXCL8 to induce shedding of CD62L or to up-regulate the expression of the adhesion molecules CD11a, CD11b, or CD15 on human neutrophils. In addition, the potency of CXCL8 to induce neutrophil degranulation and its effect on phagocytosis remained unaltered upon removal of 5 or 8 NH 2 -terminal residues. However, NH 2 -terminal truncation strongly potentiated CXCL8-induced actin polymerization. CXCL8(6-77) and CXCL8(9-77) showed a comparable capacity to induce Ca 2+ signaling in human neutrophils and to direct in vitro neutrophil migration. Strikingly, the ability of CXCL8(9-77) to recruit neutrophils into the peritoneal cavity of mice was significantly enhanced compared to CXCL8(6-77). These results suggest that NH 2 -terminal truncation influences specific biological activities of CXCL8 and indicate that CXCL8(9-77) may be the most potent neutrophil-attracting CXCL8 form in vivo., (©2020 Society for Leukocyte Biology.)

Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4 + T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4 + T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling.

Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε., (© 2019 John Wiley & Sons Ltd.)

Activation of naive T cells by antigen-presenting cells (APCs) is an essential step in mounting an adaptive immune response. It is known that antigen recognition and T cell receptor (TCR) signaling depend on forces applied by the T cell actin cytoskeleton, but until recently, the underlying mechanisms have been poorly defined. Here, we review recent advances in the field, which show that specific actin-dependent structures contribute to the process in distinct ways. In essence, T cell priming involves a tug-of-war between the cytoskeletons of the T cell and the APC, where the actin cytoskeleton serves as a mechanical intermediate that integrates force-dependent signals. We consider each of the relevant actin-rich T cell structures separately and address how they work together at the topologically and temporally complex cell-cell interface. In addition, we address how this mechanobiology can be incorporated into canonical immunological models to improve how these models explain T cell sensitivity and antigenic specificity., (© 2020 Blumenthal and Burkhardt.)

The cytoskeleton is a central factor contributing to various hallmarks of cancer. In recent years, there has been increasing evidence demonstrating the involvement of actin regulatory proteins in malignancy, and their dysregulation was shown to predict poor clinical prognosis. Although enhanced cytoskeletal activity is often associated with cancer progression, the expression of several inducers of actin polymerization is remarkably reduced in certain malignancies, and it is not completely clear how these changes promote tumorigenesis and metastases. The complexities involved in cytoskeletal induction of cancer progression therefore pose considerable difficulties for therapeutic intervention; it is not always clear which cytoskeletal regulator should be targeted in order to impede cancer progression, and whether this targeting may inadvertently enhance alternative invasive pathways which can aggravate tumor growth. The entire constellation of cytoskeletal machineries in eukaryotic cells are numerous and complex; the system is comprised of and regulated by hundreds of proteins, which could not be covered in a single review. Therefore, we will focus here on the actin cytoskeleton, which encompasses the biological machinery behind most of the key cellular functions altered in cancer, with specific emphasis on actin nucleating factors and nucleation-promoting factors. Finally, we discuss current therapeutic strategies for cancer which aim to target the cytoskeleton., Competing Interests: Conflict of interest The authors declare that the review was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2020 Elsevier Inc. All rights reserved.)

Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction., Competing Interests: SR, LW, DM, MW, SC, PT No competing interests declared, (© 2019, Roper et al.)

α M β 2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of α M β 2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained β 2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

Ibuprofen, a medication in the nonsteroidal anti-inflammatory drug class, is widely used for treating inflammatory diseases such as osteoarthritis. It has been shown in recent years that ibuprofen has a strong effect on Ras homolog gene family, member A (RhoA) inhibition in multiple cell types. Our previous finding also demonstrated that interleukin-1β (IL-1β) increases filamentous actin (F-actin) of chondrocytes via RhoA pathway. Therefore, we hypothesized that ibuprofen may suppress the IL-1β-induced F-actin upregulation in chondrocytes by inhibiting RhoA pathway. To this end, in this study, articular chondrocytes from New Zealand White rabbits were pretreated with 500 μM ibuprofen for 2 h, then with 10 ng/ml IL-1β for 24 h. Results showed that pretreatment with ibuprofen inhibited the IL-1β-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, protected the chondrocyte phenotype from IL-1β stimulation, and inhibited the IL-1β-induced actin remodeling via RhoA signaling modulation. In conclusion, ibuprofen showed not only anti-inflammatory function, but also RhoA inhibition in articular chondrocytes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Late diplotene oocytes are characterized by an essential decrease in transcriptional activity. At this time, chromosomes condense and form a compact structure named a karyosphere. The karyosphere of grass frogs Rana temporaria is surrounded by a fibrillar karyosphere capsule (KC). One of the main protein constituents of R. temporaria KC is actin. In this study, we used antibodies against different actin epitopes to trace different forms of actin in the KC. We also investigated the effect of F-actin depolymerization on the oocyte nuclear structures and transcription of chromatin DNA and rDNA in the amplified nucleoli. It was determined that disruption of actin filaments leads to chromosome shrinkage, nucleoli fusion, and distortion of the KC structure, but does not inhibit residual transcription in both the karyosphere and the nucleoli., (© 2019 Wiley Periodicals, Inc.)

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown., (© 2019 Danquah et al.)

Immunological synapse (IS) formation between a T cell and an antigen-presenting cell is accompanied by the reorientation of the T cell centrosome toward the interface. This polarization response is thought to enhance the specificity of T cell effector function by enabling the directional secretion of cytokines and cytotoxic factors toward the antigen-presenting cell. Centrosome reorientation is controlled by polarized signaling through diacylglycerol (DAG) and protein kinase C (PKC). This drives the recruitment of the motor protein dynein to the IS, where it pulls on microtubules to reorient the centrosome. Here, we used T cell receptor photoactivation and imaging methodology to investigate the mechanisms controlling dynein accumulation at the synapse. Our results revealed a remarkable spatiotemporal correlation between dynein recruitment to the synaptic membrane and the depletion of cortical filamentous actin (F-actin) from the same region, suggesting that the two events were causally related. Consistent with this hypothesis, we found that pharmacological disruption of F-actin dynamics in T cells impaired both dynein accumulation and centrosome reorientation. DAG and PKC signaling were necessary for synaptic F-actin clearance and dynein accumulation, while calcium signaling and microtubules were dispensable for both responses. Taken together, these data provide mechanistic insight into the polarization of cytoskeletal regulators and highlight the close coordination between microtubule and F-actin architecture at the IS., Competing Interests: The authors have declared that no competing interests exist.

Dietary products may protect against inflammatory bowel disease (IBD) through mechanisms such as forming gut microbiota structures and providing substrates for microbial metabolism. Recently, many studies have been conducted on diets that potentially alleviate or suppress IBD development. To assess the efficacy of dietary oils in treating IBD, we examined the protective effects of olive oil, coconut oil, corn oil, and cottonseed oil in a dextran sodium sulfate (DSS)-induced colitis mouse model. Treatment with cottonseed oil or corn oil ameliorated the severity of DSS-induced colitis, alleviating weight loss and preventing the shortening of the intestine. Moreover, cottonseed oil or corn oil treatment significantly reduced the expression of inflammatory cytokines such as tumor necrosis factor- α , interleukin (IL)-1 β , IL-6, and IL-17, as well as the expression of oxidative stress markers, including 8-hydroxyguanosine and nitrotyrosine in colon sections, compared with vehicle treatment. Cottonseed oil treatment inhibited intestinal fibrosis by reducing the expression of α -smooth muscle actin and type I collagen, compared with vehicle treatment in mice with DSS-induced colitis. Cottonseed oil protects against intestinal inflammation and the development of intestinal fibrosis by reducing inflammatory cytokines and oxidative stress markers, and may therefore be useful as a dietary product with therapeutic benefits for IBD.

Lymphatic filariasis, a chronic disfiguring disease exhibits complex pathology. Based on different clinical manifestations, infected individuals are categorized into asymptomatic-carriers and chronic-patients. The mechanism behind differential clinical outcomes remains unclear. Roles of filaria-specific B cell responses in filariasis have been documented, whereas the contribution of B1 cell response and poly-specific IgG and IgA in the context of clinical filariasis is not deciphered. In this study, we measured the poly-specific IgG and IgA levels in different clinical categories of filariasis. Asymptomatic-carriers exhibited increased IgG4 antibodies against both filarial-antigens as well as auto-antigens compared to other clinical categories, although IgG against these auto-antigens remained lower. IgA levels against both filarial and auto-antigens were decreased in asymptomatic-carriers. A positive correlation between anti-filarial IgG4 and IgG4 against auto-antigens were observed, suggesting the synergistic role of poly-specific natural IgG4 with anti-filarial IgG4 in blocking the pathogenesis in asymptomatic microfilarial cases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The microvascular architecture of the spleen plays an important role in the immunological function of this organ. The different types of vessels are related to different reticular cells each with their own immunomodulatory functions. The present study describes an immunohistochemical and morphometric analysis of the various types of vessels in 21 human autopsy non-pathological splenic samples. On an area of 785,656.37 μm 2 for each sample, we classified and quantified the type and number of vascular structures, each according to their morphology and immunohistochemical profile, and obtained the ratios between them. The distribution of trabecular vessels and the characteristics of the venules are reviewed. In our material the so-called "cavernous perimarginal sinus" (anatomical structure previously described by Schmidt et al., 1988) was observed and interpreted as a curvilinear venule shaped by the follicle in contact with the trabecular vein. Our material comprised 261 trabeculae (containing 269 arterial sections and 508 venous sections), 30,621 CD34+ capillaries, 7739 CD271+ sheathed capillaries, 2588 CD169+ sheathed capillaries, and 31,124 CD8+ sinusoids. The total area (TA) (14,765,714.88 μm 2 ) occupied by the sinusoidal sections of the 21 cases was much higher than the TA of the capillary sections (1,700,269.83 μm 2 ). Similarly, the TA (651,985 μm 2 ) occupied by the sections of the trabecular veins was much higher than the TA of the trabecular arteries (88,594 μm 2 ). The total number of CD34+ capillaries and of sinusoids CD8+ was similar for the sum of the 21 cases, nevertheless there were large differences in each case. Statistically the hypothesis that the number of capillaries and sinusoids are present with the same frequency is discarded. In view of the absence of a numerical correlation between capillaries and sinusoids, we postulate that very possibly the arterial and the venous vascular trees are two anatomically independent structures separated by the splenic cords. We believe that this is the first work where splenic microvascularization is simultaneously approached from a morphometric and immunohistochemical point of view., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Leukocytes are constantly produced in the bone marrow and released into the circulation. Many different leukocyte subpopulations exist that exert distinct functions. Leukocytes are recruited to sites of inflammation and combat the cause of inflammation via many different effector functions. Virtually all of these processes depend on dynamic actin remodeling allowing leukocytes to adhere, migrate, phagocytose, and release granules. However, actin dynamics are not possible without actin-binding proteins (ABP) that orchestrate the balance between actin polymerization, branching, and depolymerization. The homologue of the ubiquitous ABP cortactin in hematopoietic cells is hematopoietic cell-specific lyn substrate-1, often called hematopoietic cell-specific protein-1 (HCLS1 or HS1). HS1 has been reported in different leukocytes to regulate Arp2/3-dependent migration. However, more evidence is emerging that HS1 functions go far beyond just being a direct actin modulator. For example, HS1 is important for the activation of GTPases and integrins, and mediates signaling downstream of many receptors including BCR, TCR, and CXCR4. In this review, we summarize current knowledge on HS1 functions and discuss them in a pathophysiologic context., (©2018 Society for Leukocyte Biology.)

Phosphoinositides, together with the phospholipids phosphatidylserine and phosphatidic acid, are important components of the plasma membrane acting as second messengers that, with diacylglycerol, regulate a diverse range of signaling events converting extracellular changes into cellular responses. Local changes in their distribution and membrane charge on the inner leaflet of the plasma membrane play important roles in immune cell function. Here we discuss their distribution and regulators highlighting the importance of membrane changes across the immune synapse on the cytoskeleton and the impact on the function of cytotoxic T lymphocytes.

Background: Hepatitis C virus (HCV) and HIV infections are associated with higher risk of autoimmune diseases and T-cell dysfunction., Setting: We evaluate prevalence and factors associated with the presence of autoimmune antinuclear (ANA), anti-smooth muscle actin (aSMA), and anti-liver kidney microsome (aLKM1) antibodies (Ab) in HCV/HIV-coinfected patients during the post-combined antiretroviral therapy era., Methods: A cross-sectional observational study nested in the ANRS CO13 HEPAVIH cohort (NCT number: NCT03324633). We selected patients with both ANA testing and T-cell immunophenotyping determination during the cohort follow-up and collected aLKM1 and aSMA data when available. Logistic regression models were built to determine factors associated with the presence of auto-Ab., Results: Two hundred twenty-three HCV/HIV-coinfected patients fulfilled selection criteria. Prevalence of ANA and aSMA was 43.5% and 23.2%, respectively, and both were detected in 13.3% of patients. Isolated aSMA were detected in 9.9% and aLKM1 in 2 patients. In multivariable analysis, only a low nadir CD4 T-cell count was significantly associated with ANA detection., Conclusions: ANA and aSMA detection remain frequent in HCV/HIV-coinfected patients during the post-combined antiretroviral therapy era, despite fair immune restoration. These results advocate for a close monitoring of ANA before immune checkpoint inhibitor therapy in these patients with greater caution for those with a low nadir CD4 T-cell count.

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

CD5 and CD6 are related surface receptors that limit and promote T-cell responses. Co-stimulatory effects of CD6 depend on binding a cell surface ligand, CD166, and recruitment of the intracellular adaptor proteins GADS and SLP-76 by C-terminal phosphotyrosines. We have continued to identify interactions of CD5 and CD6 to understand their roles in T-cell activation. In a screen to identify binding partners for peptides containing a cytoplasmic sequence, SDSDY conserved between CD5 and CD6, we identified ezrin radixin moesin (ERM) proteins, which link plasma membrane proteins to actin. Purified radixin FERM domain bound directly to CD5 and CD6 SDSDY peptides in a phosphorylation-dependent manner (K D = 0·5-2 μm) at 37°. In human T-cell blasts, mutation of the CD6 SDSDY sequence enhanced CD69 expression in response to CD3 monoclonal antibody. In this proximal readout, interactions of the SDSDY sequence were dominant compared with the C-terminal tyrosines of CD6. In contrast, in a more downstream readout, interleukin-2 expression, in response to immobilized CD3 and CD6 monoclonal antibodies, the C-terminal tyrosines were dominant. The data suggest that varying functional effects of CD6 and potentially CD5 depend on interactions of different cytoplasmic regions with the cytoskeleton and alter depending on the stimuli., (© 2018 The Authors. Immunology Published by John Wiley & Sons Ltd.)

Background: One of the undesirable complications that might occur after breast augmentation with silicone implants is capsular contracture. In its etiology, the relations between mast cells and myofibroblasts play an important role in collagen synthesis. Mast cells are able to activate fibroblasts into myofibroblasts, through paracrine secretions, inducing collagen production. The objectives of this study were to analyze the myofibroblast concentration through the α-SMA immunomarker and evaluate the intensity of mast cell expression against the C-Kit immunomarker., Material and Method: Sixty-four Wistar rats were used, divided into two groups (polyurethane foam and textured surface) with 32 animals in each. The animals received silicone implants on the back, below the panniculus carnosus, and after the determined period, they were killed and the capsules formed around the implants were studied. The capsules were analyzed employing the immunohistochemical technique, with the α-SMA and C-Kit immunomarkers in subgroups of 30, 50, 70 and 90 days., Results: The myofibroblast concentration was higher in the polyurethane group when compared to the textured group (30 days p = 0.105; 50 days p = 0.247; 70 days p = 0.014 and 90 days p = 0.536). The intensity of mast cell expression was more pronounced in the polyurethane group when compared to the textured group (30 days p = 0.798; 50 days p = 0.537; 70 days p = 0.094 and 90 days p = 0.536)., Conclusions: Polyurethane-coated implants induced higher concentrations of myofibroblasts and higher expression of mast cells, when compared to the textured surface implants., No Level Assigned: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

T cell antigen receptor (TCR) signaling triggers selective cytokine expression to drive T cell proliferation and differentiation required for immune defense and surveillance. The nuclear signaling events responsible for specificity in cytokine gene expression upon T cell activation are largely unknown. Here, we uncover formation of a dynamic actin filament network in the nucleus that regulates cytokine expression for effector functions of CD4 + T lymphocytes. TCR engagement triggers the rapid and transient formation of a nuclear actin filament network via nuclear Arp2/3 complex, induced by elevated nuclear Ca 2+ levels and regulated via N-Wasp and NIK. Specific interference with TCR-induced formation of nuclear actin filaments impairs production of effector cytokines and prevents generation of antigen-specific antibodies but does not interfere with immune synapse formation and cell proliferation. Ca 2+ -regulated actin polymerization in the nucleus allows CD4 + T cells the rapid conversion of TCR signals into effector functions required for T cell help., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The regulatory role of most dual specific phosphatases during T cell activation remains unknown. Here, we have studied the expression and function of phosphatases of regenerating liver (PRLs: PRL-1, PRL-2, and PRL-3) during T cell activation, as well as, the dynamic delivery of PRL-1 to the Immunological Synapse (IS). We found that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio. PRL-1 redistributed at the IS in two stages: Initially, it was transiently accumulated at scanning membranes enriched in CD3 and actin, and at later times, it was delivered at the contact site from pericentriolar, CD3ζ-containing, vesicles. Once at the established IS, PRL-1 distributed to LFA-1 and CD3ε sites. Remarkably, PRL-1 was found to regulate actin dynamics during IS assembly and the secretion of IL-2. Moreover, pharmacological inhibition of the catalytic activity of the three PRLs reduced the secretion of IL-2. These results provide evidence indicating a regulatory role of PRL-1 during IS assembly and highlight the involvement of PRLs in immune responses by mature T cells.