Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. However, the methodology to directly assess the biology of primary HSCs in human liver specimens is yet to be established. In this study, we aimed to establish a robust methodology to analyse primary HSCs in human liver specimens with flow cytometry (FCM). We first applied FCM to HSCs directly isolated from liver tissues with Nycodenz density gradients. Then, we analysed HSCs in frozen/thawed liver perfusate samples and liver tissues. We also compared the phenotype of HSCs in primary biliary cholangitis (PBC) and those in healthy counterparts. We found that HSCs were substantially smaller and less dense than normal lymphocytes in the FCM analysis. By carefully defining the FCM gating strategy, we were able to establish the approach to analyse both quiescent HSCs (qHSCs) and activated HSCs (aHSCs) in human liver specimens. Importantly, we found that co-expression of CD14 and CD56 within CD45 non-immune cells permits the detection of qHSCs, whereas CD68 and CD40 within CD45 non-immune cells were valuable for assessing aHSCs. Furthermore, we found that aHSCs in PBC upregulated the expression of multiple markers associated with antigen-presentation capacity. Our established approach with FCM will be valuable for the direct analysis of qHSCs and aHSCs with FCM in various human liver specimens. Our FCM analysis of aHSCs in PBC suggested their involvement in the local immune responses.

Cutaneous allergen sensitization (CAS) is a primary driver of atopic dermatitis (AD) and a key initiator of the "atopic march", which can lead to systemic conditions such as food allergy and anaphylaxis. The type 2 cytokine interleukin-13 (IL-13) is an important regulator of high-affinity IgE antibodies, yet the precise cellular targets and mechanisms by which it orchestrates systemic allergic responses remain incompletely understood. Here, we evaluated the role of IL-13 in a murine CAS model that links skin inflammation to systemic anaphylaxis. Using cell-specific deletions of the IL-13 receptor α1 subunit (Il13ra1), we identify conventional dendritic cells (cDCs), and not T or B cells, as the essential targets of IL-13 for generating high-affinity IgE. Single-cell transcriptomics reveal that IL-13 signaling acts specifically in a cDC2 subset characterized by high expression of CX3CR1, Clec10a (CD301a), and CD301b (Mgl2). Licensing by IL-13 endows these cDC2 with superior antigen-presenting capacity, characterized by the upregulation of MHC class II and costimulatory molecules, including CD301a, CD301b, and ICOSL. These mature cDC2s are mobilized from the periphery to the spleen by a CX3CR1-dependent mechanism, where they are uniquely equipped to induce the differentiation of IL-13-producing T follicular helper (TFH13) cells. This cascade results in robust germinal center reactions and production of pathogenic, high-affinity IgE. Our findings define an IL-13-cDC2 axis that functions as a critical regulator of the atopic march, providing a mechanistic rationale for the clinical efficacy of IL-13-targeted therapies in allergic diseases.

Abstract Introduction Peripheral helper T (Tph) cells can contribute to the pathogenesis of human autoimmune diseases. Although Tph cells are recognized as the major B-cell helpers in inflamed joints of rheumatoid arthritis (RA), the mechanisms underlying their maintenance, activation, and involvement in tissue inflammation remain poorly understood. Methods We applied multiple sequencing technologies, including scRNA-seq, CITE-seq, scTCR-seq, scBCR-seq and scATAC-seq, on cells from synovial tissue (ST), synovial fluid and peripheral blood from RA patients. ST sections were utilized for spatial transcriptomics data acquisition. In vitro validation experiments, such as T cell-B cell coculture and gene editing, were also performed. Results We recently demonstrated that Tph cells comprise two distinct subsets in RA: stem-like Tph (S-Tph) and effector Tph (E-Tph) cells (Masuo et al., Sci Immunol., 2025). S-Tph cells showed self-renewal capacity and were mainly found within tertiary lymphoid structures (TLSs) together with B cells. By contrast, E-Tph cells expressed various effector molecules capable of activating macrophages. Most E-Tph cells were located outside TLSs and interacted with proinflammatory macrophages. S-Tph cells were able to differentiate into E-Tph cells when cocultured with B cells, while concurrently inducing potent immunoglobulin production by B cells. Extending our T cell analysis, scRNA-seq of B cells revealed the presence of naive, memory, GC-like, ABCs, and ASCs. Among these B cell subsets, the frequency of S-Tph cells in ST correlated most strongly with that of GC-like B cells. Conclusion These findings indicate that interactions between S-Tph and B cells within TLSs of RA synovium are crucial for sustaining Tph population by promoting S-Tph self-renewal and differentiation into E-Tph cells. Our study offers a rationale to target S-Tph cells for the treatment of RA characterized by persistent tissue inflammation with an expectation to reduce global Tph responses and TLS formation. Funding Source Japan Agency for Medical Research and Development grant Topic Categories Immune Mechanisms of Human Disease (HUM)

Primary sclerosing cholangitis (PSC) is a chronic autoimmune cholestatic liver disease characterized by progressive inflammation and fibrosis, but the role of neutrophils in PSC remains poorly understood. To define neutrophil states in the PSC liver microenvironment, we performed single-cell RNA sequencing of leukocytes isolated from liver perfusate obtained from healthy donor grafts and explanted PSC livers. We identified a marked expansion of transcriptionally diverse neutrophil subclusters in PSC liver perfusate. Importantly, PSC livers were enriched for four neutrophil subclusters: RHOB-high, immediate early gene-high, heat shock protein-high, and CXCL8-high liver neutrophil-like subclusters. These PSC-associated neutrophil subclusters showed increased activity of the TNF and IL-17 signaling pathways, consistent with current views of PSC pathogenesis. They also displayed activation of inflammatory transcriptional regulators, including CEBPB, JUN, FOS, and RELA. Pseudotime analysis suggested that RHOB and immediate early gene subsets were derived from immature neutrophils, whereas liver neutrophil-like and heat shock protein-high subsets exhibited more mature and tissue-adapted features. Together, these data reveal previously unrecognized heterogeneity of liver neutrophils in PSC and identify inflammatory neutrophil programs that may contribute to sustained hepatic inflammation and represent potential therapeutic targets.

B cells play a critical role in tumor immunity, with their presence associated with improved prognosis in various cancers, including endometrial cancer (EC). However, the nature of the B-cell response within the tumor microenvironment (TME) remains incompletely understood. In this study, we conducted single-cell analyses of B cells and CD4+ T cells in the TME of EC. We found that the TME of EC harbored abundant plasmablasts and plasma cells (PCs), which were rare in normal endometria. PCs primarily expressed either IgG or IgA, and a high abundance of IgG in TME was associated with better overall survival. B-cell receptor (BCR) repertoire analysis revealed a clonal expansion of IgG+ B cells, coinciding with an increased presence of T follicular helper (Tfh) cells in the TME. Notably, Tfh cells shared T-cell receptor clones with cycling CD4+ T cells, indicating local proliferation. BCR repertoire analysis also suggested that IgG+ PCs differentiate from IFN-responding B cells and double-negative B cells in the TME. Additionally, recombinant oligoclonal IgG antibodies were found to recognize antigens expressed by tumor cells as well as normal endometrial cells. Collectively, our study shows that the clonal expansion of IgG+ B cells, along with the Tfh cell response, is associated with a better outcome in EC.

Studies using mouse models of viral infection have shown that viral antigen-reactive regulatory T cells play multiple context-dependent roles, including attenuating the immune response against the pathogen and limiting inflammatory damage. By contrast, evidence for the presence and function of viral antigen-specific Tregs in humans remains very limited. Here, we investigated whether BNT162b2 mRNA COVID-19 vaccination activates spike (S)-reactive Tregs in healthy adults. We performed an integrated analysis combining scRNA-seq, TCR repertoire profiling, and flow cytometry of activation-induced marker (AIM) expressing S-reactive CD4+ T cells isolated from PBMCs cultured with trimeric S protein. We found that CD39+ cells co-expressing CTLA4 and TIGIT within the S-reactive AIM+ CD4+ T cell population were preferentially enriched for Tregs relative to their CD39- counterparts. In subjects vaccinated with BNT162b2 mRNA vaccine, the frequencies of CD39+CTLA4+ and CD39+TIGIT+ S-reactive Treg-like cells were significantly increased following vaccination. Using scRNA-seq, we identified two FoxP3+ Treg clusters among S-reactive CD4+ T cells: one characterized by IFN-activated gene signatures and another by high HLA class II, CD39 and CTLA4 expression. Both subsets were transcriptionally activated following vaccination, with upregulation of genes associated with T cell activation and inflammatory cytokines including TNF and IFNG. Consistent with these findings, flow cytometry detected increased proportions of CD39+ and FoxP3+ S-reactive Tregs expressing IFN-γ and/or TNF-α following vaccination. Collectively, these findings support a model in which BNT162b2 mRNA vaccination activates both an S-reactive effector T cell response and a Treg response, suggesting Treg co-induction may be critical for balancing antiviral immunity with immunoregulation.

Vaccines effectively stimulate protective immune responses in healthy individuals, but the precise roles of germinal center (GC) and follicular helper T (TFH) cells in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine responses are not fully understood. This study used a conditional loss-of-function mouse model to investigate antibody responses to the Wuhan spike protein, specifically eliminating newly developed TFH cells during either the primary or memory phase. Our findings demonstrated that TFH-mediated GC responses are essential for primary vaccination. However, after booster immunization, memory B-cell responses were effectively regulated through extrafollicular mechanisms, independent of TFH cells. Ablating IL-4 receptor signaling in B cells attenuated antibody production in both the primary and memory phases, highlighting the critical role of IL-4 for optimal humoral immunity. We identified a unique population of IL-4-expressing memory T (IL-4+Tm) cells, characterized by CD27, GATA3, and IRF4 expression, that is strongly associated with these extrafollicular memory B-cell responses, capable of neutralizing SARS-CoV-2 variants. Furthermore, Omicron-based booster immunization recovered the immunity against emerging variants under TFH-deficient conditions. These results suggest that IL-4+Tm cells are an alternative pathway to sustain memory responses when GC function is impaired, particularly in immunocompromised states. Our study advances the understanding of memory T-cell-mediated humoral responses to SARS-CoV-2, offering insights for future vaccine strategies.

Cutaneous allergen sensitization (CAS) underlies atopic dermatitis (AD) and leads to various allergic symptoms, including food allergy and anaphylaxis. IL-13 expression by T follicular helper T (TFH) has been reported to be involved in generating high-affinity IgE antibodies and causing systemic anaphylaxis.1, 2 However, the mechanisms by which IL-13 triggers IgE-mediated allergic responses remain poorly defined. In the present study, we elucidate the role of IL-13 in the CAS-mediated mechanism by which high-affinity IgE antibodies are produced when the same allergen is introduced at a distal site in the secondary sensitization. The CAS model system using mice lacking the cell lineage-specific IL-13 receptor (IL-13R) demonstrated that dendritic cells (DCs), but not T or B cells, are critical in the high-affinity IgE-mediated anaphylactic response. The IL-13 signal in type 2 conventional DCs (cDC2s) enhanced the expression of MHC class II and CD301b, which was essential for the recall of type 2 responses, inducing the production of high-affinity IgE antibodies. Similar IL-13R-expressing DCs were identified in allergic rhinitis and food allergy patients with a history of AD. These findings strongly suggest the importance of DC-specific IL-13 signaling in CAS-induced allergic reactions associated with the atopic march, which is common in human AD patients.

Stress can trigger acute inflammation by increasing the pro-inflammatory cytokine interleukin (IL)-17 for injury and infection, while inducing the production of the immunosuppressive hormone glucocorticoids (GCs). However, the mechanism through which stress-induced GCs enhance acute inflammation by directly regulating the development of IL-17-producing helper T (Th17) cells remains unclear. Here, we demonstrate that GCs promote Th17 cell differentiation and survival in both mice and humans. Stress-induced GCs augment the expansion of Th17 cells expressing low levels of TCF1, a negative regulator of IL-17 expression. In addition, GCs promote Th17 cell differentiation by enhancing glycolysis. Stress-induced GCs also increase IL-17 production and neutrophil recruitment in the intestine upon bacterial antigen stimulation. Moreover, the expansion of Th17 cells mediated by stress-induced GCs exacerbates acute colitis by promoting IL-17 production and neutrophil recruitment. Thus, stress promotes acute inflammation by enhancing the differentiation of Th17 cells through GCs, which may contribute to self-defense against infections.

Neuromyelitis optica (NMO) is an acute inflammatory demyelinating disease of the CNS. The presence of astrocyte-targeted AQP4-immunoglobulin G (IgG) in peripheral blood is a major factor in its diagnosis. Previous studies show that AQP4-IgG contributes directly to CNS inflammation and that B cells play a central pathogenic role in NMO. However, where and how the B-cell response is altered remains controversial. In this study, we used high-parameter flow cytometry to carry out a comprehensive analysis of the immune cell populations in the CSF samples obtained from first-episode acute-phase NMO patients, compared with those from patients with acute-phase multiple sclerosis and other neurological diseases. Among 10 immune cell populations defined in the analysis, the frequency only of B cells and antibody-secreting cells (ASC) was higher in the CSF of acute-phase NMO compared with other neurological diseases. Detailed assessments of B-cell and ASC subsets in the CSF revealed differences in the dominant subsets between NMO and multiple sclerosis. In NMO, a series of CD21lo B-cell subsets, including 'activated' naïve B, double-negative and switched memory subsets, considered as ASC precursors, were dominant. A majority of these CD21lo B-cell subsets expressed CD69 and CXCR3, suggesting their CNS residency. An increase of CD21lo B-cell subsets was also observed in the CSF of treatment-refractory NMO patients. Furthermore, two B-helper T-cell subsets, T peripheral helper type 1 and T follicular helper type 1 cells, both highly expressing CD69 and CXCR3, were enriched in the CSF of NMO patients, suggesting their interactions with ASC precursors in the CNS. In vitro culture experiments using blood samples from patients with NMO showed that CD21lo B cells included AQP4-IgG-producing cells and displayed a high propensity to differentiate into ASCs. We also found that CD21lo B-cell subsets in NMO upregulated the expression of C5a receptors, and C5a signals promoted their differentiation into ASCs. ASCs derived from CD21lo B cells expressed high levels of CXCR3 and CD138. The increase in CD21lo B-cell subsets was significantly correlated with the annual relapse rate. Collectively, our study strongly suggests that the mechanism to promote the generation of CD21lo B cells, probably via the extrafollicular pathway, becomes activated during the acute phase of NMO and that the generated CD21lo B-cell subsets contribute to the pathogenesis. Targeting CD21lo B-cell subsets might be useful for the development of novel therapeutic approaches.

Human CD4 + T cells play a central role in the pathogenesis of autoimmune diseases, but their immunoregulatory mechanisms driving pathogenesis remain to be elucidated. We show that human T peripheral helper cells (T PH cells) regulate peripheral immune responses via insulin-like growth factor–like family member 2 (IGFL2), an inflammatory factor found exclusively in primates. Single-cell RNA sequencing of seropositive rheumatoid arthritis (RA) synovium showed that IGFL2 is specifically expressed by CD4 + T cells, predominantly T PH cells. IGFL2 promotes transforming growth factor–β–induced CXCL13 production in CD4 + T cells, activates nuclear factor κB signaling, and induces monocyte gene signatures like those of pathogenic macrophages. CRISPR-Cas9 knockout of IGFL2 in synovial T PH cells suppressed this gene signature in cocultured monocytes. Blood IGFL2 protein levels correlated with RA disease severity and could be used as a potential biomarker. These findings highlight the involvement of IGFL2 in RA pathogenesis, emphasizing how human T PH cells regulate local immune responses via IGFL2.

ABSTRACT:Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is an orphan disease characterized by the proliferation and infiltration of EBV-infected T/natural killer (NK) cells into multiple organs. Although CAEBV is a heterogeneous disease with diverse clinical courses, its pathogenesis remains poorly understood. In this study, we explored the molecular mechanisms underlying CAEBV by performing a comprehensive multiomics analysis, including genome, transcriptome, epigenome, and single-cell transcriptome and surface proteome analyses, of 65 patients with CAEBV. Methylation analysis identified 2 distinct subtypes of NK cell-type CAEBV based on the CpG island methylator phenotype (CIMP). In CIMP-positive CAEBV, regions associated with enhancer of zeste homolog 2 binding sites and histone H3 lysine 27 trimethylation exhibited increased DNA hypermethylation, resulting in downregulation of tumor suppressor and antiherpesvirus genes. CIMP-positive CAEBV had a particularly poor prognosis and displayed a "neoplastic" phenotype with a DNA methylation pattern similar to that of extranodal NK/T-cell lymphoma, a higher tumor mutation burden, and frequent copy number alterations. In addition, both in vitro and in vivo functional assays demonstrated that 5-azacytidine, a hypomethylating agent, was a potentially effective agent for high-risk CIMP-positive CAEBV. Finally, we established a method to effectively detect EBV-infected cells in single-cell analysis, suggesting that EBV-infected NK cells have tissue-resident properties and that innate and adaptive immunity to EBV is compromised in patients with CAEBV. The present findings provide insight into the complex molecular features of CAEBV and suggest potential molecular therapies.

CD4+ tissue-resident memory T (TRM) cells contribute to host defense and to the pathogenesis of chronic inflammatory diseases, but the molecules that direct their differentiation are unknown. We found that the transcription factor hepatic leukemia factor (HLF) could direct the tissue residency program and function of CD4+ TRM cells. HLF simultaneously up-regulated tissue retention receptors, down-regulated tissue egress receptors, and promoted proinflammatory CD4+ TRM cells by inducing Bhlhe40, and all of these processes were associated with changes in chromatin accessibility. Genetic deletion of Hlf inhibited CD4+ TRM cell generation and ameliorated airway tissue inflammation in vivo. HLF+ CD4+ TRM cells isolated from inflamed airway tissue in humans had a tissue residency signature and expressed inflammatory cytokines. We conclude that HLF may act as a central regulator of proinflammatory CD4+ TRM cell development and function.

To address the increasing importance of studying immunology in humans, rather than a reliance on model systems, back-to-back symposia highlighting advances in human immunology were held in Osaka and Kyoto in Japan.

Peripheral helper T (TPH) cells can play pathogenic roles in human autoimmune diseases. TPH cells are proposed to be the major B cell helpers in inflamed joints in rheumatoid arthritis (RA), but whether and how TPH cells are engaged in tissue inflammation remains unclear. We demonstrate that TPH cells comprise two subsets in RA: stem-like TPH (S-TPH) and effector TPH (E-TPH) cells. These two subsets differed in transcriptome, epigenome, B cell helper capacity, spatial localization, and cell interactions. S-TPH cells displayed self-renewal capacity and were mainly found within tertiary lymphoid structures (TLSs) in synovial tissue together with B cells. S-TPH cells potently induced B cells to produce immunoglobulins. By contrast, E-TPH cells expressed effector molecules and colocalized with proinflammatory macrophages and CD8+ T cells outside TLSs. S-TPH cells could differentiate into E-TPH cells upon TCR stimulation and coculture with B cells. Collectively, our study shows that S-TPH cells play a central role in promoting TPH responses by undergoing self-renewal and seeding E-TPH cells.

Abstract With the incorporation of immune checkpoint inhibitors into the treatment of endometrial cancer (EC), a deeper understanding of the tumor immune microenvironment is critical. Tertiary lymphoid structures (TLSs) are considered favorable prognostic factors for EC, but the significance of their spatial distribution remains unclear. B cell receptor repertoire analysis performed using six TLS samples located at various distances from the tumor showed that TLSs in distal areas had more shared B cell clones with tumor-infiltrating lymphocytes. To comprehensively investigate the distribution of TLSs, we developed an artificial intelligence model to detect TLSs and determine their spatial locations in whole-slide images. Our model effectively quantified TLSs, and TLSs were detected in 69% of the patients with EC. We identified them as proximal or distal to the tumor margin and demonstrated that patients with distal TLSs (dTLSs) had significantly prolonged overall survival and progression-free survival (PFS) across multiple cohorts [hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.36–0.88; p = 0.01 for overall survival; HR, 0.58; 95% CI, 0.40–0.84; p = 0.004 for PFS]. When analyzed by molecular subtype, patients with dTLSs in the copy-number-high EC subtype had significantly longer PFS (HR, 0.51; 95% CI, 0.29–0.91; p = 0.02). Moreover, patients with dTLSs had a higher response rate to immune checkpoint inhibitors (87.5 vs. 41.7%) and a trend toward improved PFS. Our findings indicate that the functions and prognostic implications of TLSs may vary with their locations, and dTLSs may serve as prognostic factors and predictors of treatment efficacy. This may facilitate personalized therapy for patients with EC.

Germinal center (GC) reactions are tightly regulated to generate high-affinity antibodies. Although IL10+ Foxp3- follicular T cells have recently been described as contributing to the suppression of GC reactions, their differentiation, localization, and heterogeneity remain incompletely understood. Additionally, it remains unclear whether IL10+ Foxp3- follicular T cells represent a transient status or an independent subset. To address these gaps, we performed integrative single-cell analysis of transcriptomes, epigenomes, surface proteomes, and TCR repertoires in human tonsillar CD4+ T cells. Unbiased clustering revealed IL10+ Foxp3- follicular T cells as a transcriptionally and epigenetically unique subset. This subset exhibited features of both T follicular helper (Tfh) and T regulatory type 1 (Tr1) cells, and accordingly, hereafter, we call them T follicular regulatory type 1 (Tfr1) cells. Analysis using imaging mass cytometry and spatial RNA-TCR sequencing demonstrated their presence within GCs in humans. Bioinformatic analysis suggested that Tfr1 cells differentiate from GC-Tfh cells upon strong TCR stimulation, a finding corroborated by mouse in vivo experiments and time-series single-cell RNA-TCR sequencing of human in vivo CD4+ T cells. Of note, our bioinformatic analysis suggested that Tfr1 cells receive strong TCR signals from ICOS-Lhigh GC-B cells, likely representing high-affinity GC-B cells. Finally, we show that Tfr1 cells acquire a resident memory phenotype following an effector phase. Together, our findings suggest that high-affinity ICOS-Lhigh GC-B cells transform follicular T cells from GC-Tfh cells to Tfr1 cells, which likely become memory cells and reside in the lymphoid organ to support effective antibody production.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5′ single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4 + T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type–specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type–resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.