CD74 supports accumulation and function of regulatory T cells in tumors.

Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.

HLA-DR and CD74 Expression and the Immune Microenvironment in Renal Cell Carcinoma.

BACKGROUND/AIM: Expression of human leukocyte antigen (HLA) class I and II and CD74, which functions as a chaperone of MHC class II, play essential roles in T-cell recognition. The aim of this study was to elucidate the association between the HLAs and CD74, and their correlation with the infiltrated immune cells in renal cell carcinoma (RCC). MATERIALS AND METHODS: We retrospectively investigated the expression of HLA-A/B/C, HLA-DR, and CD74 in 38 patients with advanced RCC (T3/T4), and evaluated their correlations with CD4 and CD8-positive T-cell infiltration using immunohistochemistry. RESULTS: The expression of HLA-A/B/C, HLA-DR, and CD74 on cancer cells was observed in 37, 20, and 31 patients, respectively. The density of CD8- and CD4-positive T cells showed a positive correlation with HLA-DR expression. The density of CD4-positive lymphocytes was significantly associated with CD74 expression. CONCLUSION: The expression of HLA-DR, rather than CD74, on cancer cells was potentially associated with the anti-cancer immune microenvironment.

Surface Proteomics Reveals CD72 as a Target for In Vitro-Evolved Nanobody-Based CAR-T Cells in KMT2A/MLL1-Rearranged B-ALL.

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Anti-CD74 IgA autoantibodies in radiographic axial spondyloarthritis: a longitudinal Swedish study.

OBJECTIVES: Antibodies against anti-CD74 are related to axial spondyloarthritis (axSpA). The objectives were (i) to study IgA anti-CD74 in radiographic (r)-axSpA patients in the Backbone cohort and to calculate the sensitivity and specificity of anti-CD74, (ii) to study the fluctuation of IgA anti-CD74 levels in prospectively collected samples, and (iii) to explore the relation between IgA anti-CD74 and radiographic spinal changes. METHODS: IgA anti-CD74 was analysed by ELISA in 155 patients with r-axSpA and age- and sex-matched controls. BASDAI, ASDAS, BASFI and BASMI were assessed and spinal radiographs were scored for r-axSpA-related changes with mSASSS. Previously donated samples, before inclusion in the Backbone study, were identified in the Medical Biobank of Northern Sweden. RESULTS: A total of 155 patients comprising 69% men and 31% women, age [mean (s.d.)] 55.5 (11.4) years and 152 (98.1%) HLA-B27 positive, were included. The plasma level of IgA anti-CD74 was significantly higher in the patients [median (interquartile range), 12.9 (7.9-17.9) U/ml] compared with controls [10.9 (7.2-14.6) U/ml, P = 0.003]. IgA anti-CD74 was above the cut-off level of 20 U/ml in 36/155 (23.2%) patients and in 15/151 (9.9%) controls (P = 0.002). Multivariable logistic regression analyses revealed ≥1 syndesmophyte associated with IgA anti-CD74 (odds ratio 5.64; 95% CI: 1.02, 35.58; P = 0.048) adjusted for hsCRP, smoking, BMI, sex and age. No distinct pattern of IgA anti-CD74 over time was revealed. CONCLUSION: Plasma levels of IgA anti-CD74 were increased in r-axSpA and independently associated with radiographic spinal changes, which suggests that IgA anti-CD74 could play a role in the pathogenies of r-axSpA.

CD40 and CD72 expression and prognostic values among children with systemic lupus erythematosus: a case-control study.

Systemic lupus erythematosus (SLE) is a chronic disease with proven interactions between immune system components, including both humoral- and cell-mediated immunity, as well as co-stimulatory and inhibitory molecules such as CD40 and CD72. Here, we investigated CD40 and CD72 expression on B cells of SLE children and assessed their prognostic values. We conducted a preliminary case-control study in Mansoura University Children's Hospital, Egypt from September 2018 to January 2020 including 27 SLE children and 27 healthy controls. We assessed cases during initial flare and after remission. Flow cytometry analysis was carried out for all participants for CD40 and CD72 expression of B cells. During flare, SLE cases had statistically significant higher CD40 and lower CD72 expression in comparison with controls (p < 0.001). After remission, the number of CD40+ B cells significantly decreased (p < 0.001), while the number of CD72+ B cells significantly increased (p < 0.001) in comparison with flare. We reported non-significant positive correlations between CD40 expression and SLE Disease Activity Index (SLEDAI; p = 0.347 during flare and p = 0.653 after remission) and negative correlations between CD72 expression and SLEDAI (p = 0.34 during flare and p = 0.044 after remission). No significant differences were detected between renal histopathology classes with regard to CDs expression on B cells (p = 0.45 for CD40 and p = 0.63 for CD72). In conclusion, CD40+ B cells and CD72+ B cells could be considered as markers of paediatric SLE flare and remission, respectively.

The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

Cutting Edge: ST8Sia6-Generated α-2,8-Disialic Acids Mitigate Hyperglycemia in Multiple Low-Dose Streptozotocin-Induced Diabetes.

The immune system contains a series of checks and balances that maintain tolerance and prevent autoimmunity. Sialic acid-binding Ig-type lectins (Siglecs) are cell surface receptors found on immune cells and inhibit inflammation by recruiting protein tyrosine phosphatases to ITIMs. Islet-resident macrophages express Siglec-E, and Siglec-E expression decreases on islet-resident macrophages as insulitis progresses in the NOD mouse. The sialyltransferase ST8Sia6 generates α-2,8-disialic acids that are ligands for Siglec-E in vivo. We hypothesized that engaging Siglec-E through ST8Sia6-generated ligands may inhibit the development of immune-mediated diabetes. Constitutive overexpression of ST8Sia6 in pancreatic ß cells mitigated hyperglycemia in the multiple low-dose streptozotocin model of diabetes, demonstrating that engagement of this immune receptor facilitates tolerance in the setting of inflammation and autoimmune disease.

The sialoglycan-Siglec-E checkpoint axis in dexamethasone-induced immune subversion in glioma-microglia transwell co-culture system.

Dexamethasone (Dex) is considered as the main steroid routinely used in the standard therapy of brain tumor-induced edema. Strong immunosuppressive effects of Dex on effector systems of the immune system affect the patients' antitumor immunity and may thereby worsen the prognosis. Siglecs and their interacting sialoglycans have been described as a novel glyco-immune checkpoint axis that promotes cancer immune evasion. Despite the aberrant glycosylation in cancer is described, mechanisms involved in regulation of immune checkpoints in gliomas are not fully understood. The aim of this study was to investigate the effect of Dex on the Siglec-sialic acid interplay and determine its significance in immune inversion in monocultured and co-cultured microglia and glioma cells. Both monocultured and co-cultured in transwell system embryonic stem cell-derived microglia (ESdM) and glioma GL261 cells were exposed to Dex. Cell viability, immune inversion markers, and interaction between sialic acid and Siglec-E were detected by flow cytometry. Cell invasion was analyzed by scratch-wound migration assay using inverted phase-contrast microscopy. Exposure to Dex led to significant changes in IL-1ß, IL-10, Iba-1, and Siglec-E in co-cultured microglia compared to naïve or monocultured cells. These alterations were accompanied by increased α2.8-sialylation and Siglec-E fusion protein binding to co-cultured glioma cell membranes. This study suggests that the interplay between sialic acids and Siglecs is a sensitive immune checkpoint axis and may be crucial for Dex-induced dampening of antitumor immunity. The targeting of sialic acid-Siglec glyco-immune checkpoint can be a novel therapeutic method in glioma therapy.

AK002, a Humanized Sialic Acid-Binding Immunoglobulin-Like Lectin-8 Antibody that Induces Antibody-Dependent Cell-Mediated Cytotoxicity against Human Eosinophils and Inhibits Mast Cell-Mediated Anaphylaxis in Mice.

INTRODUCTION: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. METHODS: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. RESULTS: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. CONCLUSION: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.

CD22 and CD72 cooperatively contribute to the development of the reverse Arthus reaction model.

BACKGROUND: Local type III hypersensitivity reactions are acute inflammatory events induced by immune complex (IC) deposition. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function. OBJECTIVE: To elucidate the roles of CD22 and CD72 in the development of IgG-mediated type III hypersensitivity reactions. METHOD: The reverse Arthus reaction model in the skin was induced in mice lacking CD22 (CD22-/-), CD72 (CD72-/-), and both of them (CD22-/-/CD72-/-). Edema at 4h and hemorrhage at 8h after IC challenge were evaluated. Inflammatory cell infiltration and cytokine and chemokine expression were also examined. RESULTS: Edema and hemorrhage were significantly reduced in CD22-/-/CD72-/- mice compared with wild-type mice. The loss of both membrane proteins resulted in a greater decrease in edema at 4h, but not hemorrhage at 8h, than the loss of each protein alone. Infiltration of neutrophils, macrophages, and T cells, and the expression of TNF-α, IL-6, MIP-1α, and CCR5 mRNA were also diminished in the knockout mice compared to wild-type mice, and most significantly reduced in CD22-/-/CD72-/- mice. Regulatory T (Treg) cells in the spleen were significantly increased in all knockout mice at 4h. Significant differences in the severity of edema and hemorrhage between wild-type and knockout mice were lost when Treg cells were depleted in the knockout mice. CONCLUSION: These results demonstrate that CD22 and CD72 expression contribute to the development of the reverse Arthus reaction model and CD22 and CD72 might be therapeutic targets for human IC-mediated diseases.

Immune stimulation of rainbow trout reveals divergent regulation of MH class II-associated invariant chain isoforms.

Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1ß transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.

Renal proximal tubular epithelial cells exert immunomodulatory function by driving inflammatory CD4+ T cell responses.

In immune-mediated glomerular diseases like crescentic glomerulonephritis (cGN), inflammatory CD4+ T cells accumulate within the tubulointerstitial compartment in close contact to proximal and distal tubular epithelial cells and drive renal inflammation and tissue damage. However, whether renal epithelial cell populations play a role in the pathogenesis of cGN by modulating CD4+ T cell responses is less clear. In the present study, we aimed to investigate the potential of renal epithelial cells to function as antigen-presenting cells, thereby stimulating CD4+ T cell responses. Using a FACS-based protocol that allowed comparative analysis of cortical epithelial cell populations, we showed that particularly proximal tubular epithelial cells (PTECs) express molecules linked with antigen-presenting cell function, including major histocompatibility complex class II (MHCII), CD74, CD80, and CD86 in homeostasis and nephrotoxic nephritis, a murine model of cGN. Protein expression was visualized at the PTEC single cell level by imaging flow cytometry. Interestingly, we found inflammation-dependent regulation of epithelium-expressed CD74, CD80, and CD86, whereas MHCII expression was not altered. Antigen-specific stimulation of CD4+ T cells by PTECs in vitro supported CD4+ T cell survival and induced CD4+ T cell activation, proliferation, and inflammatory cytokine production. In patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis, MHCII and CD74 were expressed by both proximal and distal tubules, whereas CD86 was predominantly expressed by proximal tubules. Thus, particularly PTECs have the potential to induce an inflammatory phenotype in CD4+ T cells in vitro, which might also play a role in the pathology of immune-mediated kidney disease.

Single-Cell RNA Sequencing Identifies Candidate Renal Resident Macrophage Gene Expression Signatures across Species.

BACKGROUND: Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients. METHODS: As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell-negative CD45+ innate immune cells in mouse, rat, pig, and human kidney tissue. RESULTS: We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species. CONCLUSIONS: Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.

What to do with HLA-DO/H-2O two decades later?

The main objective of antigen processing is to orchestrate the selection of immunodominant epitopes for recognition by CD4 T cells. To achieve this, MHC class II molecules have evolved with a flexible peptide-binding groove in need of a bound peptide. Newly synthesized MHC-II molecules bind a class II invariant chain (Ii) upon synthesis and are shuttled to a specialized compartment, where they encounter exogenous antigens. Ii serves multiple functions, one of which is to maintain the shape of the MHC-II groove so that it can readily bind exogenous antigens upon dissociation of the Ii peptide in MHC- II compartment. MIIC contains processing enzymes, one or both accessory molecules, HLA-DM/H2-M (DM) and HLA-DO/H2-O (DO), and optimal denaturing conditions. In a process known as "editing," DM facilitates the dissociation of the invariant chain peptide, CLIP, for exchange with exogenous antigens. Despite the availability of mechanistic insights into DM functions, understanding how DO contributes to epitope selection has proven to be more challenging. The current dogma assumes that DO inhibits DM, whereas an opposing model suggests that DO fine-tunes the epitope selection process. Understanding which of these, or potentially other models of DO function is important, as DO variants have been linked to autoimmunity, cancer, and the generation of broadly neutralizing antibodies to viruses. This review therefore attempts to evaluate experimental evidence in support of these hypotheses, with an emphasis on the less discussed model, and to explore intriguing questions about the importance of DO in biology.

A universal anti-cancer vaccine: Chimeric invariant chain potentiates the inhibition of melanoma progression and the improvement of survival.

For many years, clinicians and scientists attempt to develop methods to stimulate the immune system to target malignant cells. Recent data suggest that effective cancer vaccination requires combination immunotherapies to overcome tumor immune evasion. Through presentation of both MHC-I and II molecules, DCs-based vaccine platforms are effective in generating detectable CD4 and CD8 T cell responses against tumor-associated antigens. Several platforms include DC transfection with mRNA of the desired tumor antigen. These DCs are then delivered to the host and elicit an immune response against the antigen of interest. We have recently established an mRNA genetic platform which induced specific CD8+ cytotoxic T cell response by DC vaccination against melanoma. In our study, an MHC-II mRNA DCs vaccine platform was developed to activate CD4+ T cells and to enhance the anti-tumor response. The invariant chain (Ii) was modified and the semi-peptide CLIP was replaced with an MHC-II binding peptide sequences of melanoma antigens. These chimeric MHC-II constructs are presented by DCs and induce proliferation of tumor specific CD4+ T cells. When administered in combination with the MHC-I platform into tumor bearing mice, these constructs were able to inhibit tumor growth, and improve mouse survival. Deciphering the immunological mechanism of action, we observed an efficient CTLs killing in addition to higher levels of Th1 and Th2 subsets in the groups immunized with a combination of the MHC-I and MHC-II constructs. These universal constructs can be applied in multiple combinations and offer an attractive opportunity to improve cancer treatment.

Immunoregulatory Siglec ligands are abundant in human and mouse aorta and are up-regulated by high glucose.

AIM: Inflammation is a driving force in development of atherosclerosis, and hyperglycemia is a significant risk factor for angiopathy. Siglec-9, expressed on human neutrophils and macrophages, engages specific glycan ligands on tissues to diminish ongoing inflammation. MATERIALS AND METHOD: Siglec-9 ligands on human aorta were characterized and the effects of high glucose exposure on the expression of ligands for Siglec-9 on human umbilical vein endothelial cells (HUV-EC-C) in vitro and ligands for the comparable siglec (Siglec-E) on mouse aorta in vivo were studied. KEY FINDINGS: Siglec-9 ligands were expressed broadly on human aorta, as well as on HUV-EC-C. Siglec-9 ligands on HUV-EC-C were sharply up-regulated under high glucose exposure in vitro, as were Siglec-E ligands on the aortas of hyperglycemic mice. Exposure of HUV-EC-C to high-glucose resulted in consistent inhibitory changes in co-cultured macrophages including increased apoptosis and decreased phagocytosis. Control of Siglec-9 ligand expression on HUV-EC-C was downstream of changes in an enzyme involved in their biosynthesis, UDP-galactose-4-epimerase (GALE) and increased cellular N-acetylgalactosamine. The alteration of GALE was associated with the regulatory microRNA hsa-let-7f. SIGNIFICANCE: We conclude that exposure to high-glucose results in up-regulation of immune inhibitory Siglec-9 sialoglycan ligands on aorta and HUV-EC-C cells downstream of altered GALE and GalNAc expression, resulting in up-regulation of apoptosis and decrease of phagocytic activity of macrophages. Changes in Siglec-9 sialoglycan ligand expression on vascular endothelial cells may be a natural response to the initial steps of atherosclerosis and might be a potential target to regulate inflammation in diabetic angiopathy.

Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 in patients with eosinophilic disorders: Receptor expression and targeting using chimeric antibodies.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.

Ligand Recognition Determines the Role of Inhibitory B Cell Co-receptors in the Regulation of B Cell Homeostasis and Autoimmunity.

B cells express various inhibitory co-receptors including CD22, CD72, and Siglec-G. These receptors contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region. Although many of the inhibitory co-receptors negatively regulate BCR signaling by activating SH2-containing protein tyrosine phosphatase 1 (SHP-1), different inhibitory co-receptors have distinct functional properties. CD22, Siglec-G, and CD72 preferentially regulate tonic signaling in conventional B cells, B-1 cell homeostasis, and development of lupus-like disease, respectively. CD72 recognizes RNA-related lupus self-antigen Sm/RNP as a ligand. This ligand recognition recruits CD72 to BCR in Sm/RNP-reactive B cells thereby suppressing production of anti-Sm/RNP autoantibody involved in the pathogenesis of lupus. In contrast, Siglec-G recognizes α2,3 as well as α2,6 sialic acids whereas CD22 recognizes α2,6 sialic acid alone. Because glycoproteins including BCR are dominantly glycosylated with α2,3 sialic acids in B-1 cells, Siglec-G but not CD22 recruits BCR as a ligand specifically in B-1 cells, and regulates B-1 cell homeostasis by suppressing BCR signaling in B-1 cells. Thus, recognition of distinct ligands determines functional properties of different inhibitory B cell co-receptors.

Smoking promotes exacerbated inflammatory features in dendritic cells of Chilean rheumatoid arthritis patients.

BACKGROUND: The dual potential to promote tolerance or inflammation when facing self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. There is an association between smoking and DCs maturation in patients with rheumatoid arthritis (RA). However, ethnicity is a key factor in autoimmune disorders. AIM: To evaluate phenotypic and functional alterations of DCs obtained from Chilean patients with RA as compared to healthy controls (HC). In second term, to compare the inflammatory behaviour of DCs between smoker and non-smoker patients. MATERIAL AND METHODS: Monocyte-derived DCs and T-cells were obtained from blood samples isolated from 30 HC and 32 RA-patients, 14 of which were currently smokers and 18 non-smokers. Several maturation surface markers were evaluated in DCs, including HLA-DR, CD40, CD80, CD83 and CD86. Furthermore, autologous co-cultures of DCs and T-cells were carried out and then T-cell proliferation, and expansion of Th1, Th17 and Tregs were analysed. RESULTS: Compared with HC, RA-patients displayed increased HLA-DR expression in DCs, which was manifested mainly in patients with moderate-to- high disease activity scores (DAS28). Furthermore, RA-patients presented a stronger Th17-expansion and a correlation between DAS28 and Th1-expansion. Both effects were mainly observed in patients in remission or with a low DAS28. Moreover, smoker RA-patients displayed enhanced HLA-DR and CD83 expression in DCs as well as an exacerbated Th17-expansion and a correlation between DAS28 and Th1-expansion. CONCLUSIONS: These findings suggest that smoking enhances the inflammatory behaviour of DCs and the consequent Th1 and Th17-mediated response in patients with RA.