Dynamic single-cell transcriptomic analysis of peripheral blood mononuclear cells identifies novel biomarkers for autoimmune encephalitis.

Anti-CD20 B cell depletion is associated with elevated mucosal-originating circulating regulatory IgA B cells in multiple sclerosis.

Therapy with anti-CD20 monoclonal antibodies is highly efficacious in various autoimmune diseases including multiple sclerosis (MS), a prototypic autoimmune inflammatory disease of the central nervous system (CNS). However, which B cell subsets and altered B cell-regulating factors mediate the clinical efficacy of anti-CD20 treatment is unclear. To address this gap in knowledge, we performed longitudinal high-dimensional single-cell transcriptomic and proteomic profiling of blood, cerebrospinal fluid (CSF), and intestinal samples from people with MS (pwMS), combined with immune profiling in a preclinical autoimmune encephalomyelitis model during anti-CD20 therapy. CSF analyses were performed using previously published datasets. Multisite intestinal profiling was conducted in one anti-CD20-treated patient with MS and one control participant. By applying algorithm-guided analyses of flow cytometry, single-cell transcriptomic, and immune receptor repertoire data, we found that anti-CD20 B cell depletion was associated with increased frequencies of regulatory mucosal-derived IgA-producing B cells in the periphery and CSF, together with increased B cell receptor clonal overlap between mucosal and systemic compartments, indicating enhanced trafficking of IgA B cells from gut mucosal tissues to the systemic circulation and the CNS. Moreover, we demonstrated that higher levels of B cell-activating factor and a proliferation-inducing ligand were related to favorable outcomes in pwMS during anti-CD20 treatment. Together, our findings suggest that mucosal immune regulatory mechanisms may be harnessed by anti-CD20 B cell depletion, opening previously unknown therapeutic avenues for MS.

Integrated peripheral immune profiling reveals B-cell dysregulation and CD8 effector signatures in chronic inflammatory demyelinating polyneuropathy

Background Chronic inflammatory demyelinating polyneuropathy (CIDP) is an immune-mediated peripheral neuropathy with heterogeneous and often incomplete responses to current immunotherapies, but the underlying immune basis remains poorly defined. Although CIDP shares features of immune-mediated demyelination with multiple sclerosis (MS), the two diseases affect distinct anatomical compartments and exhibit divergent therapeutic responses, suggesting fundamentally different underlying immune programs. Here, we address this gap by defining the peripheral immune architecture of CIDP using an integrated, multi-modal approach. Methods Peripheral blood was obtained from 20 patients with CIDP and 20 age- and sex-matched healthy controls. Single-cell RNA sequencing was performed in a discovery subset and integrated with publicly available MS peripheral blood datasets to provide a cross-disease reference framework. The single-cell analysis was designed as an exploratory discovery step to identify candidate immune signatures. Transcriptomic, pathway, and ligand–receptor analyses were complemented by cytokine profiling and flow-cytometric validation in the full cohort. Results CIDP exhibited broad inflammatory activation with preferential enrichment of type I interferon and inflammasome-related programs compared with MS. Despite reduced B-cell frequencies, CIDP showed transcriptional enrichment of germinal center–associated programs, indicating a dissociation between cell number and activation state. In parallel, CD8 effector T cells demonstrated enhanced cytotoxicity and cytoskeletal remodeling programs, supported by increased expression of actin-regulatory genes and strengthened intercellular signaling interactions. In contrast, MS showed greater enrichment of integrin–talin–vinculin signaling pathways in B cells and CD4 T-cell subsets, consistent with trafficking-related immune mechanisms. Together, these findings indicate a coordinated immune axis linking B-cell dysregulation and cytotoxic CD8 T-cell activation in CIDP. Conclusions Integrated peripheral immune profiling identified candidate CIDP-associated immune signatures including dysregulated B-cell activation despite numerical reduction and a prominent cytotoxic CD8 T-cell program within a type I interferon– and inflammasome-skewed inflammatory milieu. These findings provide an exploratory framework for understanding peripheral immune dysregulation in CIDP and warrant further translational studies in larger, treatment-stratified cohorts.

Single-cell analysis suggests coordinated immune-cell redistribution between blood and cerebrospinal fluid in early multiple sclerosis

Despite immune-cell infiltration being a hallmark of multiple sclerosis (MS), the interplay between the periphery and central nervous system immune responses is still incompletely characterized. We performed single-cell transcriptomic and V(D)J sequencing of paired blood and cerebrospinal fluid (CSF) immune cells from treatment-naive relapsing-remitting women with MS and compared them to age– and sex-matched healthy controls. Across major immune lineages, we identified coordinated, compartment-specific immune alterations, with enrichment of activated and memory lymphocyte populations in the CSF and concomitant depletion of related populations in peripheral blood, suggesting their recruitment from blood to CSF. Clonally expanded CD4 memory T-cells, together with activated, expanded IgM-positive B-cells, accumulated predominantly in the CSF of people with MS compared to healthy subjects. Interestingly, tissue-primed cytotoxic populations and CXCR3-associated memory populations were depleted from the CSF of MS, implying their recruitment to the target tissue in early disease. These findings reveal coordinated, compartment-specific immune changes in early MS and provide a systems-level view of immune-cell trafficking between peripheral and central nervous system compartments.

Distinct peripheral B-cell immunophenotypes define clinical heterogeneity in childhood-onset systemic lupus erythematosus.

BACKGROUND B-cell dysregulation is central to the pathogenesis of childhood-onset systemic lupus erythematosus (cSLE), yet the B-cell immunophenotypes across disease states remain incompletely defined. This study aimed to investigate the mechanistic basis and clinical relevance of B-cell-mediated immune dysregulation in cSLE. METHODS Peripheral blood samples from 64 cSLE patients and 40 age- and sex-matched healthy controls (HCs) were analyzed using flow cytometry. The subsets were compared across disease states, including lupus low disease activity state (LLDAS), active disease, and clinical flare. Diagnostic performance was evaluated using logistic regression and receiver operating characteristic analyses. RESULTS Compared with HCs, patients with cSLE exhibited reduced unswitched memory B cells and expanded pathogenic subsets, including double-negative (DN) B cells, age-associated B cells (ABCs), short-lived plasma cells (SLPCs), and long-lived plasma cells (LLPCs) (all P < 0.05). LLPCs showed the strongest disease association [area under the curve (AUC) = 0.855] and a combined model further improved discrimination (AUC = 0.936). Active disease was characterized by expanded DN B cells, ABCs, SLPCs, and LLPCs (all P < 0.05). Clinical flare involved selective enrichment of ABCs, DN B cells, and plasma cells without further increase in total B cells. Of note, LLPCs remained elevated in LLDAS compared with HCs (P < 0.01). ABCs were negatively correlated with complement C3 levels (r =  - 0.26, P = 0.04). CONCLUSIONS cSLE is characterized by distinct peripheral B-cell immunophenotypes across different clinical states, marked by expansion of ABCs and plasma-cell populations. Persistent LLPC enrichment during clinical quiescence suggests incomplete immunological restoration and may contribute to disease heterogeneity and relapse susceptibility.

Hepatic CD8+TOX+ T-cells are a hallmark of autoimmune hepatitis

Characterizing Memory T Cells Associated with Systemic Lupus Erythematosus Pathogenesis

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease associated with a severe morbidity and mortality. Around 70% of SLE patients follow a relapse-remitting pattern of disease characterized by flares of disease activity, followed by prolonged periods of disease quiescence. Memory CD4+ T cell subsets have been shown to play an important role in driving the autoantibody production which causes flares in SLE, however the precise T cell changes that accompany flares are unknown. CITE-seq and TCR-seq were performed to assess the transcriptomic profiles of CD4+ memory T cells in flaring and quiescent SLE patients. CD4+ memory T cells were isolated from PBMCs by negative selection using magnetic sorting, stained with oligo-conjugated antibodies against surface proteins for subset classification, and subsequently partitioned, barcoded, and sequenced. We examined samples from 15 distinct patients at 2 separate clinical visits spaced one year apart, yielding 30 samples. The longitudinal nature of our data allows us to inspect transcriptional changes both between and within patients. Integrated analysis of 30 samples identified 10 immune cell clusters (Figure 1A). At baseline, flaring patients (n=9) were significantly enriched for Tfh, Th2, Th17 cells, and a Treg subset, while quiescent patients (n=6) had increased Th1 cells. TCR repertoire analyses at baseline revealed a higher proportion of expanded clonotypes in flaring patients, which was not seen in quiescent patients. Interestingly, we also found that there was a higher proportion of expanded clonotypes at follow-up in various subsets of interest, particularly in flaring patients that later became quiescent, suggesting tissue egress and recirculation following resolution of inflammation. Clonal overlap among subsets was markedly greater in flaring patients, suggesting shared antigen specificity and differentiation from common progenitors. More specifically, we identified 2 functionally deviated/exhausted Treg subsets (ISGhi/ISGlo) (Figure 1B) and, at baseline, found notable clonal overlap between the ISGhi Treg subset and Th2/17 cells and between the ISGlo subset and Tfh/Tph cells in flaring patients, which was absent in quiescent patients (Figure 1C). This suggests that there are 2 distinct subsets of cells with shared antigen exposure and/or functional plasticity; one that is exposed to an IFN-rich environment in the tissue, and another that is more involved in T-B cell interactions within lymphoid compartments. We found abnormal Treg subsets with features of exhaustion and functional deviation that shared antigen specificity with other T helper cells. Their increased prevalence during flares suggests that dysregulated immunoregulation may contribute to SLE pathogenesis.