Biology subjects

Rao, D. A.

Publications and source records attributed to Rao, D. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Rethinking the Benign Nature of Class II Lupus Nephritis

Introduction: Class II lupus nephritis (LN) is considered clinically benign, yet up to 50% of patients progress to more severe disease and no molecular characterization exists. We aimed to define the single-cell landscape of Class II LN and identify cellular and transcriptional features associated with kidney outcome. Methods: We performed single-cell RNA sequencing on kidney biopsies from fifteen Class II LN patients (eight de novo, seven regressed from prior proliferative or membranous disease) and six healthy controls, integrated with 155 LN and 30 healthy-control samples from the Accelerating Medicines Partnership in SLE spanning proliferative, membranous, and mixed LN. Findings were correlated with 52-week renal response and supported by urinary proteomics. Results: Despite histologically minimal changes, Class II LN exhibited marked immune cell expansion comparable to proliferative and membranous LN, including CCL3+ CCL4+ intermediate monocytes producing TNF, FOLR2+ SIGLEC1+ macrophages producing TGF{beta} and PDGF, and CD69+ CD8+ effector memory T cells producing IFN{gamma}. Fibroblasts and myofibroblasts were significantly expanded and displayed divergent transcriptional programs: pro-fibrotic and inflammatory myofibroblast modules were associated with worse 52-week outcomes, while interferon-responsive and matrix-remodeling fibroblast programs were associated with improvement. Pathogenic and protective fibroblast populations received overlapping immune-derived signals, suggesting that fibroblast transcriptional state, rather than the identity of incoming signals, shapes the stromal response. Baseline chronicity index captured this stromal heterogeneity and was the clinical parameter most associated with outcome. Urinary proteomics aligned Class II with proliferative rather than membranous disease. Conclusions: Class II LN harbors substantial molecular activity not captured by routine histology. Fibroblast transcriptional programs and baseline chronicity index are candidate correlates of kidney outcome that warrant validation in larger, prospectively followed cohorts.

genomics↗

bioRxiv2026

Persistent circulating autoreactive PD1⁺TIGIT⁺ peripheral helper T cells reflect synovial lymphoid activity and poor response to conventional disease-modifying anti-rheumatic drugs in early rheumatoid arthritis

ObjectiveIn the first year after onset of the autoimmune disease RA (RA), 40-60% do not achieve remission on conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs). To understand how autoreactive T cells may contribute to unstable or non-remission, we studied CD4+ T cells, including those recognising citrullinated (Cit) vimentin in participants with RA. MethodsTwo cohorts of drug-naive new-onset participants with RA were treated with csDMARDs. Disease activity score (DAS28-CRP) and peripheral blood (PB) mononuclear cells were collected longitudinally. In HLA-DR-shared epitope+ cohort 1 (n=21), T cells were assessed with a 17-marker spectral flow panel, incorporating HLA-DRB1*04:01/01:01- VimentinCit6459-71 or HLA-DRB1*04:04-VimentinCit7166-78 tetramers. Changes in T cell subsets over time were assessed in remitting and non-remitting participants using a generalized linear mixed model with a negative binomial distribution. In cohort 2 (n=26), the transcriptome of disaggregated synovial tissue (ST) and PB CD4+ T cells were analysed at baseline, and ST biopsy spatial proteomics at baseline and 6 months. ResultsCD4CXCR5-PD1+ peripheral helper T cells (Tph), including Cit-vimentin-reactive Tph, CD4CCR7+CXCR5-PD1+ stem-like Tph and TIGIT+PD1+ Tph were increased with moderate/high DAS28-CRP at any time point. Remission outcome was associated with low CD4 follicular helper T cell (Tfh) and Cit-vimentin-reactive Tfh. In non-remitting participants, Tph/fh infiltrated germinal-centre-like ST aggregates. This decreased in remission. Circulating TIGIT+ Tph genes reflected B lymphoid activation and lymph node egress, while in ST they reflected local differentiation. ConclusionPersistently high circulating TIGIT+ Tph and Tfh, including Cit-vimentin specificities, reflecting antigen-presenting B-cell interactions, are associated with reduced response to csDMARDs in recent-onset RA. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LITph and Tfh cells interacting with B cells are implicated in active ACPA+ RA C_LIO_LICitrullinated (Cit)-vimentin is an important neutrophil extracellular trap-derived target of ACPA C_LI What this study addsO_LIHigh circulating TIGIT+ Tph, Cit-vimentin-autoreactive Tph and Tfh associate with failure to reach remission on conventional synthetic DMARDs within the first year in early HLA-DR shared-epitope+ RA C_LIO_LITph/Tfh and adjacent regulatory T cells surround follicular B-cells in lymphoid aggregates in synovial tissue in non-remission C_LIO_LICirculating TIGIT+ Tph bear a transcriptional signature of lymphoid tissue expansion and lymph node egress as compared to functional differentiation and antigen experience in synovial tissue C_LI How this study might affect research, practice or policyO_LIWhen a remission target is not achieved in HLA-DR shared-epitope+ RA patients during the first year of treatment, high circulating TIGIT+ Tph implicate autoreactive T-B-lymphoid expansion in synovial tissue. C_LI

immunology↗

bioRxiv2026

Mucosal-associated invariant T cells support IL-15-dependent Treg response in skin injury and promote resolution of skin inflammation

Mucosal-associated invariant T (MAIT) cells are enriched at barrier sites, but their role in autoimmune skin inflammation remains unknown. Using cutaneous lupus as a model, we identify MAIT cells as protective regulators of skin inflammation and as critical upstream modulators of regulatory T cells (Treg). Topical MAIT cell activation with 5-OP-RU induced durable resolution of spontaneous skin lesions in MRL/lpr mice and suppressed TLR7-driven skin inflammation. MAIT cell activation selectively expanded and activated Treg populations in both healthy and lupus-like skin, while suppressing effector T cell cytokine production and cytotoxic programs. This MAIT-Treg axis was also activated in UV light-driven barrier injury in healthy murine and human skin, where MAIT cells were required for UV-elicited Treg expansion and function. In lupus-like skin, local MAIT cell activation restored the defective UVB-induced Treg response and limited CD8+ T cell expansion. Mechanistically, CCR2+ monocyte-derived antigen-presenting cells and IL-15 signaling were required for MAIT cell-driven Treg accumulation and therapeutic benefits of MAIT cells in inflamed skin. These studies identify a MAIT-IL-15-Treg axis that links barrier injury sensing to immune regulation, which is disrupted in cutaneous lupus, and nominate therapeutic MAIT cell activation as an unappreciated strategy for restoring immune homeostasis in inflamed skin.

immunology↗

bioRxiv2025

Deep profiling of lupus nephritis kidneys reveals dynamic changes in myeloid cells associated with disease progression

ObjectivesLupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aim to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. MethodsSingle-cell RNA-sequencing (scRNA-seq) was used to profile dissociated kidney samples from 156 LN patients and 30 healthy individuals. Spatial transcriptomics (ST), utilizing a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, was applied to kidney samples acquired from 6 LN patients and 2 healthy controls. ResultsWe generated a full catalog of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the NIH chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4+ T, GZMK+CD8+ T, B and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are indicated to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. ConclusionsWe suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.

immunology↗

bioRxiv2025

Single Cell Analysis of pBMCs of Psoriasis patients reveals distinct CD4+ T cell phenotypes associated with response to IL-23 blockade.

The use of IL-23 inhibitors (IL-23i) for psoriatic diseases has resulted in significant improvement in disease symptoms for many patients. The extent of disease improvement following IL-23 blockade varies across patients with psoriasis; however, the immunologic factors associated with a good or poor response to IL-23 blockade remain unclear. Here, we utilized peripheral blood mononuclear cells (pBMCs) collected through the MINIMA clinical trial (NCT04271540) and applied single-cell RNA sequencing to profile circulating immune populations from 27 patients with psoriasis or psoriatic arthritis, aiming to identify cellular features associated with a response to IL-23 blockade. We identified populations of CD4+ T cells whose abundance in circulation before treatment was associated with improved skin disease following IL-23i treatment. Circulating CD4+ T cells that demonstrate transcriptomic features of Th1-like cells were associated with better psoriasis skin improvement and had transcriptomic signatures resembling T cells identified in psoriasis lesional skin. Further, decrease in levels of IL-17A and IFN{gamma} in serum each correlated with improvement in PASI levels. These results suggest that immune cell features detectable in blood may be informative in identifying patients with psoriasis who are likely to have a robust clinical response to IL-23 blockade.

immunology↗

bioRxiv2025

Spatial Transcriptomics Identify T Cell-Driven Mechanisms of Kidney Damage in Immune Checkpoint Inhibitor-Associated Acute Interstitial Nephritis

IntroductionImmune checkpoint inhibitor-associated acute interstitial nephritis (ICI-AIN) is the most common finding on histopathology among patients with ICI-associated acute kidney injury (ICI-AKI). Patients with ICI-AIN often have T cell-dominant infiltration of the kidney and high tissue levels of CXCR3 ligands like CXCL9, 10, and 11; however, the mechanisms of inflammation in ICI-AIN are not well-understood. MethodsWe applied a sub-cellular spatial transcriptomics platform (Xenium Prime 5K) to compare the cellular composition of kidney biopsy tissue from patients with ICI-AIN with ICI-treated patients with acute tubular necrosis (ICI-ATN). ResultsAcross 8 kidney biopsy specimens (4 with ICI-AIN, 4 with ICI-ATN), we analyzed 332,000 cells, comprising kidney parenchymal cells and infiltrating immune cells. Using a spatially-aware cellular neighborhood-based classification, we identified cellular niches corresponding to each part of the nephron, in addition to unique fibrotic and inflammatory niches. Gene pathway analysis identified interferon-gamma (IFN-{gamma})/STAT1 signaling as strongly increased in ICI-AIN compared to ICI-ATN. While all inflammatory niches were overrepresented in ICI-AIN, CD8+ T cell infiltration and proinflammatory myeloid cells were the dominant immune niches. Spatial niche crosstalk analysis revealed that CD8+ T cell-derived IFN-{gamma} likely induced a proinflammatory program in myeloid cells, with increased production of CXCL9, 10, and 11. Furthermore, IFN-{gamma} signaling in ICI-AIN was associated with reduced oxidative phosphorylation in kidney tubular niches. ConclusionsSpatial transcriptomics reveal novel insights into key differences in the pathophysiology of ICI-AIN versus ICI-ATN. IFN-{gamma}-producing CD8+ T cells are likely key drivers of ICI-AIN and should be investigated as future therapeutic targets. Translational StatementSpatial transcriptomics may provide insight into differences in the cellular and spatial composition of immune checkpoint inhibitor-associated acute interstitial nephritis (ICI-AIN) and ICI-associated acute tubular necrosis (ICI-ATN). Using the novel Xenium 5K platform, we demonstrate that IFN-{gamma}-producing CD8+ T cells are central to the pathogenesis of ICI-AIN, and that CD8+ T cell-derived IFN-{gamma} likely induces a proinflammatory state in myeloid cells, with increased tissue production of CXCL9, 10, and 11. We then show that a reduction in oxidative phosphorylation and an IFN-{gamma}-driven influx of proinflammatory myeloid cells may further drive kidney damage in ICI-AIN. Many of these pathways represent potential druggable targets, and our findings may therefore inform future therapeutic approaches for ICI-AIN.

immunology↗

bioRxiv2025

Pre-treatment naive T cells are associated with severe irAE following PD-1/CTLA4 checkpoint blockade for melanoma

Immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-CTLA-4 antibodies are used to induce an immune response against many types of tumors. However, ICIs often also induce autoimmune responses, referred to as immune-related adverse events (irAEs), which occur unpredictably and at varying levels of severity in ICI-treated patients. The immunologic factors that predispose patients to the development of severe irAE are largely unclear. Here, we utilized high dimensional mass cytometry immunophenotyping of longitudinal blood samples from patients with metastatic melanoma treated with combination anti-PD-1/CTLA4 ICI therapy in the context of a clinical trial to characterize alterations in immune profiles induced by combination ICI therapy and to identify immune features associated with development of severe irAEs. Deep T cell profiling highlighted that ICI therapy induces prominent expansions of activated, CD38hi CD4+ and CD8+ T cells, which are frequently bound by the therapeutic anti-PD-1 antibody, as well as substantial changes in regulatory T cell phenotypes. However, neither the baseline frequency nor the extent of expansion of these cell populations was associated with development of severe irAEs. Rather, single cell-association testing revealed naive CD4+ T cell abundance pre-treatment as significantly associated with the development of severe irAEs. Biaxial gating of naive CD4+ T cells confirmed a significant positive association of naive CD4+ T cell proportion and development of a severe irAE and with the number of irAEs developed in this cohort. Results from this broad profiling study indicate the abundance of naive CD4+ T cells as a predictive feature for the development of severe irAEs following combination anti-PD-1/CTLA4 ICI therapy.

immunology↗

bioRxiv2025

Functional and dysfunctional T regulatory cell states in human tissues in RA and other autoimmune arthritic diseases

Regulatory T cells (Tregs), characterized by FOXP3 expression, are essential for maintaining immune homeostasis by controlling inflammation. However, in autoimmune diseases such as rheumatoid arthritis (RA), impaired Treg function contributes to immune dysregulation and disease pathology. While most studies of human Tregs have focused on blood, here we analyzed Tregs in synovial tissues from RA patients using single cell RNA sequencing (scRNAseq). We identified two predominant Treg states, CD25hiCXCR6pos Tregs with strong suppressive function, and CD25loAREGpos Tregs, a dysfunctional state exclusively enriched in synovial tissues but not in blood. Computational and in vitro analyses revealed that cortisol induced AREG expression, suppressed glycolysis, and impaired the suppressive function of CD25loAREGpos Tregs. In turn, AREG promoted an IL-33+ inflammatory phenotype in synovial fibroblasts. Importantly, we found that TNFR2 engagement can prevent or reverse this dysfunctional Treg state. In contrast to CD25loAREGpos Tregs, CD25hiCXCR6pos Tregs were highly suppressive, showed coordinated abundance with macrophages in synovial tissue, and functionally interacted with membrane-bound TNF expressed by macrophages, which promoted their functional suppressive state. These two Treg subsets were similarly found in the synovial tissue in Juvenile Idiopathic Arthritis (JIA), another inflammatory arthritic disorder, indicating conserved mechanisms across arthritic diseases. Together, our findings define distinct pathways driving divergent functional and dysfunctional Treg states in inflamed tissues and point to interventions that may prevent or reverse the development of the dysfunctional state.

immunology↗

bioRxiv2025

A CD57+ cytotoxic CD8 T cell subset associated with fibrotic lung disease in systemic sclerosis

Interstitial lung disease (ILD) is a major cause of morbidity and mortality in systemic sclerosis (SSc); however, the immunopathologic mechanisms driving lung disease in SSc are unclear. T cells have been implicated as a likely driver of lung injury in SSc. Here, we have evaluated T cells in blood and lungs of patients with SSc-ILD and identified a specific population of cytotoxic CD8 T cells that is expanded in SSc-ILD patients. Cytotoxic effector memory CD8 T cells marked by CD57 expression are preferentially expanded in SSc-ILD patients compared to SSc patients without ILD and controls and show prominent clonal expansion. These CD57+ T effector memory (TEM) cells differ from T effector memory cells re-expressing CD45RA (TEMRA) transcriptomically and functionally, with cytotoxic function that is enhanced by CD155 engagement of the costimulatory receptor CD226. Analyses of cells from ILD lungs indicate endothelial cells as a likely source of CD155 to activate CD57+ cytotoxic T cells. Together, the results implicate a CD57+ cytotoxic CD8 T cell population as a potential mediator of lung injury in SSc-ILD.

immunology↗

bioRxiv2025

Clonal relationships between Tph and Tfh cells in patients with SLE and in murine lupus

Pathologic T cell-B cell interactions drive disease in systemic lupus erythematosus (SLE). The T cells that activate B cell responses include T peripheral helper (Tph) and T follicular helper (Tfh) cells, yet the developmental and clonal relationships between these B cell-helper T cell populations are unclear. Here we use T cell receptor (TCR) profiling to demonstrate substantial clonal overlap between Tph and Tfh cells in the circulation of patients with SLE. Expanded Tph and Tfh cell clones persist over the course of 1 year in patients with a new diagnosis of SLE, and clones are observed to shift both from Tfh to Tph cells and from Tph to Tfh cells over time. High resolution analysis of cells sorted as Tph cells (CXCR5- PD-1hi) from SLE patients revealed considerable heterogeneity among these cells and highlighted a subpopulation of cells with transcriptomic features of activated B cell-helper T cells. This cell population, marked by expression of TOX and CXCL13, was found in both sorted Tph and Tfh cells, and was clonally linked in these two populations. Analysis of B cell-helper T cells in murine pristane-induced lupus demonstrated similar populations of Tph and Tfh cells in both lung and spleen with strong clonal overlap. T cell-specific loss of Bcl6 prevented accumulation of Tfh cells and reduced accumulation of Tph cells in pristane-treated mice, indicating a role for Bcl6 in the survival and expansion of both populations. Together, these observations demonstrate a shared developmental path among pathologically expanded Tph and Tfh cells in SLE. The persistence of expanded Tph and Tfh cells clones over time may explain the lack of stable tolerance induction by immunosuppressive medications or by B cell depletion.

immunology↗

bioRxiv2025

A GPVI-platelet-neutrophil-NET axis drives systemic sclerosis

Systemic sclerosis (SSc) is immune-mediate inflammatory disease characterized by progressive tissue fibrosis. We observed that circulating neutrophils from patients with diffuse SSc exhibit an activated phenotype, a finding echoed in blood and skin transcriptomes. Neutrophil depletion abrogated experimental SSc induced by cutaneous injection of hypochlorous acid (HOCl) or bleomycin (BLM), and adoptive transfer of HOCl and BLM neutrophils induced skin and lung fibrosis in healthy mice, establishing neutrophils as necessary and sufficient for fibrosis. We noted that SSc patients exhibited platelet activation, a phenotype that preceded neutrophil activation in mice, suggesting an upstream role. Indeed, platelet depletion abrogated neutrophil activation and tissue fibrosis, and exposure to HOCl or BLM platelets conferred upon wild-type neutrophils the capacity to induce skin and lung fibrosis via neutrophil extracellular traps (NETs). Genetic and therapeutic blockade of the platelet collagen receptor GPVI attenuated platelet and neutrophil activation, reduced circulating NETs, and protected animals from skin and lung fibrosis. These findings identify the GPVI-platelet-neutrophil-NET as a new source of therapeutic targets in SSc.

immunology↗

bioRxiv2024

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.

genomics↗

bioRxiv2023

Deep immunophenotyping reveals circulating activated lymphocytes in individuals at risk for rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease with currently no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in At-Risk populations prior to clinical disease onset is crucial to establishing effective prevention strategies. Here, we applied mass cytometry to deeply characterize the immunophenotypes in blood from At-Risk individuals identified through the presence of serum antibodies to citrullinated protein antigens (ACPA) and/or first-degree relative (FDR) status (n=52), as compared to established RA (n=67), and healthy controls (n=48). We identified significant cell expansions in At-Risk individuals compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5low naive B cell population was expanded in ACPA-positive FDRs. Further, we developed an "RA immunophenotype score" classification method based on the degree of enrichment of cell states relevant to established RA patients. This score significantly distinguished At-Risk individuals from controls. In all, we systematically identified activated lymphocyte phenotypes in At-Risk individuals, along with immunophenotypic differences among both ACPA+ and ACPA-FDR At-Risk subpopulations. Our classification model provides a promising approach for understanding RA pathogenesis with the goal to further improve prevention strategies and identify novel therapeutic targets.

genomics↗

bioRxiv2023

The Chromatin Landscape of Pathogenic Transcriptional Cell States in Rheumatoid Arthritis

Synovial tissue inflammation is the hallmark of rheumatoid arthritis (RA). Recent work has identified prominent pathogenic cell states in inflamed RA synovial tissue, such as T peripheral helper cells; however, the epigenetic regulation of these states has yet to be defined. We measured genome-wide open chromatin at single cell resolution from 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identified 24 chromatin classes and predicted their associated transcription factors, including a CD8+ GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating an RA tissue transcriptional atlas, we found that the chromatin classes represented superstates corresponding to multiple transcriptional cell states. Finally, we demonstrated the utility of this RA tissue chromatin atlas through the associations between disease phenotypes and chromatin class abundance as well as the nomination of classes mediating the effects of putatively causal RA genetic variants.

immunology↗

bioRxiv2023

Clonal associations of lymphocyte subsets and functional states revealed by single cell antigen receptor profiling of T and B cells in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is an autoimmune disease initiated by antigen-specific T cells and B cells, which promote synovial inflammation through a complex set of interactions with innate immune and stromal cells. To better understand the phenotypes and clonal relationships of synovial T and B cells, we performed single-cell RNA and repertoire sequencing on paired synovial tissue and peripheral blood samples from 12 donors with seropositive RA ranging from early to chronic disease. Paired transcriptomic-repertoire analyses highlighted 3 clonally distinct CD4 T cells populations that were enriched in RA synovium: T peripheral helper (Tph) and T follicular helper (Tfh) cells, CCL5+ T cells, and T regulatory cells (Tregs). Among these cells, Tph cells showed a unique transcriptomic signature of recent T cell receptor (TCR) activation, and clonally expanded Tph cells expressed an elevated transcriptomic effector signature compared to non-expanded Tph cells. CD8 T cells showed higher oligoclonality than CD4 T cells, and the largest CD8 T cell clones in synovium were highly enriched in GZMK+ cells. TCR analyses revealed CD8 T cells with likely viral-reactive TCRs distributed across transcriptomic clusters and definitively identified MAIT cells in synovium, which showed transcriptomic features of TCR activation. Among B cells, non-naive B cells including age-associated B cells (ABC), NR4A1+ activated B cells, and plasma cells, were enriched in synovium and had higher somatic hypermutation rates compared to blood B cells. Synovial B cells demonstrated substantial clonal expansion, with ABC, memory, and activated B cells clonally linked to synovial plasma cells. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate RA synovium.

immunology↗

bioRxiv2022

Non-coding autoimmune risk variant accelerates T peripheral helper cell development via ICOS

Fine-mapping and functional studies implicate rs117701653, a common non-coding variant in the CD28/CTLA4/ICOS locus, as a contributor to risk for rheumatoid arthritis and type 1 diabetes. Using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated allele reduces affinity for the inhibitory chromosomal regulator SMCHD1 to drive expression of inducible T-cell costimulator (ICOS), enhancing memory CD4+ T cell ICOS expression in individuals bearing the risk allele. 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 and circulating plasmablasts, suggesting a causal link. Indeed, ICOS ligation accelerates T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13, as does carriage of the rs117701653 risk allele. Thus, mechanistic dissection of a causal non-coding variant in human autoimmunity discloses a new pathway through which ICOS regulates Tph abundance.

genetics↗

bioRxiv2021

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

ObjectivesNeutrophils are typically the most abundant leukocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. MethodsWe 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. ResultsBlood neutrophils from healthy donors and patients with active arthritis exhibited largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1,600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN{gamma}), as well as to tumor necrosis factor, interleukin 6, and hypoxia, in both humans and mice. Mass cytometry also found healthy and arthritic donor blood neutrophils largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of Fc{gamma}RI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN{gamma} and prolonged culture. ConclusionsCirculating neutrophils from arthritis patients resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN{gamma} response and aging as complementary drivers of the synovial neutrophil phenotype. KEY MESSAGESO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LINeutrophils are central in the effector phase of inflammatory arthritis but their phenotypic heterogeneity in inflamed synovial fluid is poorly understood. C_LI What does this study add?O_LIRNA-seq and mass cytometry identify a hallmark phenotype of neutrophils in synovial fluid consisting of upregulated ICAM-1, HLA-DR, PD-L1, Fc receptors and CXCR4. C_LIO_LITranscriptomics highlight an IFN{gamma} response signature conserved across humans and mice. C_LIO_LIIn vitro experiments implicate aging and IFN{gamma} as complementary factors orchestrating the synovial fluid neutrophil phenotype. C_LI How might this impact on clinical practice or future developments?O_LIUnderstanding the specific features of neutrophils in the arthritic joint may disclose opportunities for safe therapeutic targeting of this lineage. C_LI

immunology↗

bioRxiv2021

Longitudinal immune cell profiling in early systemic lupus erythematosus

ObjectiveTo investigate the immune cell profiling and their longitudinal changes in systemic lupus erythematosus (SLE). MethodsWe employed mass cytometry with three different 38-39 marker panels (Immunophenotyping, T cell/monocyte, and B cell) in cryopreserved peripheral blood mononuclear cells (PBMCs) from nine patients with early SLE, 15 patients with established SLE, and 14 non-inflammatory controls. We used machine learning-driven clustering, FlowSOM (Flow Self-Organizing Maps) and dimensional reduction with tSNE (t-distributed Stochastic Neighbor Embedding) to identify unique cell populations in early and established SLE. For the nine early SLE patients, longitudinal mass cytometry analysis was applied to PBMCs at three time points (at enrollment, six months post-enrollment, and one year post-enrollment). Serum samples were also assayed for 65 cytokines by Luminex multiplex assay, and associations between cell types and cytokines/chemokines assessed. ResultsT peripheral helper cells (Tph cells), T follicular helper cells (Tfh cells) and several Ki67+ proliferating subsets (ICOS+ Ki67+ CD8 T cells, Ki67+ regulatory T cells, CD19int Ki67hi plasmablasts, and Ki67hi PU.1hi monocytes) were increased in early SLE. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from early SLE patients revealed that Tfh cells and CXCL10 decreased at one year post-enrollment. CXCL13 correlated positively with several of the expanded cell populations in early SLE. ConclusionsTwo major helper T cell subsets and unique Ki67+ proliferating immune cell subsets were expanded in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

immunology↗