Machine learning-driven immunophenotypic stratification of mixed connective tissue disease corroborating the clinical heterogeneity.

OBJECTIVES: To stratify patients with mixed connective tissue disease (MCTD) based on their immunophenotype. METHODS: We analyzed the immunophenotype and transcriptome of 24 immune cell subsets from patients with MCTD, systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), and systemic sclerosis (SSc) from our functional genome database, ImmuNexUT (https://www.immunexut.org/). MCTD patients were stratified by employing machine learning models including Random Forest, trained by immunophenotyping data from SLE, IIM, and SSc patients. Transcriptomes were analyzed with gene set variation analysis (GSVA) and clinical features of MCTD subgroups were compared. RESULTS: This study included 215 patients, including 22 patients with MCTD. Machine learning models, constructed to classify SLE, IIM, and SSc patients based on immunophenotyping, were applied to MCTD patients, resulting in 16 classified as SLE-immunophenotype and 6 as non-SLE-immunophenotype. Among MCTD patients, patients with the SLE-immunophenotype had higher proportions of Th1 cells [2.85% (interquartile range (IQR) 1.54-3.91) vs 1.33% (IQR 0.99-1.74) p= 0.027] and plasmablasts [6.35% (IQR 4.17-17.49) vs 2.00% (IQR 1.20-2.80) p= 0.010]. Notably, the number of SLE-related symptoms was higher in patients with the SLE-immunophenotype [2.0 (IQR 1.0-2.0) vs 1.0 (IQR1.0-1.0) p= 0.038]. Moreover, GSVA scores of interferon-α and -γ responses were significantly higher in patients with the SLE-immunophenotype in central memory CD8+ T cells, while hedgehog signalling was higher in non-SLE-immunophenotype patients in 5 cell subsets. CONCLUSION: This study describes the stratification of MCTD patients based on immunophenotyping, suggesting the presence of distinct immunological processes behind the clinical subtypes of MCTD.

Age-associated CD4+ T cells with B cell-promoting functions are regulated by ZEB2 in autoimmunity.

Aging is a significant risk factor for autoimmunity, and many autoimmune diseases tend to onset during adulthood. We conducted an extensive analysis of CD4+ T cell subsets from 354 patients with autoimmune disease and healthy controls via flow cytometry and bulk RNA sequencing. As a result, we identified a distinct CXCR3midCD4+ effector memory T cell subset that expands with age, which we designated "age-associated T helper (THA) cells." THA cells exhibited both a cytotoxic phenotype and B cell helper functions, and these features were regulated by the transcription factor ZEB2. Consistent with the highly skewed T cell receptor usage of THA cells, gene expression in THA cells from patients with systemic lupus erythematosus reflected disease activity and was affected by treatment with a calcineurin inhibitor. Moreover, analysis of single-cell RNA sequencing data revealed that THA cells infiltrate damaged organs in patients with autoimmune diseases. Together, our characterization of THA cells may facilitate improved understanding of the relationship between aging and autoimmune diseases.

B cell receptor repertoire abnormalities in autoimmune disease.

B cells play a crucial role in the immune response and contribute to various autoimmune diseases. Recent studies have revealed abnormalities in the B cell receptor (BCR) repertoire of patients with autoimmune diseases, with distinct features observed among different diseases and B cell subsets. Classically, BCR repertoire was used as an identifier of distinct antigen-specific clonotypes, but the recent advancement of analyzing large-scale repertoire has enabled us to use it as a tool for characterizing cellular biology. In this review, we provide an overview of the BCR repertoire in autoimmune diseases incorporating insights from our latest research findings. In systemic lupus erythematosus (SLE), we observed a significant skew in the usage of VDJ genes, particularly in CD27+IgD+ unswitched memory B cells and plasmablasts. Notably, autoreactive clones within unswitched memory B cells were found to be increased and strongly associated with disease activity, underscoring the clinical significance of this subset. Similarly, various abnormalities in the BCR repertoire have been reported in other autoimmune diseases such as rheumatoid arthritis. Thus, BCR repertoire analysis holds potential for enhancing our understanding of the underlying mechanisms involved in autoimmune diseases. Moreover, it has the potential to predict treatment effects and identify therapeutic targets in autoimmune diseases.

Multimodal repertoire analysis unveils B cell biology in immune-mediated diseases.

OBJECTIVES: Despite the involvement of B cells in the pathogenesis of immune-mediated diseases (IMDs), biological mechanisms underlying their function are scarcely understood. To overcome this gap, here we constructed and investigated a large-scale repertoire catalogue of five B cell subsets of patients with IMDs. METHODS: We mapped B cell receptor regions from RNA sequencing data of sorted B cell subsets. Our dataset consisted of 595 donors under IMDs and health. We characterised the repertoire features from various aspects, including their association with immune cell transcriptomes and clinical features and their response to belimumab treatment. RESULTS: Heavy-chain complementarity-determining region 3 (CDR-H3) length among naïve B cells was shortened among autoimmune diseases. Strong negative correlation between interferon signature strength and CDR-H3 length was observed in naïve B cells and suggested the role for interferon in premature B cell development. VDJ gene usage was skewed especially in plasmablasts and unswitched-memory B cells of patients with systemic lupus erythematosus (SLE). We developed a scoring system to quantify this skewing, and it positively correlated with peripheral helper T cell transcriptomic signatures and negatively correlated with the amount of somatic hyper mutations in plasmablasts, suggesting the association of extrafollicular pathway. Further, this skewing led to high usage of IGHV4-34 gene with 9G4 idiotypes in unswitched-memory B cells, which showed a prominent positive correlation with disease activity in SLE. Gene usage skewing in unswitched-memory B cells was ameliorated after belimumab treatment. CONCLUSIONS: Our multimodal repertoire analysis enabled us the system-level understanding of B cell abnormality in diseases.

Distinct transcriptome architectures underlying lupus establishment and exacerbation.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple immune cells. To elucidate SLE pathogenesis, it is essential to understand the dysregulated gene expression pattern linked to various clinical statuses with a high cellular resolution. Here, we conducted a large-scale transcriptome study with 6,386 RNA sequencing data covering 27 immune cell types from 136 SLE and 89 healthy donors. We profiled two distinct cell-type-specific transcriptomic signatures: disease-state and disease-activity signatures, reflecting disease establishment and exacerbation, respectively. We then identified candidate biological processes unique to each signature. This study suggested the clinical value of disease-activity signatures, which were associated with organ involvement and therapeutic responses. However, disease-activity signatures were less enriched around SLE risk variants than disease-state signatures, suggesting that current genetic studies may not well capture clinically vital biology. Together, we identified comprehensive gene signatures of SLE, which will provide essential foundations for future genomic and genetic studies.

Efficacy of canakinumab on AA amyloidosis in late-onset NLRP3-associated autoinflammatory disease with an I574F somatic mosaic mutation.

There have been hundreds of reports on mutations in the NLRP3 gene related to NLRP3-associated autoinflammatory disease, but few of these mutations have occurred as both germline and somatic mosaic mutations. In this case-based review, we report a 68-year-old man with an NLRP3-associated autoinflammatory disease. He developed secondary amyloidosis, including a renal and colorectal presentation in his 50 s. Sequencing of the NLRP3 gene revealed an I574F somatic mosaic mutation, which has up to now only been reported in germline mutations. The patient was treated with canakinumab, which had great efficacy not only on the NLRP3-mediated inflammation, but also on the chronic renal failure and proteinuria provoked by secondary renal amyloidosis. To evaluate the effectiveness of canakinumab, we conducted a literature research on renal amyloidosis related to NLRP3-associated autoinflammatory disease treated with canakinumab. Although our patient had a relatively long medical history and greater amounts of proteinuria than other reported cases, canakinumab had great efficacy on renal impairment, in similar to other reported cases. Along with the first report of a late-onset I574F somatic mosaic mutation in NLRP3-associated autoinflammatory disease, this report demonstrates the effectiveness of canakinumab on renal amyloidosis, probably through the way that IL-1ß blockade minimizes podocyte injury.