Reproducible single cell annotation of programs underlying T-cell subsets, activation states, and functions.

T-cells recognize antigens and induce specialized gene expression programs (GEPs) enabling functions including proliferation, cytotoxicity, and cytokine production. Traditionally, different classes of helper T-cells express mutually exclusive responses - for example, Th1, Th2, and Th17 programs. However, new single-cell RNA sequencing (scRNA-Seq) experiments have revealed a continuum of T-cell states without discrete clusters corresponding to these subsets, implying the need for new analytical frameworks. Here, we advance the characterization of T-cells with T-CellAnnoTator (TCAT), a pipeline that simultaneously quantifies pre-defined GEPs capturing activation states and cellular subsets. From 1,700,000 T-cells from 700 individuals across 38 tissues and five diverse disease contexts, we discover 46 reproducible GEPs reflecting the known core functions of T-cells including proliferation, cytotoxicity, exhaustion, and T helper effector states. We experimentally characterize several novel activation programs and apply TCAT to describe T-cell activation and exhaustion in Covid-19 and cancer, providing insight into T-cell function in these diseases.

Non-coding autoimmune risk variant defines role for ICOS in T peripheral helper cell development.

Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4+ T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.

The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity.

Liver failure causes breakdown of the Blood CNS Barrier (BCB) leading to damages of the Central-Nervous-System (CNS), however the mechanisms whereby the liver influences BCB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BCB-integrity. To study BCB-integrity, we developed light-sheet imaging for three-dimensional analysis. We show that liver- or muscle-specific knockout of Hfe2/Rgmc induces BCB-breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits in mice. Soluble HFE2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells, triggering BCB-disruption. HFE2 administration in female mice with experimental autoimmune encephalomyelitis, a model for multiple sclerosis, prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BCB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BCB-dysfunction.

The intestinal microbiota modulates the transcriptional landscape of iNKT cells at steady-state and following antigen exposure.

Invariant Natural Killer T (iNKT) cells are unconventional T cells that respond to microbe-derived glycolipid antigens. iNKT cells exert fast innate effector functions that regulate immune responses in a variety of contexts, including during infection, cancer, or inflammation. The roles these unconventional T cells play in intestinal inflammation remain poorly defined and vary based on the disease model and species. Our previous work suggested that the gut microbiota influenced iNKT cell functions during dextran sulfate sodium-induced colitis in mice. This study, shows that iNKT cell homeostasis and response following activation are altered in germ-free mice. Using prenatal fecal transplant in specific pathogen-free mice, we show that the transcriptional signatures of iNKT cells at steady state and following αGC-mediated activation in vivo are modulated by the microbiota. Our data suggest that iNKT cells sense the microbiota at homeostasis independently of their T cell receptors. Finally, iNKT cell transcriptional signatures are different in male and female mice. Collectively, our findings suggest that sex and the intestinal microbiota are important factors that regulate iNKT cell homeostasis and responses. A deeper understanding of microbiota-iNKT cell interactions and the impact of sex could improve the development of iNKT cell-based immunotherapies.

Immunosuppression causes dynamic changes in expression QTLs in psoriatic skin.

Psoriasis is a chronic, systemic inflammatory condition primarily affecting skin. While the role of the immune compartment (e.g., T cells) is well established, the changes in the skin compartment are more poorly understood. Using longitudinal skin biopsies (n = 375) from the "Psoriasis Treatment with Abatacept and Ustekinumab: A Study of Efficacy"(PAUSE) clinical trial (n = 101), we report 953 expression quantitative trait loci (eQTLs). Of those, 116 eQTLs have effect sizes that were modulated by local skin inflammation (eQTL interactions). By examining these eQTL genes (eGenes), we find that most are expressed in the skin tissue compartment, and a subset overlap with the NRF2 pathway. Indeed, the strongest eQTL interaction signal - rs1491377616-LCE3C - links a psoriasis risk locus with a gene specifically expressed in the epidermis. This eQTL study highlights the potential to use biospecimens from clinical trials to discover in vivo eQTL interactions with therapeutically relevant environmental variables.

Elevated Levels of Interferon-α Act Directly on B Cells to Breach Multiple Tolerance Mechanisms Promoting Autoantibody Production.

OBJECTIVE: Elevated levels of serum interferon-α (IFNα) and the disruption of B cell tolerance are central to systemic lupus erythematosus (SLE) immunopathogenesis; however, the relationship between these 2 processes remains unclear. The purpose of this study was to investigate the impact of elevated IFNα levels on B cell tolerance mechanisms in vivo and determine whether any changes observed were due to the direct effect of IFNα on B cells. METHODS: Two classical mouse models of B cell tolerance were used in conjunction with an adenoviral vector encoding IFNα to mimic the sustained elevations of IFNα seen in SLE. The role of B cell IFNα signaling, T cells, and Myd88 signaling was determined using B cell-specific IFNα receptor-knockout, CD4+ T cell-depleted, or Myd88-knockout mice, respectively. Flow cytometry, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cell cultures were used to study the effects of elevated IFNα on the immunologic phenotype. RESULTS: Elevation of serum IFNα disrupts multiple B cell tolerance mechanisms and leads to autoantibody production. This disruption was dependent upon B cell expression of IFNα receptor. Many of the IFNα-mediated alterations also required the presence of CD4+ T cells as well as Myd88, suggesting that IFNα acts directly on B cells to modify their response to Myd88 signaling and their ability to interact with T cells. CONCLUSION: The results provide evidence that elevated IFNα levels act directly on B cells to facilitate autoantibody production and further highlight the importance of IFN signaling as a potential therapeutic target in SLE.

Making inroads to precision medicine for the treatment of autoimmune diseases: Harnessing genomic studies to better diagnose and treat complex disorders.

Precision Medicine is an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle. Autoimmune diseases are those in which the body's natural defense system loses discriminating power between its own cells and foreign cells, causing the body to mistakenly attack healthy tissues. These conditions are very heterogeneous in their presentation and therefore difficult to diagnose and treat. Achieving precision medicine in autoimmune diseases has been challenging due to the complex etiologies of these conditions, involving an interplay between genetic, epigenetic, and environmental factors. However, recent technological and computational advances in molecular profiling have helped identify patient subtypes and molecular pathways which can be used to improve diagnostics and therapeutics. This review discusses the current understanding of the disease mechanisms, heterogeneity, and pathogenic autoantigens in autoimmune diseases gained from genomic and transcriptomic studies and highlights how these findings can be applied to better understand disease heterogeneity in the context of disease diagnostics and therapeutics.

Single-cell eQTL models reveal dynamic T cell state dependence of disease loci.

Non-coding genetic variants may cause disease by modulating gene expression. However, identifying these expression quantitative trait loci (eQTLs) is complicated by differences in gene regulation across fluid functional cell states within cell types. These states-for example, neurotransmitter-driven programs in astrocytes or perivascular fibroblast differentiation-are obscured in eQTL studies that aggregate cells1,2. Here we modelled eQTLs at single-cell resolution in one complex cell type: memory T cells. Using more than 500,000 unstimulated memory T cells from 259 Peruvian individuals, we show that around one-third of 6,511 cis-eQTLs had effects that were mediated by continuous multimodally defined cell states, such as cytotoxicity and regulatory capacity. In some loci, independent eQTL variants had opposing cell-state relationships. Autoimmune variants were enriched in cell-state-dependent eQTLs, including risk variants for rheumatoid arthritis near ORMDL3 and CTLA4; this indicates that cell-state context is crucial to understanding potential eQTL pathogenicity. Moreover, continuous cell states explained more variation in eQTLs than did conventional discrete categories, such as CD4+ versus CD8+, suggesting that modelling eQTLs and cell states at single-cell resolution can expand insight into gene regulation in functionally heterogeneous cell types.

Genome editing to define the function of risk loci and variants in rheumatic disease.

Discoveries in human genetic studies have revolutionized our understanding of complex rheumatic and autoimmune diseases, including the identification of hundreds of genetic loci and single nucleotide polymorphisms that potentially predispose individuals to disease. However, in most cases, the exact disease-causing variants and their mechanisms of action remain unresolved. Functional follow-up of these findings is most challenging for genomic variants that are in non-coding genomic regions, where the large majority of common disease-associated variants are located, and/or that probably affect disease progression via cell type-specific gene regulation. To deliver on the therapeutic promise of human genetic studies, defining the mechanisms of action of these alleles is essential. Genome editing technology, such as CRISPR-Cas, has created a vast toolbox for targeted genetic and epigenetic modifications that presents unprecedented opportunities to decipher disease-causing loci, genes and variants in autoimmunity. In this Review, we discuss the past 5-10 years of progress in resolving the mechanisms underlying rheumatic disease-associated alleles, with an emphasis on how genomic editing techniques can enable targeted dissection and mechanistic studies of causal autoimmune risk variants.

Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease.

Multimodal T cell profiling can enable more precise characterization of elusive cell states underlying disease. Here, we integrated single-cell RNA and surface protein data from 500,089 memory T cells to define 31 cell states from 259 individuals in a Peruvian tuberculosis (TB) progression cohort. At immune steady state >4 years after infection and disease resolution, we found that, after accounting for significant effects of age, sex, season and genetic ancestry on T cell composition, a polyfunctional type 17 helper T (TH17) cell-like effector state was reduced in abundance and function in individuals who previously progressed from Mycobacterium tuberculosis (M.tb) infection to active TB disease. These cells are capable of responding to M.tb peptides. Deconvoluting this state-uniquely identifiable with multimodal analysis-from public data demonstrated that its depletion may precede and persist beyond active disease. Our study demonstrates the power of integrative multimodal single-cell profiling to define cell states relevant to disease and other traits.

Impaired B cell anergy is not sufficient to breach tolerance to nuclear antigen in Vκ8/3H9 lupus-prone mice.

BACKGROUND: Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population. METHODS: Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy. RESULTS: c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice. CONCLUSION: Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production.

TYK2 inhibition reduces type 3 immunity and modifies disease progression in murine spondyloarthritis.

Spondyloarthritis (SpA) represents a family of inflammatory diseases of the spine and peripheral joints. Ankylosing spondylitis (AS) is the prototypic form of SpA in which progressive disease can lead to fusion of the spine. Therapeutically, knowledge of type 3 immunity has translated into the development of IL-23- and IL-17A-blocking antibodies for the treatment of SpA. Despite being able to provide symptomatic control, the current biologics do not prevent the fusion of joints in AS patients. Thus, there is an unmet need for disease-modifying drugs. Genetic studies have linked the Janus kinase TYK2 to AS. TYK2 is a mediator of type 3 immunity through intracellular signaling of IL-23. Here, we describe and characterize a potentially novel small-molecule inhibitor of TYK2 that blocked IL-23 signaling in vitro and inhibited disease progression in animal models of SpA. The effect of the inhibitor appears to be TYK2 specific, using TYK2-inactive mice, which further revealed a duality in the induction of IL-17A and IL-22 by IL-23. Specifically, IL-22 production was TYK2/JAK2/STAT3 dependent, while IL-17A was mostly JAK2 dependent. Finally, we examined the effects of AS-associated TYK2 SNPs on TYK2 expression and function and correlated them with AS disease progression. This work provides evidence that TYK2 inhibitors have great potential as an orally delivered therapeutic for SpA.

Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci.

Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1-3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR-Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.

Fast, sensitive and accurate integration of single-cell data with Harmony.

The emerging diversity of single-cell RNA-seq datasets allows for the full transcriptional characterization of cell types across a wide variety of biological and clinical conditions. However, it is challenging to analyze them together, particularly when datasets are assayed with different technologies, because biological and technical differences are interspersed. We present Harmony (https://github.com/immunogenomics/harmony), an algorithm that projects cells into a shared embedding in which cells group by cell type rather than dataset-specific conditions. Harmony simultaneously accounts for multiple experimental and biological factors. In six analyses, we demonstrate the superior performance of Harmony to previously published algorithms while requiring fewer computational resources. Harmony enables the integration of ~106 cells on a personal computer. We apply Harmony to peripheral blood mononuclear cells from datasets with large experimental differences, five studies of pancreatic islet cells, mouse embryogenesis datasets and the integration of scRNA-seq with spatial transcriptomics data.

Multimodal single-cell approaches shed light on T cell heterogeneity.

Single-cell methods have revolutionized the study of T cell biology by enabling the identification and characterization of individual cells. This has led to a deeper understanding of T cell heterogeneity by generating functionally relevant measurements - like gene expression, surface markers, chromatin accessibility, T cell receptor sequences - in individual cells. While these methods are independently valuable, they can be augmented when applied jointly, either on separate cells from the same sample or on the same cells. Multimodal approaches are already being deployed to characterize T cells in diverse disease contexts and demonstrate the value of having multiple insights into a cell's function. But, these data sets pose new statistical challenges for integration and joint analysis.

Early progression to active tuberculosis is a highly heritable trait driven by 3q23 in Peruvians.

Of the 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5-15% will develop active tuberculosis (TB). Approximately half will progress to active TB within the first 18 months after infection, presumably because they fail to mount an effective initial immune response. Here, in a genome-wide genetic study of early TB progression, we genotype 4002 active TB cases and their household contacts in Peru. We quantify genetic heritability ([Formula: see text]) of early TB progression to be 21.2% (standard error 0.08). This suggests TB progression has a strong genetic basis, and is comparable to traits with well-established genetic bases. We identify a novel association between early TB progression and variants located in a putative enhancer region on chromosome 3q23 (rs73226617, OR = 1.18; P = 3.93 × 10-8). With in silico and in vitro analyses we identify rs73226617 or rs148722713 as the likely functional variant and ATP1B3 as a potential causal target gene with monocyte specific function.

The presence of anti-nuclear antibodies alone is associated with changes in B cell activation and T follicular helper cells similar to those in systemic autoimmune rheumatic disease.

BACKGROUND: Diagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA+ individuals to determine whether they share any of the cellular immunologic features seen in SARD. METHODS: Healthy ANA- controls and ANA+ (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString. RESULTS: A number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA+ subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA+ subjects and subjects with UCTD clustered  with patients with early SARD, rather than ANA- healthy controls. CONCLUSIONS: ANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD.

Apoptotic cell-induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins.

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

Multiple tolerance defects contribute to the breach of B cell tolerance in New Zealand Black chromosome 1 congenic mice.

Lupus is characterized by a loss of B cell tolerance leading to autoantibody production. In this study, we explored the mechanisms underlying this loss of tolerance using B6 congenic mice with an interval from New Zealand Black chromosome 1 (denoted c1(96-100)) sufficient for anti-nuclear antibody production. Transgenes for soluble hen egg white lysozyme (sHEL) and anti-HEL immunoglobulin were crossed onto this background and various tolerance mechanisms examined. We found that c1(96-100) mice produced increased levels of IgM and IgG anti-HEL antibodies compared to B6 mice and had higher proportions of germinal center B cells and long-lived plasma cells, suggesting a germinal center-dependent breach of B cell anergy. Consistent with impaired anergy induction, c1(96-100) double transgenic B cells showed enhanced survival and CD86 upregulation. Hematopoietic chimeric sHEL mice with a mixture of B6 and c1(96-100) HEL transgenic B cells recapitulated these results, suggesting the presence of a B cell autonomous defect. Surprisingly, however, there was equivalent recruitment of B6 and c1(96-100) B cells into germinal centers and differentiation to splenic plasmablasts in these mice. In contrast, there were increased proportions of c1(96-100) T follicular helper cells and long-lived plasma cells as compared to their B6 counterparts, suggesting that both B and T cell defects are required to breach germinal center tolerance in this model. This possibility was further supported by experiments showing an enhanced breach of anergy in double transgenic mice with a longer chromosome 1 interval with additional T cell defects.