42-parameter mass cytometry panel to assess cellular and functional phenotypes of leukocytes in bronchoalveolar lavage of Rhesus macaque.

This Optimized Multiparameter Immunofluorescence Panel (OMIP) reports on the development of a mass cytometry panel for broad immunophenotyping of leukocytes from bronchoalveolar lavage from rhesus macaques. Using this panel, we were able to identify myeloid populations such as macrophages, neutrophils, monocytes, myeloid and plasmacytoid DCs, basophils and lymphoid cell lineages including B cells, natural killer (NK) cells, mucosal associated invariant T (MAIT) cells, γδ T cells, CD4 T cells, CD8 β T cells, CD8 T cells, and innate lymphoid cells (ILCs). We also included markers for defining memory, differentiation (CCR7, CD28, CD45RA), homing potential (CXCR3), cytotoxic potential (perforin, granzyme B, granzyme K), cell activation/differentiation (HLA-DR, CD69, IgD) and effector function (CD154, IFN-γ, TNF, IL-2, IL-17A, IL-6, IL-1β, CCL4 and CD107a).

PD-L1 and PD-1 Are Associated with Clinical Outcomes and Alveolar Immune Cell Activation in Acute Respiratory Distress Syndrome.

The relationship between the PD-L1 (Programmed Death-Ligand 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 [ = 59] and ARDS2 [ = 78]) or plasma samples alone (ARDS3 [ = 149]) collected from subjects with ARDS and tested for associations with mortality using multiple regression.

Interconnected lineage trajectories link conventional and natural killer (NK)-like exhausted CD8 T cells beneficial in type 1 diabetes.

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.

Impact on in-depth immunophenotyping of delay to peripheral blood processing.

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.

Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (T) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGITKLRG1 T with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGITKLRG1 T is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGITKLRG1 CD8 T population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive , together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells.

A first-in-human, open-label Phase 1b and a randomised, double-blind Phase 2a clinical trial in recent-onset type 1 diabetes with AG019 as monotherapy and in combination with teplizumab.

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.

Inflammatory bone marrow signaling in pediatric acute myeloid leukemia distinguishes patients with poor outcomes.

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/β, 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.

Chemokines, soluble PD-L1, and immune cell hyporesponsiveness are distinct features of SARS-CoV-2 critical illness.

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.

Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

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.

A simple strategy for sample annotation error detection in cytometry datasets.

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.

The many faces of islet antigen-specific CD8 T cells: clues to clinical outcome in type 1 diabetes.

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.

A distinct GM-CSF T helper cell subset requires T-bet to adopt a T1 phenotype and promote neuroinflammation.

Elevation of granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing T helper (T) cells has been associated with several autoimmune diseases, suggesting a potential role in the pathogenesis of autoimmunity. However, the identity of GM-CSF–producing T 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 CD45RACD4 T cells in blood of healthy individuals including a population of GM-CSF–producing cells, known as TGM, that lacked expression of signature transcription factors and cytokines of established T lineages.

Autoreactive CD8+ T cell exhaustion distinguishes subjects with slow type 1 diabetes progression.

Although most patients with type 1 diabetes (T1D) retain some functional insulin-producing islet β cells at the time of diagnosis, the rate of further β 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 β cells.

Cytometry TOF identifies alveolar macrophage subtypes in acute respiratory distress syndrome.

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.

Tartrate-Resistant Acid Phosphatase Deficiency in the Predisposition to Systemic Lupus Erythematosus.

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.

Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells.

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.

Contrasting mechanisms of interferon-α inhibition by intravenous immunoglobulin after induction by immune complexes versus Toll-like receptor agonists.

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.

Increased ribonuclease expression reduces inflammation and prolongs survival in TLR7 transgenic mice.

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.

Type I IFN system in the development and manifestations of SLE.

Purpose Of Review: Type I interferon (IFN-I) is strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we focus on new developments in pathways of IFN-I stimulation, the role of IFN-I in syndromes associated with lupus-like diseases, the utility of IFN-I signatures as biomarkers, and the progress of therapeutic agents targeting IFN-I pathways in SLE.

Recent Findings: Immune complexesimmune complex are a dominant driver of IFN-I production by activating toll-like receptors (TLRs) in plasmacytoid dendritic cells (pDC) in SLE.

TNF-α and TGF-β counter-regulate PD-L1 expression on monocytes in systemic lupus erythematosus.

Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes. We previously found that the normal program for expression of a negative costimulatory molecule programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated the mechanism for PD-L1 dysregulation on lupus monocytes.

Plasmacytoid dendritic cells and C1q differentially regulate inflammatory gene induction by lupus immune complexes.

Immune complexes (ICs) play a pivotal role in causing inflammation in systemic lupus erythematosus (SLE). Yet, it remains unclear what the dominant blood cell type(s) and inflammation-related gene programs stimulated by lupus ICs are. To address these questions, we exposed normal human PBMCs or CD14(+) isolated monocytes to SLE ICs in the presence or absence of C1q and performed microarray analysis and other tests for cell activation.

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature.

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögren's syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log(10) odds (LOD) score of 3.