ABSTRACT
The relationship between the Programmed Death-Ligand 1 (PD-L1)/Programmed Death-1 (PD-1) pathway, lung inflammation, and clinical outcomes in acute respiratory distress syndrome (ARDS) is poorly understood. We sought to determine whether PD-L1/PD-1 in the lung or blood is associated with ARDS and associated severity. We measured soluble PD-L1 (sPD-L1) in plasma and lower respiratory tract samples (ARDS1 (n = 59) and ARDS2 (n = 78)) or plasma samples alone (ARDS3 (n = 149)) collected from subjects with ARDS and tested for associations with mortality using multiple regression. We used mass cytometry to measure PD-L1/PD-1 expression and intracellular cytokine staining in cells isolated from bronchoalveolar lavage fluid (BALF) (n = 18) and blood (n = 16) from critically-ill subjects with or without ARDS enrolled from a fourth cohort. Higher plasma levels of sPD-L1 were associated with mortality in ARDS1, ARDS2, and ARDS3. In contrast, higher levels of sPD-L1 in the lung were either not associated with mortality (ARDS2) or were associated with survival (ARDS1). Alveolar PD-1POS T cells had more intracellular cytokine staining compared with PD-1NEG T cells. Subjects without ARDS had a higher ratio of PD-L1POS alveolar macrophages to PD-1POS T cells compared with subjects with ARDS. We conclude that sPD-L1 may have divergent cellular sources and/or functions in the alveolar vs. blood compartments given distinct associations with mortality. Alveolar leukocyte subsets defined by PD-L1/PD-1 cell-surface expression have distinct cytokine secretion profiles, and the relative proportions of these subsets are associated with ARDS.
ABSTRACT
Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.
Subject(s)
CD8-Positive T-Lymphocytes , Diabetes Mellitus, Type 1 , Killer Cells, Natural , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/genetics , Humans , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/genetics , CD57 Antigens/metabolism , Cell Lineage/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-alpha/metabolism , Female , Male , AdultABSTRACT
CD4+CD25hiCD127lo/-FOXP3+ regulatory T cells (Tregs) play a key role in preventing autoimmunity. In autoimmune type 1 diabetes (T1D), adoptive transfer of autologous polyclonal Tregs has been shown to be safe in adults in phase 1 clinical trials. We explored factors contributing to efficacy of autologous polyclonal expanded Tregs (expTregs) in a randomized phase 2 multi-center, double-blind, clinical trial (Sanford/Lisata Therapeutics T-Rex phase 2 trial, ClinicalTrials.gov NCT02691247). One hundred ten treated children and adolescents with new-onset T1D were randomized 1:1:1 to high-dose (20 × 106 cells/kilogram) or low-dose (1 × 106 cells/kilogram) treatments or to matching placebo. Cytometry as well as bulk and single-cell RNA sequencing were performed on selected expTregs and peripheral blood samples from participants. The single doses of expTregs were safe but did not prevent decline in residual ß cell function over 1 year compared to placebo (P = 0.94 low dose, P = 0.21 high dose), regardless of age or baseline C-peptide. ExpTregs were highly activated and suppressive in vitro. A transient increase of activated memory Tregs was detectable 1 week after infusion in the high-dose cohort, suggesting effective transfer of expTregs. However, the in vitro fold expansion of expTregs varied across participants, even when accounting for age, and lower fold expansion and its associated gene signature were linked with better C-peptide preservation regardless of Treg dose. These results suggest that a single dose of polyclonal expTregs does not alter progression in T1D; instead, Treg quality may be an important factor.
Subject(s)
Diabetes Mellitus, Type 1 , T-Lymphocytes, Regulatory , Humans , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/therapy , T-Lymphocytes, Regulatory/immunology , Child , Adolescent , Male , Female , Double-Blind Method , Child, Preschool , Transplantation, AutologousABSTRACT
Peripheral blood mononuclear cell (PBMC) immunophenotyping is crucial in tracking activation, disease state, and response to therapy in human subjects. Many studies require the shipping of blood from clinical sites to a laboratory for processing to PBMC, which can lead to delays that impact sample quality. We used an extensive cytometry by time-of-flight (CyTOF) immunophenotyping panel to analyze the impacts of delays to processing and distinct storage conditions on cell composition and quality of PBMC from seven adults across a range of ages, including two with rheumatoid arthritis. Two or more days of delay to processing resulted in extensive red blood cell contamination and increased variability of cell counts. While total memory and naïve B- and T-cell populations were maintained, 4-day delays reduced the frequencies of monocytes. Variation across all immune subsets increased with delays of up to 7 days in processing. Unbiased clustering analysis to define more granular subsets confirmed changes in PBMC composition, including decreases of classical and non-classical monocytes, basophils, plasmacytoid dendritic cells, and follicular helper T cells, with each subset impacted at a distinct time of delay. Expression of activation markers and chemokine receptors changed by Day 2, with differential impacts across subsets and markers. Our data support existing recommendations to process PBMC within 36 h of collection but provide guidance on appropriate immunophenotyping experiments with longer delays.
Subject(s)
Immunophenotyping , Leukocytes, Mononuclear , Humans , Immunophenotyping/methods , Adult , Male , Female , Leukocytes, Mononuclear/immunology , Middle Aged , Flow Cytometry/methods , Time Factors , Monocytes/immunology , Aged , Blood Preservation/methods , B-Lymphocytes/immunologyABSTRACT
Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.
Subject(s)
Abatacept , Alleles , Arthritis, Rheumatoid , CD8-Positive T-Lymphocytes , Receptors, Immunologic , Humans , Abatacept/therapeutic use , Abatacept/pharmacology , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/genetics , Male , Female , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/drug effects , Adult , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , HLA Antigens/genetics , HLA Antigens/immunology , Middle Aged , Antirheumatic Agents/therapeutic use , Genetic Predisposition to Disease , T-Cell ExhaustionABSTRACT
AIMS/HYPOTHESIS: We hypothesised that islet beta cell antigen presentation in the gut along with a tolerising cytokine would lead to antigen-specific tolerance in type 1 diabetes. We evaluated this in a parallel open-label Phase 1b study using oral AG019, food-grade Lactococcus lactis bacteria genetically modified to express human proinsulin and human IL-10, as a monotherapy and in a parallel, randomised, double-blind Phase 2a study using AG019 in combination with teplizumab. METHODS: Adults (18-42 years) and adolescents (12-17 years) with type 1 diabetes diagnosed within 150 days were enrolled, with documented evidence of at least one autoantibody and a stimulated peak C-peptide level >0.2 nmol/l. Participants were allocated to interventions using interactive response technology. We treated 42 people aged 12-42 years with recent-onset type 1 diabetes, 24 with Phase 1b monotherapy (open-label) and 18 with Phase 2a combination therapy. In the Phase 2a study, after treatment of the first two open-label participants, all people involved were blinded to group assignment, except for the Data Safety Monitoring Board members and the unblinded statistician. The primary endpoint was safety and tolerability based on the incidence of treatment-emergent adverse events, collected up to 6 months post treatment initiation. The secondary endpoints were pharmacokinetics, based on AG019 detection in blood and faeces, and pharmacodynamic activity. Metabolic and immune endpoints included stimulated C-peptide levels during a mixed meal tolerance test, HbA1c levels, insulin use, and antigen-specific CD4+ and CD8+ T cell responses using an activation-induced marker assay and pooled tetramers, respectively. RESULTS: Data from 24 Phase 1b participants and 18 Phase 2a participants were analysed. No serious adverse events were reported and none of the participants discontinued AG019 due to treatment-emergent adverse events. No systemic exposure to AG019 bacteria, proinsulin or human IL-10 was demonstrated. In AG019 monotherapy-treated adults, metabolic variables were stabilised up to 6 months (C-peptide, insulin use) or 12 months (HbA1c) post treatment initiation. In participants treated with AG019/teplizumab combination therapy, all measured metabolic variables stabilised or improved up to 12 months and CD8+ T cells with a partially exhausted phenotype were significantly increased at 6 months. Circulating preproinsulin-specific CD4+ and CD8+ T cells were detected before and after treatment, with a reduction in the frequency of preproinsulin-specific CD8+ T cells after treatment with monotherapy or combination therapy. CONCLUSIONS/INTERPRETATION: Oral delivery of AG019 was well tolerated and safe as monotherapy and in combination with teplizumab. AG019 was not shown to interfere with the safety profile of teplizumab and may have additional biological effects, including changes in preproinsulin-specific T cells. These preliminary data support continuing studies with this agent alone and in combination with teplizumab or other systemic immunotherapies in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03751007, EudraCT 2017-002871-24 FUNDING: This study was funded by Precigen ActoBio.
Subject(s)
Diabetes Mellitus, Type 1 , Adult , Adolescent , Humans , Interleukin-10 , C-Peptide , CD8-Positive T-Lymphocytes/metabolism , Proinsulin , Double-Blind MethodABSTRACT
High levels of the inflammatory cytokine IL-6 in the bone marrow are associated with poor outcomes in pediatric acute myeloid leukemia (pAML), but its etiology remains unknown. Using RNA-seq data from pre-treatment bone marrows of 1489 children with pAML, we show that > 20% of patients have concurrent IL-6, IL-1, IFNα/ß, and TNFα signaling activity and poorer outcomes. Targeted sequencing of pre-treatment bone marrow samples from affected patients (n = 181) revealed 5 highly recurrent patterns of somatic mutation. Using differential expression analyses of the most common genomic subtypes (~60% of total), we identify high expression of multiple potential drivers of inflammation-related treatment resistance. Regardless of genomic subtype, we show that JAK1/2 inhibition reduces receptor-mediated inflammatory signaling by leukemic cells in-vitro. The large number of high-risk pAML genomic subtypes presents an obstacle to the development of mutation-specific therapies. Our findings suggest that therapies targeting inflammatory signaling may be effective across multiple genomic subtypes of pAML.
Subject(s)
Bone Marrow , Leukemia, Myeloid, Acute , Humans , Child , Bone Marrow/metabolism , Interleukin-6/metabolism , Signal Transduction/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolismABSTRACT
Critically ill patients manifest many of the same immune features seen in coronavirus disease 2019 (COVID-19), including both "cytokine storm" and "immune suppression." However, direct comparisons of molecular and cellular profiles between contemporaneously enrolled critically ill patients with and without severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited. We sought to identify immune signatures specifically enriched in critically ill patients with COVID-19 compared with patients without COVID-19. We enrolled a multisite prospective cohort of patients admitted under suspicion for COVID-19, who were then determined to be SARS-CoV-2-positive (n = 204) or -negative (n = 122). SARS-CoV-2-positive patients had higher plasma levels of CXCL10, sPD-L1, IFN-γ, CCL26, C-reactive protein (CRP), and TNF-α relative to SARS-CoV-2-negative patients adjusting for demographics and severity of illness (Bonferroni P value < 0.05). In contrast, the levels of IL-6, IL-8, IL-10, and IL-17A were not significantly different between the two groups. In SARS-CoV-2-positive patients, higher plasma levels of sPD-L1 and TNF-α were associated with fewer ventilator-free days (VFDs) and higher mortality rates (Bonferroni P value < 0.05). Lymphocyte chemoattractants such as CCL17 were associated with more severe respiratory failure in SARS-CoV-2-positive patients, but less severe respiratory failure in SARS-CoV-2-negative patients (P value for interaction < 0.01). Circulating T cells and monocytes from SARS-CoV-2-positive subjects were hyporesponsive to in vitro stimulation compared with SARS-CoV-2-negative subjects. Critically ill SARS-CoV-2-positive patients exhibit an immune signature of high interferon-induced lymphocyte chemoattractants (e.g., CXCL10 and CCL17) and immune cell hyporesponsiveness when directly compared with SARS-CoV-2-negative patients. This suggests a specific role for T-cell migration coupled with an immune-checkpoint regulatory response in COVID-19-related critical illness.
Subject(s)
COVID-19 , Respiratory Insufficiency , B7-H1 Antigen , Chemokines , Critical Illness , Humans , Prospective Studies , SARS-CoV-2 , Tumor Necrosis Factor-alphaABSTRACT
Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen-agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort-specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry-based T-cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D.
Subject(s)
Diabetes Mellitus, Type 1 , Biomarkers/analysis , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 1/therapy , Flow Cytometry/methods , Humans , Immunophenotyping , T-LymphocytesABSTRACT
Individuals with Down syndrome show cellular and clinical features of dysregulated aging of the immune system, including a shift from naïve to memory T cells and increased incidence of autoimmunity. However, a quantitative understanding of how various immune compartments change with age in Down syndrome remains lacking. Here, we performed deep immunophenotyping of a cohort of individuals with Down syndrome across the life span, selecting for autoimmunity-free individuals. We simultaneously interrogated age- and sex-matched healthy controls and people with type 1 diabetes as a representative autoimmune disease. We built an analytical software, IMPACD (Iterative Machine-assisted Permutational Analysis of Cytometry Data), that enabled us to rapidly identify many features of immune dysregulation in Down syndrome shared with other autoimmune diseases. We found quantitative and qualitative dysregulation of naïve CD4+ and CD8+ T cells in individuals with Down syndrome and identified interleukin-6 as a candidate driver of some of these changes, thus extending the consideration of immunopathologic cytokines in Down syndrome beyond interferons. We used immune cellular composition to generate three linear models of aging (immune clocks) trained on control participants. All three immune clocks demonstrated advanced immune aging in individuals with Down syndrome. One of these clocks, informed by Down syndromerelevant biology, also showed advanced immune aging in individuals with type 1 diabetes. Orthologous RNA sequencingderived immune clocks also demonstrated advanced immune aging in individuals with Down syndrome. Together, our findings demonstrate an approach to studying immune aging in Down syndrome that may have implications in other autoimmune diseases.
Subject(s)
Autoimmune Diseases , Diabetes Mellitus, Type 1 , Down Syndrome , Aging , Autoimmunity/genetics , CD8-Positive T-Lymphocytes , Down Syndrome/genetics , Humans , ImmunophenotypingABSTRACT
Mislabeling samples or data with the wrong participant information can affect study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of human leukocyte antigen (HLA) class I alleles. We measured HLA-A*02 and HLA-B*07 expression in three longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples' cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample-labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.
Subject(s)
Cross-Sectional Studies , Alleles , Humans , Real-Time Polymerase Chain ReactionABSTRACT
Immune monitoring enables a better understanding of disease processes and response to therapy, but has been challenging in the setting of chronic autoimmunity because of unknown etiology, variable and protracted kinetics of the disease process, heterogeneity across patients and the complexity of immune interactions. To begin to parse this complexity, we focus here on type 1 diabetes (T1D) and CD8 T cells as a cell type that has features that are associated with different stages of disease, rates of progression and response to therapy. Specifically, we discuss the current understanding of the role of autoreactive CD8 T cells in disease outcome, which implicates particular CD8 functional subsets, rather than unique antigens or total number of autoreactive T cells. Next, we discuss how autoreactive CD8 T-cell features can be reflected in measures of global CD8 T cells, and then pull these concepts together by highlighting immune therapies recently shown to modulate both CD8 T cells and disease progression. We end by discussing outstanding questions about the role of specific subsets of autoreactive CD8 T cells in disease progression and how they may be optimally modulated to treat and prevent T1D.
Subject(s)
Diabetes Mellitus, Type 1 , Autoantigens , Autoimmunity , CD8-Positive T-Lymphocytes , Diabetes Mellitus, Type 1/therapy , HumansABSTRACT
Elevation of granulocyte-macrophage colony-stimulating factor (GM-CSF)producing T helper (TH) cells has been associated with several autoimmune diseases, suggesting a potential role in the pathogenesis of autoimmunity. However, the identity of GM-CSFproducing TH cells has not been closely examined. Using single-cell RNA sequencing and high-dimensional single-cell mass cytometry, we identified eight populations of antigen-experienced CD45RA−CD4+ T cells in blood of healthy individuals including a population of GM-CSFproducing cells, known as THGM, that lacked expression of signature transcription factors and cytokines of established TH lineages. Using GM-CSF-reporter/fate reporter mice, we show that THGM cells are present in the periphery and central nervous system in a mouse model of experimental autoimmune encephalomyelitis. In addition to GM-CSF, human and mouse THGM cells also expressed IL-2, tumor necrosis factor (TNF), IL-3, and CCL20. THGM cells maintained their phenotype through several cycles of activation but up-regulated expression of T-bet and interferon-γ (IFN-γ) upon exposure to IL-12 in vitro and in the central nervous system of mice with autoimmune neuroinflammation. Although T-bet was not required for the development of THGM cells, it was essential for their encephalitogenicity. These findings demonstrate that THGM cells constitute a distinct population of TH cells with lineage characteristics that are poised to adopt a TH1 phenotype and promote neuroinflammation.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Multiple Sclerosis/immunology , T-Box Domain Proteins/metabolism , Th1 Cells/immunology , Animals , Cell Lineage/genetics , Cell Lineage/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Gene Expression Regulation/immunology , Healthy Volunteers , Humans , Male , Mice , Mice, Transgenic , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , RNA-Seq , Single-Cell Analysis , Th1 Cells/metabolismABSTRACT
Although most patients with type 1 diabetes (T1D) retain some functional insulin-producing islet ß cells at the time of diagnosis, the rate of further ß cell loss varies across individuals. It is not clear what drives this differential progression rate. CD8+ T cells have been implicated in the autoimmune destruction of ß cells. Here, we addressed whether the phenotype and function of autoreactive CD8+ T cells influence disease progression. We identified islet-specific CD8+ T cells using high-content, single-cell mass cytometry in combination with peptide-loaded MHC tetramer staining. We applied a new analytical method, DISCOV-R, to characterize these rare subsets. Autoreactive T cells were phenotypically heterogeneous, and their phenotype differed by rate of disease progression. Activated islet-specific CD8+ memory T cells were prevalent in subjects with T1D who experienced rapid loss of C-peptide; in contrast, slow disease progression was associated with an exhaustion-like profile, with expression of multiple inhibitory receptors, limited cytokine production, and reduced proliferative capacity. This relationship between properties of autoreactive CD8+ T cells and the rate of T1D disease progression after onset make these phenotypes attractive putative biomarkers of disease trajectory and treatment response and reveal potential targets for therapeutic intervention.
Subject(s)
Autoimmunity , CD8-Positive T-Lymphocytes/immunology , Diabetes Mellitus, Type 1/immunology , Immunologic Memory , Islets of Langerhans/immunology , Lymphocyte Activation , Adolescent , Adult , CD8-Positive T-Lymphocytes/pathology , Child , Child, Preschool , Diabetes Mellitus, Type 1/pathology , Female , Humans , Infant , Islets of Langerhans/pathology , Male , Middle AgedABSTRACT
Studies in human peripheral blood monocyte-derived macrophages in vitro have shown clear evidence that multiple macrophage polarization states exist. The extent to which different alveolar macrophage (AM) polarization states exist in homeostasis or in the setting of severe injury such as acute respiratory distress syndrome (ARDS) is largely unknown. We applied single-cell cytometry TOF (CyTOF) to simultaneously measure 36 cell-surface markers on CD45+ cells present in bronchoalveolar lavage from healthy volunteers, as well as mechanically ventilated subjects with and without ARDS. Visualization of the high-dimensional data with the t-distributed stochastic neighbor embedding algorithm demonstrated wide diversity of cell-surface marker profiles among CD33+CD71+CD163+ AMs. We then used a κ-nearest neighbor density estimation algorithm to statistically identify distinct alveolar myeloid subtypes, and we discerned 3 AM subtypes defined by CD169 and PD-L1 surface expression. The percentage of AMs that were classified into one of the 3 AM subtypes was significantly different between healthy and mechanically ventilated subjects. In an independent cohort of subjects with ARDS, PD-L1 gene expression and PD-L1/PD-1 pathway-associated gene sets were significantly decreased in AMs from patients who experienced prolonged mechanical ventilation or death. Unsupervised CyTOF analysis of alveolar leukocytes from human subjects has potential to identify expected and potentially novel myeloid populations that may be linked with clinical outcomes.
Subject(s)
Flow Cytometry/methods , Macrophages, Alveolar/classification , Respiratory Distress Syndrome/pathology , Adult , Antigens, CD/immunology , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Bronchoalveolar Lavage Fluid , Case-Control Studies , Female , Humans , Immunophenotyping , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Male , Young AdultABSTRACT
OBJECTIVE: Mutations in the ACP5 gene, which encodes tartrate-resistant acid phosphatase (TRAP), cause the immuno-osseous disorder spondyloenchondrodysplasia, which includes as disease features systemic lupus erythematosus (SLE) and a type I interferon (IFN) signature. Our aims were to identify TRAP substrates, determine the consequences of TRAP deficiency in immune cells, and assess whether ACP5 mutations are enriched in sporadic cases of SLE. METHODS: Interaction between TRAP and its binding partners was tested by a yeast 2-hybrid screening, confocal microscopy, and immunoprecipitation/Western blotting. TRAP knockdown was performed using small interfering RNA. Phosphorylation of osteopontin (OPN) was analyzed by mass spectrometry. Nucleotide sequence analysis of ACP5 was performed by Sanger sequencing or next-generation sequencing. RESULTS: TRAP and OPN colocalized and interacted in human macrophages and plasmacytoid dendritic cells (PDCs). TRAP dephosphorylated 3 serine residues on specific OPN peptides. TRAP knockdown resulted in increased OPN phosphorylation and increased nuclear translocation of IRF7 and P65, with resultant heightened expression of IFN-stimulated genes and IL6 and TNF following Toll-like receptor 9 stimulation. An excess of heterozygous ACP5 missense variants was observed in SLE compared to controls (P = 0.04), and transfection experiments revealed a significant reduction in TRAP activity in a number of variants. CONCLUSION: Our findings indicate that TRAP and OPN colocalize and that OPN is a substrate for TRAP in human immune cells. TRAP deficiency in PDCs leads to increased IFNα production, providing at least a partial explanation for how ACP5 mutations cause lupus in the context of spondyloenchondrodysplasia. Detection of ACP5 missense variants in a lupus cohort suggests that impaired TRAP functioning may increase susceptibility to sporadic lupus.
Subject(s)
Lupus Erythematosus, Systemic/enzymology , Lupus Erythematosus, Systemic/etiology , Mutation , Tartrate-Resistant Acid Phosphatase/deficiency , Autoimmune Diseases/genetics , Genetic Predisposition to Disease , Humans , Macrophages , Osteochondrodysplasias/genetics , Osteopontin/metabolism , Phosphorylation , Tartrate-Resistant Acid Phosphatase/geneticsABSTRACT
Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.
Subject(s)
Autoantibodies/biosynthesis , B-Lymphocyte Subsets/immunology , Membrane Glycoproteins/metabolism , Precursor Cells, B-Lymphoid/immunology , Toll-Like Receptor 7/metabolism , Animals , Apoptosis , B-Lymphocyte Subsets/cytology , Cell Differentiation , Cell Proliferation , Cytidine Deaminase/genetics , Humans , Immunoglobulin Class Switching , Interferon Type I/metabolism , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Precursor Cells, B-Lymphoid/cytology , RNA/immunology , Receptors, Antigen, B-Cell/metabolism , Spleen/cytology , Spleen/immunology , T-Box Domain Proteins/genetics , Toll-Like Receptor 7/genetics , Up-RegulationABSTRACT
OBJECTIVE: Plasmacytoid dendritic cells (PDCs) produce high concentrations of interferon-α (IFNα) following exposure to immune complexes containing nucleic acids. We previously reported that serum from healthy donors inhibits IFNα production by PDCs in response to systemic lupus erythematosus (SLE) immune complexes, and that inhibition is mediated, in part, by IgG. IgG is the major component of intravenous immunoglobulin and is well known to exert antiinflammatory properties. Although suppression of inflammation by the sialylated subfraction of IgG has been implicated in some models, the mechanism of IFNα inhibition by IgG and the importance of sialylation have not been studied. METHODS: SLE immune complexes or synthetic Toll-like receptor (TLR) agonists were used to stimulate total or individual cell-depleted human mononuclear cell cultures in the presence or absence of IgG, Fc fragments, F(ab')2 fragments, and their sialylated or unsialylated subfractions. Cytokines were quantified by enzyme-linked immunosorbent assay. RESULTS: We identified 2 distinct mechanisms by which IgG inhibits IFNα production. First, IgG Fc fragments inhibited SLE immune complex-stimulated IFNα production via a sialic acid-independent mechanism, by inhibiting immune complex binding to Fcγ receptor IIa on PDCs. In contrast, the F(ab')2 fragment of the sialylation-enriched fraction of IgG inhibited TLR-7 or TLR-9 agonist-induced IFNα production but did not require the sialic acid residue itself. The inhibitory activity of IgG on TLR agonist-induced IFNα required monocyte production of prostaglandin E2, a potent suppressor of IFNα production by PDCs. CONCLUSION: IgG attenuates IFNα production by PDCs by both cell surface receptor and intracellular pathways, depending on the nature of the inducing stimulus.
Subject(s)
Antigen-Antibody Complex/metabolism , Dendritic Cells/metabolism , Immunoglobulin G/pharmacology , Interferon-alpha/antagonists & inhibitors , Interferon-alpha/drug effects , Toll-Like Receptors/metabolism , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/pathology , Dinoprostone/metabolism , Humans , Immunoglobulin G/administration & dosage , Injections, Intravenous , Interferon-alpha/metabolism , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Receptors, IgG/metabolism , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/metabolism , Toll-Like Receptors/agonistsABSTRACT
TLR7 activation is implicated in the pathogenesis of systemic lupus erythematosus. Mice that overexpress TLR7 develop a lupus-like disease with autoantibodies and glomerulonephritis and early death. To determine whether degradation of the TLR7 ligand RNA would alter the course of disease, we created RNase A transgenic (Tg) mice. We then crossed the RNase Tg to TLR7 Tg mice to create TLR7 × RNase double Tg (DTg) mice. DTg mice had a significantly increased survival associated with reduced activation of T and B lymphocytes and reduced kidney deposition of IgG and C3. We observed massive hepatic inflammation and cell death in TLR7 Tg mice. In contrast, hepatic inflammation and necrosis were strikingly reduced in DTg mice. These findings indicate that high concentrations of serum RNase protect against immune activation and inflammation associated with TLR7 stimulation and that RNase may be a useful therapeutic strategy in the prevention or treatment of inflammation in systemic lupus erythematosus and, possibly, liver diseases.