Interactions between Siglec-8 and endogenous sialylated cis ligands restrain cell death induction in human eosinophils and mast cells.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a sialoside-binding receptor expressed by eosinophils and mast cells that exhibits priming status- and cell type-dependent inhibitory activity. On eosinophils that have been primed with IL-5, GM-CSF, or IL-33, antibody ligation of Siglec-8 induces cell death through a pathway involving the ß2 integrin-dependent generation of reactive oxygen species (ROS) via NADPH oxidase. In contrast, Siglec-8 engagement on mast cells inhibits cellular activation and mediator release but reportedly does not impact cell viability. The differences in responses between cytokine-primed and unprimed eosinophils, and between eosinophils and mast cells, to Siglec-8 ligation are not understood. We previously found that Siglec-8 binds to sialylated ligands present on the surface of the same cell (so-called cis ligands), preventing Siglec-8 ligand binding in trans. However, the functional relevance of these cis ligands has not been elucidated. We therefore explored the potential influence of cis ligands of Siglec-8 on both eosinophils and mast cells. De-sialylation using exogenous sialidase profoundly altered the consequences of Siglec-8 antibody engagement on both cell types, eliminating the need for cytokine priming of eosinophils to facilitate cell death and enabling Siglec-8-dependent mast cell death without impacting anti-Siglec-8 antibody binding. The cell death process licensed by de-sialylation resembled that characterized in IL-5-primed eosinophils, including CD11b upregulation, ROS production, and the activities of Syk, PI3K, and PLC. These results implicate cis ligands in restraining Siglec-8 function on eosinophils and mast cells and reveal a promising approach to the selective depletion of mast cells in patients with mast cell-mediated diseases.

Siglecs as potential targets of therapy in human mast cell- and/or eosinophil-associated diseases.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of vertebrate glycan-binding cell-surface proteins. The majority mediate cellular inhibitory activity once engaged by specific ligands or ligand-mimicking molecules. As a result, Siglec engagement is now of interest as a strategy to therapeutically dampen unwanted cellular responses. When considering allergic inflammation, human eosinophils and mast cells express overlapping but distinct patterns of Siglecs. For example, Siglec-6 is selectively and prominently expressed on mast cells while Siglec-8 is highly specific for both eosinophils and mast cells. This review will focus on a subset of Siglecs and their various endogenous or synthetic sialoside ligands that regulate eosinophil and mast cell function and survival. It will also summarize how certain Siglecs have become the focus of novel therapies for allergic and other eosinophil- and mast cell-related diseases.

Extracellular Vesicle-associated GARP/TGFß:LAP Mediates "Infectious" Allo-tolerance.

Here we test the hypothesis that, like CD81-associated "latent" IL35, the transforming growth factor (TGF)ß:latency-associated peptide (LAP)/glycoprotein A repetitions predominant (GARP) complex was also tethered to small extracellular vesicles (sEVs), aka exosomes, produced by lymphocytes from allo-tolerized mice. Once these sEVs are taken up by conventional T cells, we also test whether TGFß could be activated suppressing the local immune response. Methods: C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes followed by anti-CD40L/CD154 antibody treatment on days 0, 2, and 4. On day 35, spleen and lymph nodes were extracted and isolated lymphocytes were restimulated with sonicates of CBA splenocytes overnight. sEVs were extracted from culture supernatants by ultracentrifugation (100 000g) and assayed for (a) the presence of TGFß:LAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFß:LAP and to activation from its latent form, as well as various TGFß receptors; and (c) TGFß-dependent function in 1° and 2° immunosuppression of tetanus toxoid-immunized B6 splenocytes using trans-vivo delayed-type hypersensitivity assay. Results: After tolerization, CBA-restimulated lymphocytes secreted GARP/TGFß:LAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFß:LAP was mainly associated with CD81+ exosomes. sEV-bound GARP/TGFß:LAP became active in both 1° and 2° immunosuppression, the latter requiring sEV uptake by "bystander" T cells and reexpression on the cell surface. Conclusions: Like other immune-suppressive components of the Treg exosome, which are produced in a latent form, exosomal GARP/TGFß:LAP produced by allo-specific regulatory T cells undergoes either immediate activation (1° suppression) or internalization by naive T cells, followed by surface reexpression and subsequent activation (2°), to become suppressive. Our results imply a membrane-associated form of TGFß:LAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFß:LAP along with Treg-derived GARP as part of the infectious tolerance network.

Memory Precursors and Short-Lived Effector T cell Subsets Have Different Sensitivities to TGFß.

After exposure to an antigen, CD8 T cells reach a decision point about their fate: to become either short-lived effector cells (SLECs) or memory progenitor effector cells (MPECs). SLECs are specialized in providing an immediate effector function but have a shorter lifespan and lower proliferative capacity compared to MPECs. Upon encountering the cognate antigen during an infection, CD8 T cells rapidly expand and then contract to a level that is maintained for the memory phase after the peak of the response. Studies have shown that the contraction phase is mediated by TGFß and selectively targets SLECs, while sparing MPECs. The aim of this study is to investigate how the CD8 T cell precursor stage determines TGFß sensitivity. Our results demonstrate that MPECs and SLECs have differential responses to TGFß, with SLECs being more sensitive to TGFß than MPECs. This difference in sensitivity is associated with the levels of TGFßRI and RGS3, and the SLEC-related transcriptional activator T-bet binding to the TGFßRI promoter may provide a molecular basis for increased TGFß sensitivity in SLECs.

NKG2D signaling shifts the balance of CD8 T cells from single cytokine- to polycytokine-producing effector cells.

CD8 T cells play a critical role in immunity against intracellular pathogens and cancer. A primary objective of T cell-based vaccine strategies is the induction of durable and effective immune responses. Achieving this goal involves more than simply boosting the numbers of responding T cells. Of particular interest is the induction of CD8 T cells with polycytokine capability, specifically with the ability of CD8 T cells to co-produce IFNγ, TNFα and IL-2. The presence of these polycytokine-producing CD8 T cells correlates strongly with protection against foreign pathogens and cancer. Therefore, approaches capable of inducing such polyfunctional responses are needed. NKG2D engagement on CD8 T cells has been shown to result in increased effector response. However, the manner in which NKG2D engagement results in improved CD8 T cell effector response is unclear. Here we demonstrate in vitro and in vivo that NKG2D engagement by its natural ligand, Rae-1ε, shifts the balance from single cytokine to polycytokine (IL-2, IFNγ, and TFNα) production. These data define a previously unrecognized process in which NKG2D costimulation on CD8 T cells results in improved effector responses.

Drug delivery targets and strategies to address mast cell diseases.

INTRODUCTION: Current and developing mast cell therapeutics are reliant on small molecule drugs and biologics, but few are truly selective for mast cells. Most have cellular and disease-specific limitations that require innovation to overcome longstanding challenges to selectively targeting and modulating mast cell behavior. This review is designed to serve as a frame of reference for new approaches that utilize nanotechnology or combine different drugs to increase mast cell selectivity and therapeutic efficacy. AREAS COVERED: Mast cell diseases include allergy and related conditions as well as malignancies. Here, we discuss the targets of existing and developing therapies used to treat these disease pathologies, classifying them into cell surface, intracellular, and extracellular categories. For each target discussed, we discuss drugs that are either the current standard of care, under development, or have indications for potential use. Finally, we discuss how novel technologies and tools can be used to take existing therapeutics to a new level of selectivity and potency against mast cells. EXPERT OPINION: There are many broadly and very few selectively targeted therapeutics for mast cells in allergy and malignant disease. Combining existing targeting strategies with technology like nanoparticles will provide novel platforms to treat mast cell disease more selectively.

Siglecs in allergy and asthma.

The term "allergic diseases" encompasses several common, IgE-mediated conditions that range from being annoying to those that are life-threatening. Available treatments include active avoidance of the instigating allergen and the use of a variety of oral, inhaled, intranasal, intraocular and injected agents. While most individuals with allergies do well with existing therapies, there are still unmet therapeutic needs. Siglecs (sialic acid-binding, immunoglobulin-like lectins) are a family of single-pass transmembrane I-type lectins found on various subsets of cells, especially those of the immune system. All Siglecs have extracellular domains recognizing sialoside ligands, and most contain cytoplasmic domains with inhibitory signaling activity. This review focuses on Siglecs that likely play a role in regulating allergic and asthmatic responses, and how specific Siglecs, expressed on cells such as eosinophils and mast cells, are being targeted for therapeutic benefit.

Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants.

The first-generation COVID-19 vaccines have been effective in mitigating severe illness and hospitalization, but recurring waves of infections are associated with the emergence of SARS-CoV-2 variants that display progressive abilities to evade antibodies, leading to diminished vaccine effectiveness. The lack of clarity on the extent to which vaccine-elicited mucosal or systemic memory T cells protect against such antibody-evasive SARS-CoV-2 variants remains a critical knowledge gap in our quest for broadly protective vaccines. Using adjuvanted spike protein­based vaccines that elicit potent T cell responses, we assessed whether systemic or lung-resident CD4 and CD8 T cells protected against SARS-CoV-2 variants in the presence or absence of virus-neutralizing antibodies. We found that 1) mucosal or parenteral immunization led to effective viral control and protected against lung pathology with or without neutralizing antibodies, 2) protection afforded by mucosal memory CD8 T cells was largely redundant in the presence of antibodies that effectively neutralized the challenge virus, and 3) "unhelped" mucosal memory CD8 T cells provided no protection against the homologous SARS-CoV-2 without CD4 T cells and neutralizing antibodies. Significantly, however, in the absence of detectable virus-neutralizing antibodies, systemic or lung-resident memory CD4 and "helped" CD8 T cells provided effective protection against the relatively antibody-resistant B1.351 (ß) variant, without lung immunopathology. Thus, induction of systemic and mucosal memory T cells directed against conserved epitopes might be an effective strategy to protect against SARS-CoV-2 variants that evade neutralizing antibodies. Mechanistic insights from this work have significant implications in the development of T cell­targeted immunomodulation or broadly protective SARS-CoV-2 vaccines.

Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells.

Mast cells are tissue-resident cells that contribute to allergic diseases, among others, due to excessive or inappropriate cellular activation and degranulation. Therapeutic approaches to modulate mast cell activation are urgently needed. Siglec-6 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptor selectively expressed by mast cells, making it a promising target for therapeutic intervention. However, the effects of its engagement on mast cells are poorly defined. Siglec-6 expression and endocytosis on primary human mast cells and mast cell lines were assessed by flow cytometry. SIGLEC6 mRNA expression was examined by single-cell RNAseq in esophageal tissue biopsy samples. The ability of Siglec-6 engagement or co-engagement to prevent primary mast cell activation was determined based on assessments of mediator and cytokine secretion and degranulation markers. Siglec-6 was highly expressed by all mast cells examined, and the SIGLEC6 transcript was restricted to mast cells in esophageal biopsy samples. Siglec-6 endocytosis occurred with delayed kinetics relative to the related receptor Siglec-8. Co-crosslinking of Siglec-6 with FcεRIα enhanced the inhibition of mast cell activation and diminished downstream ERK1/2 and p38 phosphorylation. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.

Cross-decoration of dendritic cells by non-inherited maternal antigen-containing extracellular vesicles: Potential mechanism for PD-L1-based tolerance in cord blood and organ transplantation.

Exposure to non-inherited maternal antigens (NIMA) during the fetal period induces lifelong split tolerance to grafts expressing these allo-antigens. In adult mice, the production of extracellular vesicles (EVs) from maternal microchimeric cells causes cross-decoration (XD) of offspring dendritic cells (DC) with NIMA and upregulation of PD-L1, contributing to NIMA tolerance. To see how this may apply to humans, we tested NIMA acquisition by fetal DCS in human cord blood. The average percentage of NIMA-XD among total DCs was 2.6% for myeloid and 4.5% for Plasmacytoid DC. These cells showed higher PD-L1 expression than their non-XD counterparts (mDC: p = .0016; pDC: p = .024). We detected CD9+ EVs bearing NIMA and PD-L1 in cord blood. To determine if this immune regulatory mechanism persists beyond the pregnancy, we analyzed NIMA-expressing kidney and liver transplant recipients. We found donor antigen XD DCs in peripheral blood and graft-infiltrating DCs. As in cord blood, the pattern of donor antigen expression was punctate, and PD-L1 expression was upregulated, likely due to both protein and miRNA acquired from EV. Our findings support a mechanism for split tolerance to NIMAs that develops during pregnancy and is recapitulated in adult transplant recipients.

Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5-primed eosinophils causes cell death via CD11b/CD18 integrin-mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8-induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8-induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5-primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.

Discovery, Function, and Therapeutic Targeting of Siglec-8.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass cell surface receptors that have inhibitory activities on immune cells. Among these, Siglec-8 is a CD33-related family member selectively expressed on human mast cells and eosinophils, and at low levels on basophils. These cells can participate in inflammatory responses by releasing mediators that attract or activate other cells, contributing to the pathogenesis of allergic and non-allergic diseases. Since its discovery in 2000, initial in vitro studies have found that the engagement of Siglec-8 with a monoclonal antibody or with selective polyvalent sialoglycan ligands induced the cell death of eosinophils and inhibited mast cell degranulation. Anti-Siglec-8 antibody administration in vivo to humanized and transgenic mice selectively expressing Siglec-8 on mouse eosinophils and mast cells confirmed the in vitro findings, and identified additional anti-inflammatory effects. AK002 (lirentelimab) is a humanized non-fucosylated IgG1 antibody against Siglec-8 in clinical development for mast cell- and eosinophil-mediated diseases. AK002 administration has safely demonstrated the inhibition of mast cell activity and the depletion of eosinophils in several phase 1 and phase 2 trials. This article reviews the discovery and functions of Siglec-8, and strategies for its therapeutic targeting for the treatment of eosinophil- and mast cell-associated diseases.

Donor HLA-DR Drives the Development of De Novo Autoimmunity Following Lung and Heart Transplantation.

Individuals harbor preexisting HLA-DR/DQ-restricted responses to collagen type V (ColV) mediated by Th17 cells under Treg control, both specific to peptides that bind to inherited HLA class II antigens. Yet after transplant, the donor-DR type somehow influences graft outcome. We hypothesized that, long after a lung or heart allograft, the particular HLA-DR type of the mismatched transplant donor transforms the specificity of the "anti-self" response. This could explain why, over long term, certain donor DRs could be more immunogenic than others. METHODS: We analyzed 7 HLA-DR15neg patients who had received a lung allograft from a DR15+ donor. To determine the mechanism of acquired specificity in self-reactivity, we analyzed the kinetics of DR1 (host) and DR15 (donor) peptide restriction in a heart transplant model using DR-transgenic mice. RESULTS: Beyond 1.5 years post-lung transplant, all patients tested had acquired DR15-restricted immune responses to ColV peptides. These responses were either unrestrained Th17 type (n = 4) or Th17 controlled by Treg arising early (<5 y) or late (>7 y) after transplant (n = 4). Treg suppression via conventional (transforming growth factor-ß [TGF-ß]) and extracellular vesicle-associated (IL-35) cytokines correlated with superior outcomes. Naïve DR1 and DR15 transgenic mice had preexisting DR-restricted responses, exclusively to ColV fragments containing DR1- or DR15-binding peptides. When HLA-DR1 transgenic recipients of a HLA-DR15 heart developed ColV reactivity post-transplant, mice that acutely rejected (20-25 d) responded only to the DR1-restricted ColV peptide epitope. In animals whose grafts survived long term, we could detect acquisition of DR from the transplant donor onto the surface of recipient dendritic cells, and immune responses against a donor DR15-restricted ColV peptide. CONCLUSIONS: These results might explain how certain donor HLA-DR types redirect host immune responses to novel peptides of critical self-antigens. Unless regulated, such responses may predispose the allograft to chronic rejection.

Infectious Tolerance as Seen With 2020 Vision: The Role of IL-35 and Extracellular Vesicles.

Originally identified as lymphocyte regulation of fellow lymphocytes, our understanding of infectious tolerance has undergone significant evolutions in understanding since being proposed in the early 1970s by Gershon and Kondo and expanded upon by Herman Waldman two decades later. The evolution of our understanding of infectious tolerance has coincided with significant cellular and humoral discoveries. The early studies leading to the isolation and identification of Regulatory T cells (Tregs) and cytokines including TGFß and IL-10 in the control of peripheral tolerance was a paradigm shift in our understanding of infectious tolerance. More recently, another potential, paradigm shift in our understanding of the "infectious" aspect of infectious tolerance was proposed, identifying extracellular vesicles (EVs) as a mechanism for propagating infectious tolerance. In this review, we will outline the history of infectious tolerance, focusing on a potential EV mechanism for infectious tolerance and a novel, EV-associated form for the cytokine IL-35, ideally suited to the task of propagating tolerance by "infecting" other lymphocytes.

NFATc3 regulation of collagen V expression contributes to cellular immunity to collagen type V and hypoxic pulmonary hypertension.

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.

Single-site, five-year experience with human eosinophil isolation by density gradient centrifugation and CD16 immunomagnetic negative separation.

OBJECTIVE: Little has been reported regarding the reliability of methods for the purification of human blood eosinophils. We retrospectively reviewed our experience with 350 consecutive eosinophil isolations. RESULTS: Between January 2014 and December 2018, we conducted 350 eosinophil purifications from 83 donors. Absolute eosinophil count (AEC), calculated from hospital complete blood counts when available (n = 289), ranged from 32 to 1352 eosinophils/µL ([Formula: see text]: 179 ± 136/µL). Eosinophil yields ranged from 0.4 to 24.4 million cells per 20 mL of blood drawn ([Formula: see text]: 3.1 ± 1.9 million eosinophils) with > 98% purity. Comparing AEC to actual yield, recovery was 87% ± 29% ([Formula: see text]) and AEC strongly correlated with yield. To explore the reproducibility of yield, a subsequent analysis was limited to those donors drawn ≥ 3 times (N = 35), and there was no difference in the average coefficient of variation for yield between allergic and non-allergic donors. Viability of isolated eosinophils was consistently > 95% and after 24 h of culture did not differ between allergic and non-allergic donors. We conclude that this immunomagnetic separation method for human eosinophil isolation from whole blood is a reliable, reproducible technique for obtaining an average of 87% yield with high purity and viability.

Eosinophil and mast cell Siglecs: From biology to drug target.

Mast cells and eosinophils are innate immune cells involved in both acute and chronic inflammatory responses. Siglecs are a family of cell surface receptors that share sialic acid binding activity. Over the past 20 years, our knowledge of the expression and function of Siglecs on cells of the immune system and others has greatly expanded, as has our understanding of their signaling, ligands, and possible roles in disease pathophysiology. Because of this, Siglecs have garnered interest as potential drug targets using strategies ranging from biologics to ligand-directed nanoparticles. This mini-review will highlight the state of our knowledge regarding human eosinophil and mast cell Siglecs, their biology, what they recognize, tools developed for in vitro and preclinical experimentation, and the status of ongoing efforts to develop drugs that engage eosinophil and mast cell Siglecs for potential therapeutic benefit.

Treg-Cell-Derived IL-35-Coated Extracellular Vesicles Promote Infectious Tolerance.

Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in TregEbi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3+ and Foxp3neg (iTr35) T cells. Furthermore, IL-35 producers, although rare, secrete Ebi3 and p35 on extracellular vesicles (EVs) targeting a 25- to 100-fold higher number of T and B lymphocytes, causing them to acquire surface IL-35. This surface IL-35 is absent when EV production is inhibited or if Ebi3 is genetically deleted in Treg cells. The unique ability of EVs to coat bystander lymphocytes with IL-35, promoting exhaustion in, and secondary suppression by, non-Treg cells identifies a novel mechanism of infectious tolerance.

Leukocyte-Associated Ig-like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses.

Leukocyte-associated Ig-like receptor 1 (LAIR1) is an ITIM-bearing collagen receptor expressed by leukocytes and is implicated in immune suppression. However, using a divalent soluble LAIR1/Fc recombinant protein to block interaction of cell surface LAIR1 with matrix collagen, we found that whereas Th1 responses were enhanced as predicted, Th17 responses were strongly inhibited. Indeed, LAIR1 on both T cells and monocytes was required for optimal Th17 responses to collagen type (Col)V. For pre-existing "natural" Th17 response to ColV, the LAIR1 requirement was absolute, whereas adaptive Th17 and Th1/17 immune responses in both mice and humans were profoundly reduced in the absence of LAIR1. Furthermore, the addition of C1q, a natural LAIR1 ligand, decreased Th1 responses in a dose-dependent manner, but it had no effect on Th17 responses. In IL-17-dependent murine organ transplant models of chronic rejection, LAIR1+/+ but not LAIR1-/- littermates mounted strong fibroproliferative responses. Surface LAIR1 expression was higher on human Th17 cells as compared with Th1 cells, ruling out a receptor deficiency that could account for the differences. We conclude that LAIR1 ligation by its natural ligands favors Th17 cell development, allowing for preferential activity of these cells in collagen-rich environments. The emergence of cryptic self-antigens such as the LAIR1 ligand ColV during ischemia/reperfusion injury and early acute rejection, as well as the tendency of macrophages/monocytes to accumulate in the allograft during chronic rejection, favors Th17 over Th1 development, posing a risk to long-term graft survival.

Identification of a Siglec-F+ granulocyte-macrophage progenitor.

In recent years multi-parameter flow cytometry has enabled identification of cells at major stages in myeloid development; from pluripotent hematopoietic stem cells, through populations with increasingly limited developmental potential (common myeloid progenitors and granulocyte-macrophage progenitors), to terminally differentiated mature cells. Myeloid progenitors are heterogeneous, and the surface markers that define transition states from progenitors to mature cells are poorly characterized. Siglec-F is a surface glycoprotein frequently used in combination with IL-5 receptor alpha (IL5Rα) for the identification of murine eosinophils. Here, we describe a CD11b+ Siglec-F+ IL5Rα- myeloid population in the bone marrow of C57BL/6 mice. The CD11b+ Siglec-F+ IL5Rα- cells are retained in eosinophil deficient PHIL mice, and are not expanded upon overexpression of IL-5, indicating that they are upstream or independent of the eosinophil lineage. We show these cells to have GMP-like developmental potential in vitro and in vivo, and to be transcriptionally distinct from the classically described GMP population. The CD11b+ Siglec-F+ IL5Rα- population expands in the bone marrow of Myb mutant mice, which is potentially due to negative transcriptional regulation of Siglec-F by Myb. Lastly, we show that the role of Siglec-F may be, at least in part, to regulate GMP viability.