NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins.

Increased recruitment of transitional and non-classical monocytes in the lung during SARS-CoV-2 infection is associated with COVID-19 severity. However, whether specific innate sensors mediate the activation or differentiation of monocytes in response to different SARS-CoV-2 proteins remain poorly characterized. Here, we show that SARS-CoV-2 Spike 1 but not nucleoprotein induce differentiation of monocytes into transitional or non-classical subsets from both peripheral blood and COVID-19 bronchoalveolar lavage samples in a NFκB-dependent manner, but this process does not require inflammasome activation. However, NLRP3 and NLRC4 differentially regulated CD86 expression in monocytes in response to Spike 1 and Nucleoprotein, respectively. Moreover, monocytes exposed to Spike 1 induce significantly higher proportions of Th1 and Th17 CD4 + T cells. In contrast, monocytes exposed to Nucleoprotein reduce the degranulation of CD8 + T cells from severe COVID-19 patients. Our study provides insights in the differential impact of innate sensors in regulating monocytes in response to different SARS-CoV-2 proteins, which might be useful to better understand COVID-19 immunopathology and identify therapeutic targets.

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia.

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eµ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eµ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eµ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

Immune synapse formation promotes lipid peroxidation and MHC-I upregulation in licensed dendritic cells for efficient priming of CD8+ T cells.

Antigen cognate dendritic cell (DC)-T cell synaptic interactions drive activation of T cells and instruct DCs. Upon receiving CD4+ T cell help, post-synaptic DCs (psDCs) are licensed to generate CD8+ T cell responses. However, the cellular and molecular mechanisms that enable psDCs licensing remain unclear. Here, we describe that antigen presentation induces an upregulation of MHC-I protein molecules and increased lipid peroxidation on psDCs in vitro and in vivo. We also show that these events mediate DC licensing. In addition, psDC adoptive transfer enhances pathogen-specific CD8+ T responses and protects mice from infection in a CD8+ T cell-dependent manner. Conversely, depletion of psDCs in vivo abrogates antigen-specific CD8+ T cell responses during immunization. Together, our data show that psDCs enable CD8+ T cell responses in vivo during vaccination and reveal crucial molecular events underlying psDC licensing.

End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network.

The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.

Activation kinetics of regulatory molecules during immunological synapse in T cells.

T cell activation through TCR stimulation leads to the formation of the immunological synapse (IS), a specialized adhesion organized between T lymphocytes and antigen presenting cells (APCs) in which a dynamic interaction among signaling molecules, the cytoskeleton and intracellular organelles achieves proper antigen-mediated stimulation and effector function. The kinetics of molecular reactions at the IS is essential to determine the quality of the response to the antigen stimulation. Herein, we describe methods based on biochemistry, flow cytometry and imaging in live and fixed cells to study the activation state and dynamics of regulatory molecules at the IS in the Jurkat T cell line CH7C17 and primary human and mouse CD4+ T lymphocytes stimulated by antigen presented by Raji and HOM2 B cell lines and human and mouse dendritic cells.

T cell activation and effector function in the human Jurkat T cell model.

In order to understand T cell function, it is necessary to completely decipher the molecular dynamics underlying T cell activation and effector function. In vitro easy-to-handle cellular models are valuable tools to study intracellular molecular mechanisms in live cells. The CD4 T cell line Jurkat (JK) has been widely employed to investigate intracellular signaling leading to T cell activation in response to T cell receptor (TCR) triggering. Here, we describe diverse, complementary protocols to evaluate the TCR- and costimulation-mediated T cell activation, as well as the immunological synapse assembly and cytokine production occurring as a consequence of successful early activation events. This in vitro model is extremely useful to address molecular mechanisms operating during T cell activation and effector function acting in diverse pathophysiological scenarios.

ReCom: A semi-supervised approach to ultra-tolerant database search for improved identification of modified peptides.

Open-search methods allow unbiased, high-throughput identification of post-translational modifications in proteins at an unprecedented scale. The performance of current open-search algorithms is diminished by experimental errors in the determination of the precursor peptide mass. In this work we propose a semi-supervised open search approach, called ReCom, that minimizes this effect by taking advantage of a priori known information from a reference database, such as Unimod or a database provided by the user. We present a proof-of-concept study using Comet-ReCom, an improved version of Comet-PTM. Comet-ReCom increased identification performance of Comet-PTM by 68%. This increased performance of Comet-ReCom to score the MS/MS spectrum comes in parallel with a significantly better assignation of the monoisotopic peak of the precursor peptide in the MS spectrum, even in cases of peptide coelution. Our data demonstrate that open searches using ultra-tolerant mass windows can benefit from using a semi-supervised approach that takes advantage from previous knowledge on the nature of protein modifications. SIGNIFICANCE: The present study introduces a novel approach to ultra-tolerant database search, which employs prior knowledge of post-translational modifications (PTMs) to improve identification of modified peptides. This method addresses the limitations related to experimental errors and precursor mass assignation of previous open-search methods. Thus, it enables the study of the biological significance of a wider variety of PTMs, including unknown or unexpected modifications that may have gone unnoticed using non-supervised search methods.

Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics.

Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.

Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation.

Inhibition of the heterodimeric amino acid carrier SLC7A5/SLC3A2 (LAT1/CD98) has been widely studied in tumor biology but its role in physiological conditions remains largely unknown. Here we show that the SLC7A5/SLC3A2 heterodimer is constitutively present at different stages of erythroid differentiation but absent in mature erythrocytes. Administration of erythropoietin (EPO) further induces SLC7A5/SLC3A2 expression in circulating reticulocytes, as it also occurs in anemic conditions. Although Slc7a5 gene inactivation in the erythrocyte lineage does not compromise the total number of circulating red blood cells (RBCs), their size and hemoglobin content are significantly reduced accompanied by a diminished erythroblast mTORC1 activity. Furthermore circulating Slc7a5-deficient reticulocytes are characterized by lower transferrin receptor (CD71) expression as well as mitochondrial activity, suggesting a premature transition to mature RBCs. These data reveal that SLC7A5/SLC3A2 ensures adequate maturation of reticulocytes as well as the proper size and hemoglobin content of circulating RBCs.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV.

BACKGROUND: Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated. METHODS: We studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles. FINDINGS: HIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1- cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. INTERPRETATION: Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines. FUNDING: NIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Post-translational modifications and stabilization of microtubules regulate transport of viral factors during infections.

Tubulin post-translational modifications (PTMs) constitute a source of diversity for microtubule (MT) functions, in addition to the different isotypes of α and ß-tubulin acting as building blocks of MTs. Also, MT-associated proteins (MAPs) confer different characteristics to MTs. The combination of all these factors regulates the stability of these structures that act as rails to transport organelles within the cell, facilitating the association of motor complexes. All these functions are involved in crucial cellular processes in most cell types, ranging from spindle formation in mitosis to the defense against incoming cellular threats during phagocytosis mediated by immune cells. The regulation of MT dynamics through tubulin PTMs has evolved to depend on many different factors that act in a complex orchestrated manner. These tightly regulated processes are particularly relevant during the induction of effective immune responses against pathogens. Viruses have proved not only to hijack MTs and MAPs in order to favor an efficient infection, but also to induce certain PTMs that improve their cellular spread and lead to secondary consequences of viral processes. In this review, we offer a perspective on relevant MT-related elements exploited by viruses.

T cell asymmetry and metabolic crosstalk can fine-tune immunological synapses.

T cell asymmetry upon specific cell-cell interactions during mammalian immunological synapse (IS) contacts requires mammalian target of rapamycin complex (mTORC) activation and chaperones, such as the eukaryotic chaperonin containing TCP1 (CCT) for protein synthesis and folding. This mechanism can control cytoskeleton dynamics, and regulate mitochondrial fate, respiration, and metabolic rates, ultimately underlying cell reprogramming events that are relevant for CD4+ T cell functional outcomes.

Folding for the Immune Synapse: CCT Chaperonin and the Cytoskeleton.

Lymphocytes rearrange their shape, membrane receptors and organelles during cognate contacts with antigen-presenting cells (APCs). Activation of T cells by APCs through pMHC-TCR/CD3 interaction (peptide-major histocompatibility complex-T cell receptor/CD3 complexes) involves different steps that lead to the reorganization of the cytoskeleton and organelles and, eventually, activation of nuclear factors allowing transcription and ultimately, replication and cell division. Both the positioning of the lymphocyte centrosome in close proximity to the APC and the nucleation of a dense microtubule network beneath the plasma membrane from the centrosome support the T cell's intracellular polarity. Signaling from the TCR is facilitated by this traffic, which constitutes an important pathway for regulation of T cell activation. The coordinated enrichment upon T cell stimulation of the chaperonin CCT (chaperonin-containing tailless complex polypeptide 1; also termed TRiC) and tubulins at the centrosome area support polarized tubulin polymerization and T cell activation. The proteasome is also enriched in the centrosome of activated T cells, providing a mechanism to balance local protein synthesis and degradation. CCT assists the folding of proteins coming from de novo synthesis, therefore favoring mRNA translation. The functional role of this chaperonin in regulating cytoskeletal composition and dynamics at the immune synapse is discussed.

Rapid Visualization of Intracellular Vesicle Events During Synaptic Stimulation.

The immune synapse (IS) enables cell-cell communication between immune cells through close contacts, as well as T-cell activation and vesicle secretion. It is sustained by fine-tuned molecular interactions of receptors at both cell sides of the IS and intracellular cytoskeletal components. The resulting intracellular polarization of different organelles, through cytoskeleton-guided vesicular traffic, is a key player in IS formation and signaling. We describe herein a method to analyze rapid changes of vesicle localization through microscopy analysis upon polarization toward the IS. These vesicles are monitored using the centrosome and its associated microtubular network or the actin-based structures as spatial references during the organization of the IS.

TIRF Microscopy as a Tool to Determine Exosome Composition.

Exosomes are extracellular vesicles (EVs) containing different biomolecules with biological activity, such as proteins, miRNA, long noncoding RNA, and DNA. EVs are efficient platforms for intercellular communication, especially during immune responses, but also in some pathological contexts, such as tumor cell growth. The precise assessment of EV content is relevant for the selection of specific vesicles with specialized biological activities, whose content is hardly visualized due to their small size. We describe herein a protocol for the determination of the content of individual EVs through microscopy imaging and user-friendly analysis using TIRF microscopy.

The Swing of Lipids at Peroxisomes and Endolysosomes in T Cell Activation.

The immune synapse (IS) is a well-known intercellular communication platform, organized at the interphase between the antigen presenting cell (APC) and the T cell. After T cell receptor (TCR) stimulation, signaling from plasma membrane proteins and lipids is amplified by molecules and downstream pathways for full synapse formation and maintenance. This secondary signaling event relies on intracellular reorganization at the IS, involving the cytoskeleton and components of the secretory/recycling machinery, such as the Golgi apparatus and the endolysosomal system (ELS). T cell activation triggers a metabolic reprogramming that involves the synthesis of lipids, which act as signaling mediators, and an increase of mitochondrial activity. Then, this mitochondrial activity results in elevated reactive oxygen species (ROS) production that may lead to cytotoxicity. The regulation of ROS levels requires the concerted action of mitochondria and peroxisomes. In this review, we analyze this reprogramming and the signaling implications of endolysosomal, mitochondrial, peroxisomal, and lipidic systems in T cell activation.

Integrated miRNA and mRNA expression profiling identifies novel targets and pathological mechanisms in autoimmune thyroid diseases.

BACKGROUND: The mechanisms underlying autoimmune thyroid disease (AITD) remain elusive. Identification of such mechanisms would reveal novel and/or better therapeutic targets. Here, we use integrated analysis of miRNAs and mRNAs expression profiling to identify potential therapeutic targets involved in the mechanisms underlying AITD. METHODS: miRNA and mRNA from twenty fresh-frozen thyroid tissues (15 from AITD patients and 5 from healthy controls) were subjected to next-generation sequencing. An anti-correlated method revealed potential pathways and disease targets, including proteins involved in the formation of primary cilia. Thus, we examined the distribution and length of primary cilia in thyroid tissues from AITD and controls using immunofluorescence and scanning electron microscopy, and parsed cilia formation in thyroid cell lines in response to inflammatory stimuli in the presence of miRNA mimics. FINDINGS: We found that the expression of miR-21-5p, miR-146b-3p, miR-5571-3p and miR-6503-3p was anti-correlated with Enolase 4 (ENO4), in-turned planar cell polarity protein (INTU), kinesin family member 27 (KIF27), parkin co-regulated (PACRG) and serine/threonine kinase 36 (STK36) genes. Functional classification of these miRNA/mRNAs revealed that their differential expression was associated with cilia organization. We demonstrated that the number and length of primary cilia in thyroid tissues was significantly lower in AITD than in control (frequency of follicular ciliated cells in controls = 67.54% vs a mean of 22.74% and 21.61% in HT and GD respectively p = 0.0001, by one-way ANOVA test). In addition, pro-inflammatory cytokines (IFNγ and TNFα) and specific miRNA mimics for the newly identified target genes affected cilia appearance in thyroid cell lines. INTERPRETATION: Integrated miRNA/gene expression analysis has identified abnormal ciliogenesis as a novel susceptibility pathway that is involved in the pathogenesis of AITD. These results reflect that ciliogenesis plays a relevant role in AITD, and opens research pathways to design therapeutic targets in AITD. FUNDING: Instituto de Salud Carlos III, Comunidad de Madrid, Grupo Español de Tumores Neuroendocrinos y Endocrinos, Ministerio de Economía y Empresa and FEDER.

Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function.

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.