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Classical dendritic cells (cDC) are professional antigen-presenting cells (APC) that regulate immunity and tolerance. Neutrophil-derived cells with properties of DCs (nAPC) are observed in human diseases and after culture of neutrophils with cytokines. Here we show that FcγR-mediated endocytosis of antibody-antigen complexes or an anti-FcγRIIIB-antigen conjugate converts neutrophils into nAPCs that, in contrast to those generated with cytokines alone, activate T cells to levels observed with cDCs and elicit CD8+ T cell-dependent anti-tumor immunity in mice. Single cell transcript analyses and validation studies implicate the transcription factor PU.1 in neutrophil to nAPC conversion. In humans, blood nAPC frequency in lupus patients correlates with disease. Moreover, anti-FcγRIIIB-antigen conjugate treatment induces nAPCs that can activate autologous T cells when using neutrophils from individuals with myeloid neoplasms that harbor neoantigens or those vaccinated against bacterial toxins. Thus, anti-FcγRIIIB-antigen conjugate-induced conversion of neutrophils to immunogenic nAPCs may represent a possible immunotherapy for cancer and infectious diseases., Neutrophils are versatile immune cells that may also serve as antigen-presenting cells (APC). Here the authors show that engaging FcγRs on neutrophils with immune complexes or an anti-FcγR-antigen conjugate induces neutrophil APC with comparable functions as classical dendritic cells, and with therapeutic potentials for cancer and infectious diseases.

Summary Background T cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19. Methods Antigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes. Findings High correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [TEMRA]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%–38% and 20%–23%, respectively, among COVID-19 patients. Conclusions Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19. Funding This study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation.

Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2–activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2–activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.

BACKGROUND T cells control viral infection and promote vaccine durability and in COVID-19 associate with mild disease. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diptheria-Pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. METHODS Antigen presenting cells (APC) loaded ex vivo with SARS-CoV-2, MMR or Tdap antigens and autologous T cells from COVID-19 convalescent and uninfected individuals, and COVID-19 mRNA vaccinated donors were co-cultured and T cell activation and phenotype were detected by IFN-γ ELISpot assays and flow cytometry. ELISA assays and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and scRNA-seq identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes. FINDINGS High correlation was observed between T cell responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap proteins in COVID-19 convalescent and vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (TEMRA) implicated in protective, anti-viral immunity and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR or Tdap vaccinated individuals by 32-38% and 20-23% respectively, among COVID-19 patients. CONCLUSIONS Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19 disease. FUNDING National Institutes of Health (R01HL065095, R01AI152522, R01NS097719), donation from Barbara and Amos Hostetter and the Chleck Foundation., Graphical Abstract, T cells critically control infection and effective vaccination. In COVID-19 convalescent or COVID-19 vaccinated individuals, memory T cells previously generated by MMR or Tdap vaccines are reactivated by SARS-CoV-2 antigens and have features of TEMRA, implicated in anti-viral immunity. Prior MMR or Tdap vaccination may protect against severe COVID-19 disease.

SummaryEndothelial-dependent mechanisms of mononuclear cell influx are not well understood. We showed that acute stimulation of murine microvascular endothelial cells expressing the tumor necrosis factor receptors TNFR1 and TNFR2 with the soluble cytokine TNF led to CXCR3 chemokine generation. The TNF receptors signaled through interferon regulatory factor-1 (IRF1) to induce interferon-β (IFN-β) and subsequent autocrine signaling via the type I IFN receptor and the transcription factor STAT1. Both TNFR2 and TNFR1 were required for IRF1-IFNβ signaling and, in human endothelial cells TNFR2 expression alone induced IFN-β signaling and monocyte recruitment. In vivo, TNFR1 was required for acute renal neutrophil and monocyte influx after systemic TNF treatment, whereas the TNFR2-IRF1-IFN-β autocrine loop was essential only for macrophage accumulation. In a chronic model of proliferative nephritis, IRF1 and renal-expressed TNFR2 were essential for sustained macrophage accumulation. Thus, our data identify a pathway in endothelial cells that selectively recruits monocytes during a TNF-induced inflammatory response.

The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-β-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ–deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.

Adhesive forces at endothelial cell-cell borders maintain vascular integrity. cAMP enhances barrier properties and controls cellular processes through protein kinase A bound to A-kinase anchoring proteins (AKAPs). It also activates exchange protein directly activated by cAMP (Epac1), an exchange factor for Ras-related protein 1 (Rap1) GTPases that promotes cadherin- and integrin-mediated adhesion through effects on the actin cytoskeleton. We demonstrate that AKAP9 facilitates the microtubule polymerization rate in endothelial cells, interacts with Epac1, and is required for Epac1-stimulated microtubule growth. AKAP9 is not required for maintaining barrier properties under steady-state conditions. Rather, it is essential when the cell is challenged to make new adhesive contacts, as is the case when Epac activation enhances barrier function through a mechanism that, surprisingly, requires integrin adhesion at cell-cell contacts. In the present study, defects in Epac-induced responses in AKAP9-silenced cells were evident despite an intact Epac-induced increase in Rap activation, cortical actin, and vascular endothelial-cadherin adhesion. We describe a pathway that integrates Epac-mediated signals with AKAP9-dependent microtubule dynamics to coordinate integrins at lateral borders.

The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis., A-kinase anchoring protein 9 (AKAP9) is a scaffold protein that binds signalling proteins and regulates microtubules. Here the authors show that during inflammation AKAP9 in T cells is required for their reactivation and retention at the inflammation site and that its deletion protects from inflammation-induced organ damage.

Endothelial cell-cell junctional proteins and cortical actin are of central importance for regulating vascular permeability. Rap1, a member of the Ras family of GTPases, is enriched at endothelial cell-cell contacts and activated by cyclic AMP (cAMP) through a PKA-independent pathway. Activation of a cAMP-inducible guanine-exchange factor for Rap, Epac, results in markedly enhanced basal endothelial barrier function by increasing cortical actin and subsequent redistribution of adherens and tight junctional molecules to cell-cell contacts. Activation of Epac also counteracts thrombin-induced hyperpermeability through down-regulation of Rho GTPase activation, suggesting cross-talk between Rap and Rho GT-Pases. Thus, Epac/Rap activation represents a new pathway for regulating endothelial cell barrier function.

© 2014 by Annual Reviews. All rights reserved. Neutrophils and neutrophil-like cells are the major pathogen-fighting immune cells in organisms ranging from slime molds to mammals. Central to their function is their ability to be recruited to sites of infection, to recognize and phagocytose microbes, and then to kill pathogens through a combination of cytotoxic mechanisms. These include the production of reactive oxygen species, the release of antimicrobial peptides, and the recently discovered expulsion of their nuclear contents to form neutrophil extracellular traps. Here we discuss these primordial neutrophil functions, which also play key roles in tissue injury, by providing details of neutrophil cytotoxic functions and congenital disorders of neutrophils. In addition, we present more recent evidence that interactions between neutrophils and adaptive immune cells establish a feed-forward mechanism that amplifies pathologic inflammation. These newly appreciated contributions of neutrophils are described in the setting of several inflammatory and autoimmune diseases.

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Objective Rheumatoid arthritis culminates in joint destruction that, in mouse models of disease, is supported by innate immune molecules, including Fcγ receptors (FcγR) and complement. However, these findings may not be predictive of the outcome in humans, given the structural differences between murine and human activating FcγR on neutrophils, a prominent component of joint exudates. The aim of this study was to examine the role of human neutrophil FcγRIIa in the development of arthritis and probe the underlying mechanism by which FcγRIIa initiates disease. Methods K/BxN mouse serum transfer–induced arthritis was examined in mice expressing human FcγRIIa on neutrophils but lacking their own activating FcγR (γ-chain–deficient mice). The role of mast cells, complement (C3 and C5a), and CD18 integrins in FcγRIIa-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies, respectively. Crosstalk between the complement receptor C5aR and FcγRIIa on neutrophils was evaluated in vitro. Results The expression of human FcγRIIa on neutrophils was sufficient to restore susceptibility to K/BxN serum–induced neutrophil recruitment, synovitis, and bone destruction in γ-chain–deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, features shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on the presence of a CD18 integrin, lymphocyte function–associated antigen 1, and C5aR. In addition, C5aR significantly enhanced FcγRIIa-mediated phagocytosis and oxidative burst in vitro. Conclusion Human and murine activating FcγR on neutrophils are not functionally equivalent, and in humans, they may play a primary role in arthritis. Crosstalk between neutrophil C5aR and FcγRIIa is essential for disease progression, thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.

The signals linking neutrophil opsonic receptors, FcγRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcγRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcγRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcγR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcγR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions.

Tissue-specific silencing of genes may be used for genetic engineering in mice and has possible therapeutic applications in humans. Current strategies in mice rely on Cre/loxP technology requiring the generation of multiple transgenic lines and breeding strategies. Here, we describe the selective silencing of CD18, a leukocyte-specific integrin in neutrophils using a micro RNA (miRNA) strategy that requires the generation of one transgenic line. CD18-specific miRNA hairpin driven by the myeloid specific human MRP8 promoter resulted in the generation of transgenic lines with 75% to 95% reduction in CD18 protein levels in neutrophils and monocytes. Minimal decreases in T cells and a partial diminution in macrophages were observed. Neutrophil CD18 silencing resulted in neutrophilia, splenomegaly, and significant defects in neutrophil trafficking with the degree of alterations correlating with the extent of CD18 silencing. Thus, our data demonstrate the utility of using miRNA approaches to silence genes in neutrophils, which are terminally differentiated cells with a short half-life that largely precludes their genetic manipulation in vitro. Furthermore, the mouse models provide a valuable tool to examine the contribution of CD18 on neutrophils to leukocyte adhesion deficiency type I (LAD-I), a complex inherited disorder in which reduced or absent CD18 expression in multiple leukocyte subsets leads to impaired innate and adaptive immune responses.

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3+ human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1α (SDF-1α)–stimulated LFA-1–ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly, silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1α- and phorbol 12-myristate 13-acetate (PMA)–induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1, whereas adhesion to VCAM-1 was reduced. Thus, PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells, whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4.

Beta2 integrins are leukocyte-specific membrane receptors that are crucial for host defense. They are best known for promoting neutrophil recruitment into inflamed tissue and pathogen phagocytosis. More recent data suggest that they also modulate neutrophil apoptosis. Neutrophils are terminally differentiated cells, which undergo constitutive apoptosis, and their apoptosis and clearance is required for the resolution of inflammation. Engagement of the beta2 integrin Mac-1 through its adhesion to its ligands, intercellular adhesion molecule-1 (ICAM-1) and fibrinogen, signals survival cues in neutrophils. However, in the presence of pro-apoptotic signals, such as tumor necrosis factor (TNF), Mac-1 engagement accelerates apoptosis. Furthermore, Mac-1-dependent phagocytosis of complement-opsonized pathogens triggers rapid neutrophil apoptosis, which is dependent on NADPH oxidase-generated reactive oxygen species and caspase activation. This is also associated with changes in the transcription profiles of pro- and anti-apoptotic genes. In this review, the beta2 integrin-dependent mechanisms that modulate the decision between life and death in neutrophils are overviewed.

Phagocytes generate reactive oxygen species, the regulation of which is important in eliminating ingested microbes while limiting tissue damage. Clustering of FcγRs results in the activation of Vav proteins, Rho/Rac guanine nucleotide exchange factors, and results in robust superoxide generation through the NADPH oxidase. In this study, studies in neutrophils isolated from mice deficient in Vav or Rac isoforms demonstrate a critical role for Vav3 in Rac2-dependent activation of the NADPH oxidase following FcγR clustering. However, studies in cytokine-primed cells revealed a strict requirement for Vav1 and Vav3 and Rac1 and Rac2 in the FcγR-mediated oxidative burst. In comparison, Vav was not essential for PMA or G protein-coupled receptor-mediated superoxide generation. The FcγR-mediated oxidative burst defect in Vav-deficient cells was linked to aberrant Rac activation as well as Rac- and actin-polymerization-independent, but PI3K-dependent, phosphorylation of the NADPH oxidase component p40(phox). In macrophages, Vav regulation of Rac GTPases was required specifically in FcγR-mediated activation of the oxidative burst, but not in phagocytosis. Thus, Vav proteins specifically couple FcγR signaling to NADPH oxidase function through a Rac-dependent as well as an unexpected Rac-independent signal that is proximal to NADPH oxidase activation and does not require actin polymerization.