Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Phosphatidylserine-positive extracellular vesicles boost effector CD8+ T cell responses during viral infection.

CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo.

Mechanosensing via a GpIIb/Src/14-3-3ζ axis critically regulates platelet migration in vascular inflammation.

Platelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ-dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI.

Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbß3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.

PI3-Kinase-γ Has a Distinct and Essential Role in Lung-Specific Dendritic Cell Development.

Development of dendritic cells (DCs) commences in the bone marrow, from where pre-DCs migrate to peripheral organs to differentiate into mature DCs in situ. However, the factors that regulate organ-specific differentiation to give rise to tissue-specific DC subsets remain unclear. Here we show that the Ras-PI3Kγ-Akt-mTOR signaling axis acted downstream of FLT3L signaling and was required for development of lung CD103(+) DCs and, to a smaller extent, for lung CD11b(+) DCs, but not related DC populations in other non-lymphoid organs. Furthermore, we show that in lymphoid organs such as the spleen, DCs depended on a similar signaling network to respond to FLT3 ligand with overlapping and partially redundant roles for kinases PI3Kγ and PI3Kδ. Thus we identified PI3Kγ as an essential organ-specific regulator of lung DC development and discovered a signaling network regulating tissue-specific DC development mediated by FLT3.

High-Fat Diet Rapidly Modifies Trafficking, Phenotype, and Function of Plasmacytoid Dendritic Cells in Adipose Tissue.

Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α4ß1 and α4ß7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism.

CCL17 Promotes Colitis-Associated Tumorigenesis Dependent on the Microbiota.

Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.

Impaired function and delayed regeneration of dendritic cells in COVID-19.

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

Predicting single-cell gene expression profiles of imaging flow cytometry data with machine learning.

High-content imaging and single-cell genomics are two of the most prominent high-throughput technologies for studying cellular properties and functions at scale. Recent studies have demonstrated that information in large imaging datasets can be used to estimate gene mutations and to predict the cell-cycle state and the cellular decision making directly from cellular morphology. Thus, high-throughput imaging methodologies, such as imaging flow cytometry can potentially aim beyond simple sorting of cell-populations. We introduce IFC-seq, a machine learning methodology for predicting the expression profile of every cell in an imaging flow cytometry experiment. Since it is to-date unfeasible to observe single-cell gene expression and morphology in flow, we integrate uncoupled imaging data with an independent transcriptomics dataset by leveraging common surface markers. We demonstrate that IFC-seq successfully models gene expression of a moderate number of key gene-markers for two independent imaging flow cytometry datasets: (i) human blood mononuclear cells and (ii) mouse myeloid progenitor cells. In the case of mouse myeloid progenitor cells IFC-seq can predict gene expression directly from brightfield images in a label-free manner, using a convolutional neural network. The proposed method promises to add gene expression information to existing and new imaging flow cytometry datasets, at no additional cost.

Type I interferon mediated induction of somatostatin leads to suppression of ghrelin and appetite thereby promoting viral immunity in mice.

Loss of appetite (anorexia) is a typical behavioral response to infectious diseases that often reduces body weight. Also, anorexia can be observed in cancer and trauma patients, causing poor quality of life and reduced prospects of positive therapeutic outcomes. Although anorexia is an acute symptom, its initiation and endocrine regulation during antiviral immune responses are poorly understood. During viral infections, plasmacytoid dendritic cells (pDCs) produce abundant type I interferon (IFN-I) to initiate first-line defense mechanisms. Here, by targeted ablation of pDCs and various in vitro and in vivo mouse models of viral infection and inflammation, we identified that IFN-I is a significant driver of somatostatin (SST). Consequently, SST suppressed the hunger hormone ghrelin that led to severe metabolic changes, anorexia, and rapid body weight loss. Furthermore, during vaccination with Modified Vaccinia Ankara virus (MVA), the SST-mediated suppression of ghrelin was critical to viral immune response, as ghrelin restrained the production of early cytokines by natural killer (NK) cells and pDCs, and impaired the clonal expansion of CD8+ T cells. Thus, the hormonal modulation of ghrelin through SST and the cytokine IFN-I is fundamental for optimal antiviral immunity, which comes at the expense of calorie intake.

The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles.

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.

Constitutive CD40 Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption.

The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr-/-) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr-/- chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr-/- (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

CD169+ macrophages are sufficient for priming of CTLs with specificities left out by cross-priming dendritic cells.

Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs), which directly prime or cross-prime MHC I-restricted cytotoxic T cells (CTLs). However, recent evidence suggests the existence of other, as-yet unidentified APCs also able to prime T cells. To identify those APCs, we used adenoviral (rAd) vectors, which do not infect DCs but selectively accumulate in CD169(+) macrophages (MPs). In mice that lack DCs, infection of CD169(+) MPs was sufficient to prime CTLs specific for all epitopes tested. In contrast, CTL responses relying exclusively on cross-presenting DCs were biased to selected strong MHC I-binding peptides only. When both DCs and MPs were absent, no CTL responses could be elicited. Therefore, CD169(+) MPs can be considered APCs that significantly contribute to CTL responses.

Constitutive crosspresentation of tissue antigens by dendritic cells controls CD8+ T cell tolerance in vivo.

Immature dendritic cells (DCs) sample tissue-specific antigens (TSAs) and process them for "crosspresentation" via major histocompatibility complex (MHC) class I and II molecules. Findings with adoptively transferred T cell receptor (TCR)-transgenic CD8+ T cells in transgenic mice expressing model TSA indicate that this process contributes to tolerance induction of CD8+ T cells, a phenomenon termed "crosstolerance." However, up to now it has been unknown whether "crosstolerance" can also control autoimmune T cells specific for physiological nontransgenic TSA. Here, we showed that a DC-specific deficiency in uptake of apoptotic material inhibits crosspresentation in vivo. This defect allowed the accumulation of fully functional autoreactive CD8+ T cells that could be activated for autoimmune attack in peripheral lymphoid organs. Thus, our data demonstrate the importance of crosstolerance induction by DCs as a vital instrument for controlling self-reactive T cells from the peripheral repertoire and preventing autoimmune disease.

Control of Homeostasis and Dendritic Cell Survival by the GTPase RhoA.

Tissues accommodate defined numbers of dendritic cells (DCs) in highly specific niches where different intrinsic and environmental stimuli control DC life span and numbers. DC homeostasis in tissues is important, because experimental changes in DC numbers influence immunity and tolerance toward various immune catastrophes and inflammation. However, the precise molecular mechanisms regulating DC life span and homeostasis are unclear. We report that the GTPase RhoA controls homeostatic proliferation, cytokinesis, survival, and turnover of cDCs. Deletion of RhoA strongly decreased the numbers of CD11b(-)CD8(+) and CD11b(+)Esam(hi) DC subsets, whereas CD11b(+)Esam(lo) DCs were not affected in conditional RhoA-deficient mice. Proteome analyses revealed a defective prosurvival pathway via PI3K/protein kinase B (Akt1)/Bcl-2-associated death promoter in the absence of RhoA. Taken together, our findings identify RhoA as a central regulator of DC homeostasis, and its deletion decreases DC numbers below critical thresholds for immune protection and homeostasis, causing aberrant compensatory DC proliferation.

Langerhans cell homeostasis in mice is dependent on mTORC1 but not mTORC2 function.

The PI3K/Akt/mTOR pathway has emerged as a critical regulator of dendritic cell (DC) development and function. The kinase mTOR is found in 2 distinct complexes, mTORC1 and mTORC2. In this study, we show that mTORC1 but not mTORC2 is required for epidermal Langerhans cell (LC) homeostasis. Although the initial seeding of the epidermis with LCs is not affected, the lack of mTORC1 activity in DCs by conditional deletion of Raptor leads to a progressive loss of LCs in the skin of mice. Ablation of mTORC2 function by deletion of Rictor results in a modest reduction of LCs in skin draining lymph nodes. In young mice Raptor-deficient LCs show an increased tendency to leave the skin, leading to a higher frequency of migratory DCs in skin draining lymph nodes, indicating that the loss of LCs results from enhanced migration. LCs lacking Raptor are smaller and display reduced expression of Langerin, E-cadherin, ß-catenin, and CCR7 but unchanged levels of MHC-II, ruling out enhanced spontaneous maturation. Ki-67 and annexin V stainings revealed a faster turnover rate and increased apoptosis of Raptor-deficient LCs, which might additionally affect the preservation of the LC network. Taken together our results show that the homeostasis of LCs strictly depends on mTORC1.

Rho-family GTPase Cdc42 controls migration of Langerhans cells in vivo.

Epidermal Langerhans cells (LCs) of the skin represent the prototype migratory dendritic cell (DC) subtype. In the skin, they take up Ag, migrate to the draining lymph nodes, and contribute to Ag transport and immunity. Different depletion models for LCs have revealed contrasting roles and contributions of this cell type. To target the migratory properties of DCs, we generated mice lacking the Rho-family GTPase Cdc42 specifically in DCs. In these animals, the initial seeding of the epidermis with LCs is functional, resulting in slightly reduced Langerhans cell numbers. However, Cdc42-deficient LCs fail to leave the skin in steady state as well as upon stimulation, as they do not enter the skin-draining afferent lymph vessels. Similarly, also other Cdc42-deficient migratory DC subsets fail to home properly to the corresponding draining lymph nodes. We used this novel mouse model, in which LCs are locked out, to demonstrate that these cells contribute substantially to priming of Ag-specific CD4 and CD8 T cell responses upon epicutaneous immunization, but could not detect a role in the induction of contact hypersensitivity to various doses of hapten.

Antigen amount dictates CD8+ T-cell exhaustion during chronic viral infection irrespective of the type of antigen presenting cell.

Chronic viral infections lead to CD8(+) T-cell exhaustion, characterized by impaired cytokine secretion and loss of proliferative capacity. While viral load and T-cell dysfunction correlate, it is currently unclear whether the quality of a cell type presenting antigen determines the degree of T-cell exhaustion or if the overall amount of antigen recognized by T cells promotes exhaustion. We found that chronic lymphocytic chorio-meningitis virus infection led to decreased CD8(+) T-cell exhaustion in DC-MHC class I (MHCI) mice, in which CD8(+) T cells can only recognize antigen on DCs. However, this increase in CD8(+) T-cell function came at the expense of fatal immunopathology. Additional antigen recognition on nonhematopoietic cells in DC-MHCI mice promoted T-cell exhaustion and avoidance of immunopathology. Likewise, increased numbers of antigen-expressing hematopoietic cells, as well as a selective elevation of the number of DCs as the only cell type presenting antigen in DC-MHCI mice, resulted in compromised T-cell function. These results favor a scenario in which the overall amount of antigen exposure, rather than the type of cell engaging with virus-specific CD8(+) T cells, is responsible for their functional exhaustion. Furthermore, exhaustion of virus-specific CD8(+) T cells leads to avoidance of life-threatening immunopathology.

Non-hematopoietic cells in lymph nodes drive memory CD8 T cell inflation during murine cytomegalovirus infection.

During human and murine cytomegalovirus (MCMV) infection an exceptionally large virus-specific CD8 T cell pool is maintained in the periphery lifelong. This anomalous response is only seen for specific subsets of MCMV-specific CD8 T cells which are referred to as 'inflationary T cells'. How memory CD8 T cell inflation is induced and maintained is unclear, though their activated phenotype strongly suggests an involvement of persistent antigen encounter during MCMV latency. To dissect the cellular and molecular requirements for memory CD8 T cell inflation, we have generated a transgenic mouse expressing an MHC class I-restricted T cell receptor specific for an immunodominant inflationary epitope of MCMV. Through a series of adoptive transfer experiments we found that memory inflation was completely dependent on antigen presentation by non-hematopoietic cells, which are also the predominant site of MCMV latency. In particular, non-hematopoietic cells selectively induced robust proliferation of inflationary CD8 T cells in lymph nodes, where a majority of the inflationary CD8 T cells exhibit a central-memory phenotype, but not in peripheral tissues, where terminally differentiated inflationary T cells accumulate. These results indicate that continuous restimulation of central memory CD8 T cells in the lymph nodes by infected non-hematopoietic cells ensures the maintenance of a functional effector CD8 T pool in the periphery, providing protection against viral reactivation events.