TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Repertoire analyses reveal T cell antigen receptor sequence features that influence T cell fate.

T cells acquire a regulatory phenotype when their T cell antigen receptors (TCRs) experience an intermediate- to high-affinity interaction with a self-peptide presented via the major histocompatibility complex (MHC). Using TCRß sequences from flow-sorted human cells, we identified TCR features that promote regulatory T cell (Treg) fate. From these results, we developed a scoring system to quantify TCR-intrinsic regulatory potential (TiRP). When applied to the tumor microenvironment, TiRP scoring helped to explain why only some T cell clones maintained the conventional T cell (Tconv) phenotype through expansion. To elucidate drivers of these predictive TCR features, we then examined the two elements of the Treg TCR ligand separately: the self-peptide and the human MHC class II molecule. These analyses revealed that hydrophobicity in the third complementarity-determining region (CDR3ß) of the TCR promotes reactivity to self-peptides, while TCR variable gene (TRBV gene) usage shapes the TCR's general propensity for human MHC class II-restricted activation.

Publisher Correction: The immune cell landscape in kidneys of patients with lupus nephritis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes.

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments.

T peripheral helper cells in autoimmune diseases.

Pathologic T cell-B cell interactions underlie many autoimmune diseases. The T cells that help B cells in autoimmune diseases vary in phenotype and include T cells that lack typical features of T follicular helper cells, such as expression of CXCR5 and BCL6. A population of PD-1hi CXCR5- T peripheral helper (Tph) cells has now been recognized in multiple autoantibody-associated diseases. Tph cells display a distinctive set of features, merging the ability to provide B cell help with the capacity to migrate to inflamed peripheral tissues. Here, we review the scope of immune-related conditions in which Tph cells have been implicated and provide a perspective on their potential contributions to pathologic B cell activation in autoimmune diseases. We discuss Tph cells as a promising therapeutic strategy in autoimmunity and consider the utility of tracking Tph cells in blood as a biomarker to quantify aberrant T cell-B cell activation in patients with autoimmune diseases.

Characterisation of choroid plexus-infiltrating T cells reveals novel therapeutic targets in murine neuropsychiatric lupus.

OBJECTIVE: Diffuse central nervous system manifestations, referred to as neuropsychiatric lupus (NPSLE), are observed in 20-40% of lupus patients and involve complex mechanisms that have not yet been adequately elucidated. In murine NPSLE models, choroid plexus (ChP)-infiltrating T cells have not been fully evaluated as drivers of neuropsychiatric disease. METHOD: Droplet-based single-cell transcriptomic analysis (single-cell RNA sequencing) and immune T-cell receptor profiling were performed on ChP tissue from MRL/lpr mice, an NPSLE mouse model, at an 'early' and 'late' disease state, to investigate the infiltrating immune cells that accumulate with NPSLE disease progression. RESULTS: We found 19 unique clusters of stromal and infiltrating cells present in the ChP of NPSLE mice. Higher resolution of the T-cell clusters uncovered multiple T-cell subsets, with increased exhaustion and hypoxia expression profiles. Clonal analysis revealed that the clonal CD8+T cell CDR3 sequence, ASGDALGGYEQY, matched that of a published T-cell receptor sequence with specificity for myelin basic protein. Stromal fibroblasts are likely drivers of T-cell recruitment by upregulating the VCAM signalling pathway. Systemic blockade of VLA-4, the cognate ligand of VCAM, resulted in significant resolution of the ChP immune cell infiltration and attenuation of the depressive phenotype. CONCLUSION: Our analysis details the dynamic transcriptomic changes associated with murine NPSLE disease progression, and highlights its potential use in identifying prospective lupus brain therapeutic targets.

TET2-mutant clonal hematopoiesis and risk of gout.

Gout is a common inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in individuals with hyperuricemia. Acute flares are accompanied by secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition predisposing to hematologic cancers and cardiovascular disease. CHIP is associated with elevated IL-1ß, thus we investigated CHIP as a risk factor for gout. To test the clinical association between CHIP and gout, we analyzed whole exome sequencing data from 177 824 individuals in the MGB Biobank (MGBB) and UK Biobank (UKB). In both cohorts, the frequency of gout was higher among individuals with CHIP than without CHIP (MGBB, CHIP with variant allele fraction [VAF] ≥2%: odds ratio [OR], 1.69; 95% CI, 1.09-2.61; P = .0189; UKB, CHIP with VAF ≥10%: OR, 1.25; 95% CI, 1.05-1.50; P = .0133). Moreover, individuals with CHIP and a VAF ≥10% had an increased risk of incident gout (UKB: hazard ratio [HR], 1.28; 95% CI, 1.06-1.55; P = .0107). In murine models of gout pathogenesis, animals with Tet2 knockout hematopoietic cells had exaggerated IL-1ß secretion and paw edema upon administration of MSU crystals. Tet2 knockout macrophages elaborated higher levels of IL-1ß in response to MSU crystals in vitro, which was ameliorated through genetic and pharmacologic Nlrp3 inflammasome inhibition. These studies show that TET2-mutant CHIP is associated with an increased risk of gout in humans and that MSU crystals lead to elevated IL-1ß levels in Tet2 knockout murine models. We identify CHIP as an amplifier of NLRP3-dependent inflammatory responses to MSU crystals in patients with gout.

Activation of CD8+ T Cells in Chronic Obstructive Pulmonary Disease Lung.

Rationale: Despite the importance of inflammation in chronic obstructive pulmonary disease (COPD), the immune cell landscape in the lung tissue of patients with mild-moderate disease has not been well characterized at the single-cell and molecular level. Objectives: To define the immune cell landscape in lung tissue from patients with mild-moderate COPD at single-cell resolution. Methods: We performed single-cell transcriptomic, proteomic, and T-cell receptor repertoire analyses on lung tissue from patients with mild-moderate COPD (n = 5, Global Initiative for Chronic Obstructive Lung Disease I or II), emphysema without airflow obstruction (n = 5), end-stage COPD (n = 2), control (n = 6), or donors (n = 4). We validated in an independent patient cohort (N = 929) and integrated with the Hhip+/- murine model of COPD. Measurements and Main Results: Mild-moderate COPD lungs have increased abundance of two CD8+ T cell subpopulations: cytotoxic KLRG1+TIGIT+CX3CR1+ TEMRA (T effector memory CD45RA+) cells, and DNAM-1+CCR5+ T resident memory (TRM) cells. These CD8+ T cells interact with myeloid and alveolar type II cells via IFNG and have hyperexpanded T-cell receptor clonotypes. In an independent cohort, the CD8+KLRG1+ TEMRA cells are increased in mild-moderate COPD lung compared with control or end-stage COPD lung. Human CD8+KLRG1+ TEMRA cells are similar to CD8+ T cells driving inflammation in an aging-related murine model of COPD. Conclusions: CD8+ TEMRA cells are increased in mild-moderate COPD lung and may contribute to inflammation that precedes severe disease. Further study of these CD8+ T cells may have therapeutic implications for preventing severe COPD.

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis.

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Ageing and interferon gamma response drive the phenotype of neutrophils in the inflamed joint.

OBJECTIVE: Neutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. METHODS: We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. RESULTS: Blood neutrophils from healthy donors and patients with active arthritis showed largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN-γ), as well as to tumour necrosis factor, interleukin-6 and hypoxia, in both humans and mice. Mass cytometry confirmed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN-γ and prolonged culture. CONCLUSIONS: Circulating neutrophils from patients with arthritis resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN-γ response and ageing as complementary drivers of the synovial fluid neutrophil phenotype.

High incidence of proliferative and membranous nephritis in SLE patients with low proteinuria in the Accelerating Medicines Partnership.

OBJECTIVE: Delayed detection of LN associates with worse outcomes. There are conflicting recommendations regarding a threshold level of proteinuria at which biopsy will likely yield actionable management. This study addressed the association of urine protein:creatinine ratios (UPCR) with clinical characteristics and investigated the incidence of proliferative and membranous histology in patients with a UPCR between 0.5 and 1. METHODS: A total of 275 SLE patients (113 first biopsy, 162 repeat) were enrolled in the multicentre multi-ethnic/racial Accelerating Medicines Partnership across 15 US sites at the time of a clinically indicated renal biopsy. Patients were followed for 1 year. RESULTS: At biopsy, 54 patients had UPCR <1 and 221 had UPCR ≥1. Independent of UPCR or biopsy number, a majority (92%) of patients had class III, IV, V or mixed histology. Moreover, patients with UPCR <1 and class III, IV, V, or mixed had a median activity index of 4.5 and chronicity index of 3, yet 39% of these patients had an inactive sediment. Neither anti-dsDNA nor low complement distinguished class I or II from III, IV, V or mixed in patients with UPCR <1. Of 29 patients with baseline UPCR <1 and class III, IV, V or mixed, 23 (79%) had a UPCR <0.5 at 1 year. CONCLUSION: In this prospective study, three-quarters of patients with UPCR <1 had histology showing class III, IV, V or mixed with accompanying activity and chronicity despite an inactive sediment or normal serologies. These data support renal biopsy at thresholds lower than a UPCR of 1.

SerpinB1 controls encephalitogenic T helper cells in neuroinflammation.

SerpinB1, a protease inhibitor and neutrophil survival factor, was recently linked with IL-17-expressing T cells. Here, we show that serpinB1 (Sb1) is dramatically induced in a subset of effector CD4 cells in experimental autoimmune encephalomyelitis (EAE). Despite normal T cell priming, Sb1-/- mice are resistant to EAE with a paucity of T helper (TH) cells that produce two or more of the cytokines, IFNγ, GM-CSF, and IL-17. These multiple cytokine-producing CD4 cells proliferate extremely rapidly; highly express the cytolytic granule proteins perforin-A, granzyme C (GzmC), and GzmA and surface receptors IL-23R, IL-7Rα, and IL-1R1; and can be identified by the surface marker CXCR6. In Sb1-/- mice, CXCR6+ TH cells are generated but fail to expand due to enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death. Finally, anti-CXCR6 antibody treatment, like Sb1 deletion, dramatically reverts EAE, strongly indicating that the CXCR6+ T cells are the drivers of encephalitis.

Plateletpheresis-associated lymphopenia in frequent platelet donors.

More than 1 million apheresis platelet collections are performed annually in the United States. After 2 healthy plateletpheresis donors were incidentally found to have low CD4+ T-lymphocyte counts, we investigated whether plateletpheresis causes lymphopenia. We conducted a cross-sectional single-center study of platelet donors undergoing plateletpheresis with the Trima Accel, which removes leukocytes continuously with its leukoreduction system chamber. We recruited 3 groups of platelet donors based on the total number of plateletpheresis sessions in the prior 365 days: 1 or 2, 3 to 19, or 20 to 24. CD4+ T-lymphocyte counts were <200 cells per microliter in 0/20, 2/20, and 6/20 donors, respectively (P = .019), and CD8+ T-lymphocyte counts were low in 0/20, 4/20, and 11/20 donors, respectively (P < .001). The leukoreduction system chamber's lymphocyte-extraction efficiency was ∼15% to 20% for all groups. Immunophenotyping showed decreases in naive CD4+ T-lymphocyte and T helper 17 (Th17) cell percentages, increases in CD4+ and CD8+ effector memory, Th1, and regulatory T cell percentages, and stable naive CD8+ and Th2 percentages across groups. T-cell receptor repertoire analyses showed similar clonal diversity in all groups. Donor screening questionnaires supported the good health of the donors, who tested negative at each donation for multiple pathogens, including HIV. Frequent plateletpheresis utilizing a leukoreduction system chamber is associated with CD4+ and CD8+ T-cell lymphopenia in healthy platelet donors. The mechanism may be repeated extraction of these cells during plateletpheresis. The cytopenias do not appear to be harmful.

Adverse Effects of Low-Dose Methotrexate: A Randomized Trial.

Background: Low-dose methotrexate (LD-MTX) is the most commonly used drug for systemic rheumatic diseases worldwide and is the recommended first-line agent for rheumatoid arthritis. Despite extensive clinical use for more than 30 years, few data on adverse event (AE) rates derive from randomized, placebo-controlled trials, where both causality and magnitude of risk can be inferred. Objective: To investigate AE rates, risk, and risk differences comparing LD-MTX versus placebo. Design: Prespecified secondary analyses of a double-blind, placebo-controlled, randomized trial. (ClinicalTrials.gov: NCT01594333). Setting: North America. Participants: Adults with known cardiovascular disease and diabetes or metabolic syndrome. Intervention: Random allocation to LD-MTX (≤20 mg/wk) or placebo. All participants received folic acid, 1 mg/d, 6 days per week. Measurements: Risks for specific AEs of interest, as well as for all AEs, were compared across treatment groups after blinded adjudication. Results: After an active run-in period, 6158 patients were enrolled and 4786 randomly assigned to a group; median follow-up was 23 months and median dosage 15 mg/wk. Among the randomly assigned participants, 81.2% were male, median age was 65.7 years, and median body mass index was 31.5 kg/m2. Of 2391 participants assigned to LD-MTX, 2080 (87.0%) had an AE of interest, compared with 1951 of 2395 (81.5%) assigned to placebo (hazard ratio [HR], 1.17 [95% CI, 1.10 to 1.25]). The relative hazards of gastrointestinal (HR, 1.91 [CI, 1.75 to 2.10]), pulmonary (HR, 1.52 [CI, 1.16 to 1.98]), infectious (HR, 1.15 [CI, 1.01 to 1.30]), and hematologic (HR, 1.15 [CI, 1.07 to 1.23]) AEs were elevated for LD-MTX versus placebo. With the exception of increased risk for skin cancer (HR, 2.05 [CI, 1.28 to 3.28]), the treatment groups did not differ in risk for other cancer or mucocutaneous, neuropsychiatric, or musculoskeletal AEs. Renal AEs were reduced in the LD-MTX group (HR, 0.85 [CI, 0.78 to 0.93]). Limitation: The trial was done in patients without rheumatic disease who tolerated LD-MTX during an active run-in period. Conclusion: Use of LD-MTX was associated with small to moderate elevations in risks for skin cancer and gastrointestinal, infectious, pulmonary, and hematologic AEs, whereas renal AEs were decreased. Primary Funding Source: National Institutes of Health.

Circulating CXCR5-PD-1hi peripheral T helper cells are associated with progression to type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes is preceded by a period of asymptomatic autoimmunity characterised by positivity for islet autoantibodies. Therefore, T helper cell responses that induce B cell activation are likely to play a critical role in the disease process. Here, we aimed to evaluate the role of a recently described subset, C-X-C motif chemokine receptor type 5-negative, programmed cell death protein 1-positive (CXCR5-PD-1hi) peripheral T helper (Tph) cells, in human type 1 diabetes. METHODS: The phenotype of blood CXCR5-PD-1hi CD4+ T cells was analysed by multicolour flow cytometry. The frequencies of circulating CXCR5-PD-1hi T cells were analysed in a cohort of 44 children with newly diagnosed type 1 diabetes, 40 autoantibody-positive (AAb+) at-risk children and 84 autoantibody-negative healthy control children, and the findings were replicated in a separate cohort of 15 children with newly diagnosed type 1 diabetes and 15 healthy control children. RESULTS: Circulating CXCR5-PD-1hi Tph cells share several features associated with B cell helper function with circulating CXCR5+PD-1hi follicular T helper (Tfh) cells. Moreover, the frequency of circulating Tph cells was increased in children with newly diagnosed type 1 diabetes, especially in those who are positive for multiple autoantibodies. Importantly, circulating Tph cells were also increased in autoantibody-positive at-risk children who later progressed to type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our results demonstrate that circulating CXCR5-PD-1hi Tph cells are associated with progression to clinical type 1 diabetes. Consequently, Tph cells could have potential both as a biomarker of disease progression and as a target for immunotherapy in type 1 diabetes.