Direct presentation of inflammation-associated self-antigens by thymic innate-like T cells induces elimination of autoreactive CD8+ thymocytes.

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.

Peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells.

Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C+ NK cells has remained unclear. Here we found that adaptive NKG2C+ NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C+ NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C+ NK cell populations among HCMV-seropositive people.

An optimized platform for efficient siRNA delivery into human NK cells.

The molecular networks that regulate natural killer (NK) cell functions are not completely understood. Here, we present a workflow for efficient delivery of siRNA into human NK cells without compromising viability. This methodology represents a promising approach for rapidly interrogating gene functions in primary human NK cells.

Human Cord Blood B Cells Differ from the Adult Counterpart by Conserved Ig Repertoires and Accelerated Response Dynamics.

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

Recombinant multimeric dog allergen prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH 2 and TH 17 cell responses.

BACKGROUND: Allergy to dogs affects around 10% of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. METHODS: We established a mouse model of dog allergy by repeatedly administering dog dander and epithelium extracts via the intranasal route. We also assessed the efficacy of a recombinant multimeric protein containing Can f 1, f 2, f 4 and f 6 in preventing inflammatory responses to dog extracts. RESULTS: Repeated inhalation of dog extracts induced infiltration of the airways by TH 2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. Dog extracts also induced robust airway hyperresponsiveness and promoted TH 17 cell responses, which was associated with a high neutrophilic infiltration of the airways. scRNA-Seq analysis of T helper cells in the airways pinpointed a unique gene signature for TH 17 cells. Analysis of T-cell receptors depicted a high frequency of clones that were shared between TH 17, TH 2 and suppressive Treg cells, indicative of a common differentiation trajectory for these subsets. Importantly, sublingual administration of multimeric Can f 1-2-4-6 protein prior to sensitization reduced airway hyperresponsiveness and type 2-mediated inflammation in this model. CONCLUSION: Dog allergen extracts induce robust TH 2 and TH 17 cell-mediated responses in mice. Recombinant Can f 1-2-4-6 can induce tolerance to complex dog allergen extracts.

Intrinsic TNFR2 signaling in T regulatory cells provides protection in CNS autoimmunity.

TNF is a multifunctional cytokine involved in autoimmune disease pathogenesis that exerts its effects through two distinct TNF receptors, TNFR1 and TNFR2. While TNF- and TNFR1-deficient (but not TNFR2-deficient) mice show very similar phenotypes, the significance of TNFR2 signaling in health and disease remains incompletely understood. Recent studies implicated the importance of the TNF/TNFR2 axis in T regulatory (Treg) cell functions. To definitively ascertain the significance of TNFR2 signaling, we generated and validated doubly humanized TNF/TNFR2 mice, with the option of conditional inactivation of TNFR2. These mice carry a functional human TNF-TNFR2 (hTNF-hTNFR2) signaling module and provide a useful tool for comparative evaluation of TNF-directed biologics. Conditional inactivation of TNFR2 in FoxP3+ cells in doubly humanized TNF/TNFR2 mice down-regulated the expression of Treg signature molecules (such as FoxP3, CD25, CTLA-4, and GITR) and diminished Treg suppressive function in vitro. Consequently, Treg-restricted TNFR2 deficiency led to significant exacerbation of experimental autoimmune encephalomyelitis (EAE), accompanied by reduced capacity to control Th17-mediated immune responses. Our findings expose the intrinsic and beneficial effects of TNFR2 signaling in Treg cells that could translate into protective functions in vivo, including treatment of autoimmunity.

BHLHE40/41 regulate microglia and peripheral macrophage responses associated with Alzheimer's disease and other disorders of lipid-rich tissues.

Genetic and experimental evidence suggests that Alzheimer's disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages, including microglia, to damage of lipid-rich tissues like the brain. Recently, sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues, we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors, BHLHE40/41, in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia, resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing, increased cholesterol efflux and storage, and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues.

BHLHE40/41 regulate macrophage/microglia responses associated with Alzheimer's disease and other disorders of lipid-rich tissues.

Background: Genetic and experimental evidence strongly implicates myeloid cells in the etiology of AD and suggests that AD-associated alleles and genes may modulate disease risk by altering the transcriptional and cellular responses of macrophages (like microglia) to damage of lipid-rich tissues (like the brain). Specifically, recent single-cell/nucleus RNA sequencing (sc/nRNA-seq) studies identified a transcriptionally distinct state of subsets of macrophages in aging or degenerating brains (usually referred to as disease-associated microglia or DAM) and in other diseased lipid-rich tissues (e.g., obese adipose tissue, fatty liver, and atherosclerotic plaques). We collectively refer to these subpopulations as lipid-associated macrophages or LAMs. Importantly, this particular activation state is characterized by increased expression of genes involved in the phagocytic clearance of lipid-rich cellular debris (efferocytosis), including several AD risk genes. Methods: We used sc/nRNA-seq data from human and mouse microglia from healthy and diseased brains and macrophages from other lipid-rich tissues to reconstruct gene regulatory networks and identify transcriptional regulators whose regulons are enriched for LAM response genes (LAM TFs) across species. We then used gene knock-down/knock-out strategies to validate some of these LAM TFs in human THP-1 macrophages and iPSC-derived microglia in vitro, as well as mouse microglia in vivo. Results: We nominate 11 strong candidate LAM TFs shared across human and mouse networks (BHLHE41, HIF1A, ID2, JUNB, MAF, MAFB, MEF2A, MEF2C, NACA, POU2F2 and SPI1). We also demonstrate a strong enrichment of AD risk alleles in the cistrome of BHLHE41 (and its close homolog BHLHE40), thus implicating its regulon in the modulation of disease susceptibility. Loss or reduction of BHLHE40/41 expression in human THP-1 macrophages and iPSC-derived microglia, as well as loss of Bhlhe40/41 in mouse microglia led to increased expression of LAM response genes, specifically those involved in cholesterol clearance and lysosomal processing, with a concomitant increase in cholesterol efflux and storage, as well as lysosomal mass and degradative capacity. Conclusions: Taken together, this study nominates transcriptional regulators of the LAM response, experimentally validates BHLHE40/41 in human and mouse macrophages/microglia, and provides novel targets for therapeutic modulation of macrophage/microglia function in AD and other disorders of lipid-rich tissues.

Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis.

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.

Recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of Vγ1 γδTCRs.

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1-containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate-like αß T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

Cytokines and Chemokines in Cerebral Malaria Pathogenesis.

Cerebral malaria is among the major causes of malaria-associated mortality and effective adjunctive therapeutic strategies are currently lacking. Central pathophysiological processes involved in the development of cerebral malaria include an imbalance of pro- and anti-inflammatory responses to Plasmodium infection, endothelial cell activation, and loss of blood-brain barrier integrity. However, the sequence of events, which initiates these pathophysiological processes as well as the contribution of their complex interplay to the development of cerebral malaria remain incompletely understood. Several cytokines and chemokines have repeatedly been associated with cerebral malaria severity. Increased levels of these inflammatory mediators could account for the sequestration of leukocytes in the cerebral microvasculature present during cerebral malaria, thereby contributing to an amplification of local inflammation and promoting cerebral malaria pathogenesis. Herein, we highlight the current knowledge on the contribution of cytokines and chemokines to the pathogenesis of cerebral malaria with particular emphasis on their roles in endothelial activation and leukocyte recruitment, as well as their implication in the progression to blood-brain barrier permeability and neuroinflammation, in both human cerebral malaria and in the murine experimental cerebral malaria model. A better molecular understanding of these processes could provide the basis for evidence-based development of adjunct therapies and the definition of diagnostic markers of disease progression.

Adaptive Natural Killer Cells Integrate Interleukin-18 during Target-Cell Encounter.

Human cytomegalovirus (HCMV) infection induces adaptations in the natural killer (NK)-cell compartment. Expanded subsets of adaptive NK cells display potent effector functions against cellular targets, despite their apparent unresponsiveness to stimulation with classical dendritic cell-derived cytokines interleukin (IL)-12 and IL-18. However, it remains unclear whether adaptive NK cells have completely lost their ability to sense inflammation via IL-12 and IL-18 or whether these pro-inflammatory signals can be functionally integrated into defined contexts. Here, we demonstrate that adaptive NKG2C+ NK cells can be costimulated by the presence of pro-inflammatory cytokines during target cell-induced activation. Cytokine costimulation of adaptive NK cells resulted in elevated interferon (IFN)-gamma and tumor necrosis factor (TNF) production, which promoted protein expression of HLA class I and adhesion molecules as well as transcription of genes involved in antigen processing and antiviral states in endothelial bystander cells in vitro. We further show that IL-18 drove costimulation in functional assays and was sufficient for elevated cytokine production in the absence of IL-12. Hence, adaptive NKG2C+ NK cells-although poorly responsive to IL-12 and IL-18 as an isolated stimulus-integrate IL-18 as a costimulatory signal during target-cell encounter.

Interaction between Plasmodium Glycosylphosphatidylinositol and the Host Protein Moesin Has No Implication in Malaria Pathology.

Glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum origin is considered an important toxin leading to severe malaria pathology through stimulation of pro-inflammatory responses from innate immune cells. Even though the GPI-induced immune response is widely described to be mediated by pattern recognition receptors such as TLR2 and TLR4, previous studies have revealed that these two receptors are dispensable for the development of severe malaria pathology. Therefore, this study aimed at the identification of potential alternative Plasmodium GPI receptors. Herein, we have identified the host protein moesin as an interaction partner of Plasmodium GPI in vitro. Given previous reports indicating the relevance of moesin especially in the LPS-mediated induction of pro-inflammatory responses, we have conducted a series of in vitro and in vivo experiments to address the physiological relevance of the moesin-Plasmodium GPI interaction in the context of malaria pathology. We report here that although moesin and Plasmodium GPI interact in vitro, moesin is not critically involved in processes leading to Plasmodium-induced pro-inflammatory immune responses or malaria-associated cerebral pathology.

Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 µm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping.