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.

ICAMs in Immunity, Intercellular Adhesion and Communication.

Interactions among leukocytes and leukocytes with immune-associated auxiliary cells represent an essential feature of the immune response that requires the involvement of cell adhesion molecules (CAMs). In the immune system, CAMs include a wide range of members pertaining to different structural and functional families involved in cell development, activation, differentiation and migration. Among them, ß2 integrins (LFA-1, Mac-1, p150,95 and αDß2) are predominantly involved in homotypic and heterotypic leukocyte adhesion. ß2 integrins bind to intercellular (I)CAMs, actin cytoskeleton-linked receptors belonging to immunoglobulin superfamily (IgSF)-CAMs expressed by leukocytes and vascular endothelial cells, enabling leukocyte activation and transendothelial migration. ß2 integrins have long been viewed as the most important ICAMs partners, propagating intracellular signalling from ß2 integrin-ICAM adhesion receptor interaction. In this review, we present previous evidence from pioneering studies and more recent findings supporting an important role for ICAMs in signal transduction. We also discuss the contribution of immune ICAMs (ICAM-1, -2, and -3) to reciprocal cell signalling and function in processes in which ß2 integrins supposedly take the lead, paying particular attention to T cell activation, differentiation and migration.

Beneficial effect of temporary methotrexate interruption on B and T cell responses upon SARS-CoV-2 vaccination in patients with rheumatoid arthritis or psoriatic arthritis.

B and T cell responses were evaluated in patients with rheumatoid arthritis (RA) or psoriatic arthritis (PsA) after 1 or 2 weeks of methotrexate (MTX) withdrawal following each COVID-19 vaccine dose and compared with those who maintained MTX. Adult RA and PsA patients treated with MTX were recruited and randomly assigned to 3 groups: MTX-maintenance (n = 72), MTX-withdrawal for 1 week (n = 71) or MTX-withdrawal for 2 weeks (n = 73). Specific antibodies to several SARS-CoV-2 antigens and interferon (IFN)-γ and interleukin (IL)-21 responses were assessed. MTX withdrawal in patients without previous COVID-19 was associated with higher levels of anti-RBD IgG and neutralising antibodies, especially in the 2-week withdrawal group and with higher IFN-γ secretion upon stimulation with pools of SARS-CoV-2 S peptides. No increment of RA/PsA relapses was detected across groups. Our data indicate that two-week MTX interruption following COVID-19 vaccination in patients with RA or PsA improves humoral and cellular immune responses.

MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

Analysis of humoral and cellular immunity after SARS-CoV-2 vaccination in patients with multiple sclerosis treated with immunomodulatory drugs.

We analyzed immune response to SARS-CoV-2 vaccination by measuring specific IgG titers and T-cell reactivity to different SARS-CoV-2 peptides in multiple sclerosis patients taking different disease-modifying treatments. Of the 88 patients included, 72 developed any kind of immune response after vaccination. Although DMTs such as fingolimod and anti-CD20+ treatments prevented patients from developing a robust humoral response to the vaccine, most of them were still able to develop a cellular response, which could be crucial for long-term immunity. It is probably advisable that all MS patients take additional/booster doses to increase their humoral and/or cellular immune response to SARS-CoV-2.

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.

T regulatory lymphocytes specific for SARS-CoV-2 display increased functional plasticity.

The study of phenotypic and functional characteristics of immune cells involved in host response to SARS-CoV-2 is relevant for understanding COVID-19 pathogenesis and individual differences in disease progression. We have analyzed chemokine receptor expression in SARS-CoV-2-specific CD4+ T lymphocytes from vaccinated donors, and have found an increase of CCR9+ and CCR6+ cells. CCR9+ specific CD4+ cells are enriched in T regulatory (Treg) lymphocytes. These cells specifically show heterogeneous regulatory activity, associated with different profiles of CCR9/CCR6 expression, individual differences in IL-10 and IL-17 production, and variable FoxP3 and Notch4 expression. A higher heterogeneity in FoxP3 is selectively observed in convalescent individuals within vaccinated population. Accordingly, SARS-CoV-2-specific CD4+ lymphocytes from COVID-19 patients are also enriched in CCR9+ and CCR6+ cells. CCR6+ specific Treg lymphocytes are mainly increased in critically ill individuals, indicating a preferential role for these cells in lung injury pathogenesis. We provide experimental evidence for a SARS-CoV-2-specific Treg population with increased plasticity, which may contribute to the differential pathogenic response against SARS-CoV-2 among individuals, and underlie the development of autoimmune conditions following SARS-CoV-2 infection.

Reprogramming dendritic cells through the immunological synapse: A two-way street.

Dendritic cells (DCs) bridge innate and adaptive immunity. Their main function is to present antigens to prime T cells and initiate and shape adaptive responses. Antigen presentation takes place through intimate contacts between the two cells, termed immune synapses (IS). During the formation of IS, information travels towards the T-cell side to induce and tune its activation; but it also travels in reverse via engagement of membrane receptors and within extracellular vesicles transferred to the DC. Such reverse information transfer and its consequences on DC fate have been largely neglected. Here, we review the events and effects of IS-mediated antigen presentation on DCs. In addition, we discuss novel technological advancements that enable monitoring DCs interactions with T lymphocytes, the main effects of DCs undergoing productive IS (postsynaptic DCs, or psDCs), and how reverse information transfer could be harnessed to modulate immune responses for therapeutic intervention.

ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.

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.

Immunological synapse-driven transfer of extracellular vesicle microRNAs in primary lymphocytes.

Cell-to-cell communication is necessary to orchestrate effective immune responses against disease-causing agents and in homeostasis. During immune synapsis, transfer of small extracellular vesicles that contain bioactive molecules, including microRNAs, occurs from the T lymphocyte to the antigen-presenting cell. In this chapter, we describe the methodology to identify and validate specific microRNAs shuttled from T lymphocytes to B cells upon immune synapse formation, and to analyze their functional impact on post-synaptic antigen-presenting 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.

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.

Bacteria-instructed B cells cross-prime naïve CD8+ T cells triggering effective cytotoxic responses.

In addition to triggering humoral responses, conventional B cells have been described in vitro to cross-present exogenous antigens activating naïve CD8+ T cells. Nevertheless, the way B cells capture these exogenous antigens and the physiological roles of B cell-mediated cross-presentation remain poorly explored. Here, we show that B cells capture bacteria by trans-phagocytosis from previously infected dendritic cells (DC) when they are in close contact. Bacterial encounter "instructs" the B cells to acquire antigen cross-presentation abilities, in a process that involves autophagy. Bacteria-instructed B cells, henceforth referred to as BacB cells, rapidly degrade phagocytosed bacteria, process bacterial antigens and cross-prime naïve CD8+ T cells which differentiate into specific cytotoxic cells that efficiently control bacterial infections. Moreover, a proof-of-concept experiment shows that BacB cells that have captured bacteria expressing tumor antigens could be useful as novel cellular immunotherapies against cancer.

Unraveling CD69 signaling pathways, ligands and laterally associated molecules.

CD69 is an early leukocyte activation marker involved in the regulation of the immune response. Initial in vitro studies evaluated its function using monoclonal antibodies until knock-out mice were developed. Subsequently, four ligands for CD69 have been identified, namely galectin-1, S100A8/S100A9 complex, myosin light chains 9 and 12, and oxidized low-density lipoproteins. In addition, several molecules are laterally associated with and regulated by CD69, including calreticulin and two transmembrane receptors, sphingosine-1-phosphate receptor (S1P1) and the heterodimeric amino acid transporter complex SLC7A5-SLC3A2 (LAT1-CD98). Recently, CD69 engagement has been shown to induce the expression of the immunoregulatory receptor programmed cell death-1 (PD-1) in T cells. The molecular signaling induced by CD69 has been explored in different scenarios and cell types. This review provides a perspective on the molecular pathways, ligands and cellular functions known to be regulated by CD69.

Development of an Effective Immune Response in Adults With Down Syndrome After Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccination.

BACKGROUND: Immune dysregulation in individuals with Down syndrome (DS) leads to an increased risk for hospitalization and death due to coronavirus disease 2019 (COVID-19) and may impair the generation of protective immunity after vaccine administration. METHODS: The cellular and humoral responses of 55 individuals with DS who received a complete SARS-CoV-2 vaccination regime at 1 to 3 (visit [V 1]) and 6 (V2) months were characterized. RESULTS: SARS-CoV-2-reactive CD4+ and CD8+ T lymphocytes with a predominant Th1 phenotype were observed at V1 and increased at V2. Likewise, an increase in SARS-CoV-2-specific circulating Tfh (cTfh) cells and CD8+ CXCR5+ PD-1hi lymphocytes was already observed at V1 after vaccine administration. Specific immunoglobulin G (IgG) antibodies against SARS-CoV-2 S protein were detected in 96% and 98% of subjects at V1 and V2, respectively, although IgG titers decreased significantly between both time points. CONCLUSIONS: Our findings show that DS individuals develop an effective immune response to usual regimes of SARS-CoV-2 vaccination.

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.

NLRC4-mediated activation of CD1c+ DC contributes to perpetuation of synovitis in rheumatoid arthritis.

The individual contribution of specific myeloid subsets such as CD1c+ conventional DC (cDC) to perpetuation of rheumatoid arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDC in patients with RA and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional, and functional characteristics of CD1c+ and CD141+ cDC and monocytes from the blood and synovial fluid of patients with RA. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC was associated with the presence of this DC subset in the synovial membrane in patients with RA. Moreover, synovial CD1c+ cDC are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFN-γ+IL-17+CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ receptors and NLRC4 as a potential molecular mechanism mediating pathogenic activation, CD64 upregulation, and functional specialization of CD1c+ cDC in response to dsDNA-IgG in patients with RA.

Alarming and Calming: Opposing Roles of S100A8/S100A9 Dimers and Tetramers on Monocytes.

Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).

Extracellular vesicles from Listeria monocytogenes-infected dendritic cells alert the innate immune response.

Communication through cell-cell contacts and extracellular vesicles (EVs) enables immune cells to coordinate their responses against diverse types of pathogens. The function exerted by EVs in this context depends on the proteins and nucleic acids loaded into EVs, which elicit specific responses involved in the resolution of infection. Several mechanisms control protein and nucleic acid loading into EVs; in this regard, acetylation has been described as a mechanism of cellular retention during protein sorting to exosomes. HDAC6 is a deacetylase involved in the control of cytoskeleton trafficking, organelle polarity and cell migration, defense against Listeria monocytogenes (Lm) infection and other immune related functions. Here, we show that the protein content of dendritic cells (DCs) and their secreted EVs (DEVs) vary during Lm infection, is enriched in proteins related to antiviral functions compared to non-infected cells and depends on HDAC6 expression. Analyses of the post-translational modifications revealed an alteration of the acetylation and ubiquitination profiles upon Lm infection both in DC lysates and DEVs. Functionally, EVs derived from infected DCs upregulate anti-pathogenic genes (e.g. inflammatory cytokines) in recipient immature DCs, which translated into protection from subsequent infection with vaccinia virus. Interestingly, absence of Listeriolysin O in Lm prevents DEVs from inducing this anti-viral state. In summary, these data underscore a new mechanism of communication between bacteria-infected DC during infection as they alert neighboring, uninfected DCs to promote antiviral responses.