Cell Repertoire Research Articles

Early-life thymectomy leads to an increase of granzyme-producing γδ T cells in children with congenital heart disease.

Congenital heart disease (CHD) is the most common birth defect in newborns, often requiring cardiac surgery with concomitant thymectomy that is known to increase disease susceptibility later in life. Studies of γδ T cells, which are one of the dominant T cells in the early fetal human thymus, are rare. Here, we provide a comprehensive analysis of the γδ T cell compartment via flow cytometry and next-generation sequencing in children and infants with CHD, who underwent cardiac surgery shortly after birth. A perturbation of the γδ T cell repertoire is evident, and Vδ1 T cell numbers are reduced. However, those cells that are present, do retain cytotoxicity. In contrast, GZMA+CD28+CD161hi innate effector Vγ9Vδ2 T cells are found in higher proportions. TCR-seq identifies an increase in TRDJ3+ γδ T cell clones in children with CHD, but not in a confirmatory group of neonates prior to CHD surgery, which overall points to a persistence of fetal-derived effector γδ T cells in children with CHD.

Genome streamlining of Pseudomonas putida B6-2 for bioremediation.

Microbial transformation is a favored approach for environmental remediation. However, the effectiveness of microbial remediation has been limited by the lack of chassis cells with satisfactory contaminant degradation performance. Pseudomonas putida B6-2, with a wide substrate spectrum and high solvent tolerance, is a chassis strain with great potential for application in environmental remediation. Here, guided by bioinformatic analyses and genome-scale metabolic model (GEM) predictions, we successfully optimized P. putida B6-2 by rationally reducing its nonessential genetic components and generating a more robust genome-streamlined strain, P. putida BGR4. Several improvements were observed compared with the original P. putida B6-2 strain, including a 1.4 × 105-fold increase in electroporation efficiency, an 8.3-fold increase in conjugation efficiency, improved glycerol utilization capability, and increased phenol utilization after heterologous expression of the phenol monooxygenase encoded by dmpKLMNOP. Additionally, P. putida BGR4 exhibited enhanced tolerance to several stressors, including starvation, oxidative stress, and DNA damage. Transcriptomic analysis revealed that genome streamlining led to the upregulation of genes involved in the "carbon metabolism" and "tricarboxylic acid cycle" pathways in P. putida BGR4, which likely contributed to the superior phenotype of P. putida BGR4 in terms of carbon source utilization and contaminant degradation capabilities. Furthermore, the absence of four prophages was identified as a potential cause of the enhanced stress resistance observed in P. putida BGR4. Overall, we developed a combined genome-streamlining strategy involving bioinformatic analyses and GEM predictions and generated a more robust chassis strain, P. putida BGR4, which expands the repertoire of chassis cells for environmental remediation.IMPORTANCEDespite the development of many chassis cells, there is still a lack of robust chassis cells with satisfactory contaminant degradation performance. Targeted genome streamlining is an effective way to provide powerful chassis cells. However, genome streamlining does not always lead to the improved phenotypes of genome-streamlined chassis cells. In this research, a novel procedure that combined bioinformatic analyses and GEM predictions was proposed to guide genome streamlining and predict the effects of genome streamlining. This genome streamlining procedure was successfully applied to Pseudomonas putida B6-2, which was a chassis cell with great potential for application in environmental remediation and resulted in the generation of a more robust chassis cell, P. putida BGR4, thereby providing a superior chassis cell for efficient and sustainable environmental remediation and a valuable framework for guiding the genome streamlining of strains for other applications.

CTIM-18. HIGH LEVEL OF MUTATIONAL LOAD AND DIVERSITY OF TUMOR INFILTRATING T LYMPHOCYTES ARE FAVORABLE PROGNOSTIC MARKERS FOR CANCER SURVIVAL IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT) has shown remarkable efficacy in some patients with solid tumors, resulting in durable complete responses. A key mechanism behind this success is the targeting of neoantigens by T cells. There is culminating evidence that tumor-infiltrating lymphocytes (TILs) have a major effect on the clinical attributes of human cancer and can influence the tumor response to various therapy regimens. We have been sequencing RNA isolated from patient’s tumors to evaluate their mutational burden and identify tumor-specific neoantigens. Additionally, we have identified the entire repertoire of tumor-infiltrating lymphocytes in patients with recurrent medulloblastoma. METHODS Total tumor RNA was extracted from resected brain tumor specimens and after we applied in-house bulk RNA-Seq and TCR-Seq techniques using rapid amplification of cDNA ends (RACE) technology with the addition of a common adapter at the 5’ end. RESULTS & CONCLUSIONS Analysis of the 22 tumor tissues revealed that the frequency of non-synonymous mutations varied dramatically more than 10-fold among patients with recurrent medulloblastoma undergoing adoptive cellular therapy (Re-MATCH protocol, FDA IND BB-14058). Patients with shorter overall survival exhibited non-synonymous mutation frequencies between 0.113 and 0.335 mutations per megabase (Mb). Children with prolonged overall survival had higher mutation frequencies, ranging from 0.6 to 1.1 mutations per Mb. These findings highlight the potential of using mutational burden as a biomarker for patient selection and treatment customization, ultimately aiming to improve outcomes in ACT for recurrent medulloblastoma. Up to 2000 unique TCR clones were found among TIL populations in patients who responded well to ACT. Patients with poor outcomes had significantly lower TCR diversity, with only 10-40 unique TCR clones among TILs. The correlation between TCR diversity among TILs and clinical outcomes in pediatric brain tumors underscores the importance of a diverse T cell repertoire for the success of ACT.

Shared lung and joint T cell repertoire in early rheumatoid arthritis driven by cigarette smoking

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic...

The role of polyreactive memory B cells in systemic lupus erythematosus.

In systemic lupus erythematosus (SLE), the production of autoantibodies is a crucial characteristic, and B cells play a significant role in its pathogenesis. B cells are the immune cells most associated with the genetic predispositions of SLE, and recent clinical studies showing that anti-CD19 CAR-T cell therapy induces drug-free remission have underscored the importance of B cells in SLE. Meanwhile, various B cell subsets exist across different stages of differentiation, from naive B cells to plasma-cells, and identifying the important subpopulations within SLE remains a critical future challenge. Years of B cell repertoire analyses have revealed the importance of polyreactive B cell receptors (BCRs) and autoantibodies that react to a various self-antigens and microbial antigens. Particularly, memory B cells with polyreactive BCRs, which play a crucial role in biological defense during the fetal stage, are characteristically differentiated in SLE. Type I interferon-mediated expression of CXCL13 and IL21 in CD4+ T cells is associated with the development of polyreactive memory B cells. The expansion of the polyreactive B cell repertoire, vital for defending against infections such as viruses, may exert an intrinsic function in SLE.

Characteristics of the IgM Repertoires in the Peripheral Blood of Early Rheumatoid Arthritis Patients.

Rheumatoid arthritis is a common autoimmune disease, but little is known about the characteristics of the B cell repertoires in the peripheral blood. In this study, the peripheral IgM repertoires of early rheumatoid arthritis (ERA) patients were analyzed by high-throughput sequencing and bioinformatics analyses. Clonal expansion was observed in IgM repertoires of ERA patients. Interestingly, a subset of the dominant clones in ERA repertoires showed self- and poly-reactivity to several autoantigens. The clones were also present in IgM repertoires of healthy adults but they were not expanded, suggesting that may stem from the natural auto-reactive B cell repertoire. Additionally, the ERA repertoires exhibited a greater extent of somatic hypermutations, particularly in the ERA dominant clones, resulting in an enrichment of amino acids important for antigen-antibody interaction. The in-depth analysis of B-cell repertoires improved our knowledge of the IgM repertoires in early rheumatoid arthritis, offering potential insights into the disease's pathogenesis.

Human Anti-Glycan Reactivity is Driven by the Selection of B cells Utilizing Private Antibody Gene Rearrangements that are Affinity Maturated in Germinal Centers.

The human antibody repertoire is broadly reactive with carbohydrate antigens represented in the universe of all living things, including both the host/self-as well as the commensal microflora-derived glycomes. Here we have used BCR receptor cloning and expression together with single-cell transcriptomics to analyze the B cell repertoire to the ubiquitous N-acetyl-D-glucosamine (GlcNAc) epitope in human cohorts and dissect the immune phylogeny of this predominant class of antibodies. We find that circulating anti-GlcNAc B cells exhibiting canonical BMem phenotypes emerge rapidly after birth and couple this observation with evidence for germinal center-dependent affinity maturation of carbohydrate-specific B cell receptors in situ during early childhood. Direct analysis of individual B cell clonotypes reveals they exhibit strikingly distinct fine-specificity profiles for palettes of GlcNAc containing moieties. These results suggest that a generalized exposure to complex environmental glycans drives the steady state anti-glycan repertoire.

PRMT5: splicing up tolerance.

Expression of tissue-restricted antigens (TRAs) within the thymus is critical for the establishment of self-tolerance; however, exact mechanisms regulating the expression of TRAs has proven more complex than previously appreciated. In this issue of the JCI, Muro et al. identify a central role for protein arginine methyltransferase 5 (PRMT5) in posttranscriptional regulation of TRAs and thereby central tolerance. Using conditional KO mice, the authors showed that thymic deficiency of Prmt5 predisposed mice to developing autoimmune diseases while enhancing antitumor efficacy. These studies provide insight into the role of PRMT5 in shaping the T cell repertoire with implications for the development of therapies.

Transcript splicing optimizes the thymic self-antigen repertoire to suppress autoimmunity.

Immunological self-tolerance is established in the thymus by the expression of virtually all self-antigens, including tissue-restricted antigens (TRAs) and cell-type-restricted antigens (CRAs). Despite a wealth of knowledge about the transcriptional regulation of TRA genes, posttranscriptional regulation remains poorly understood. Here, we show that protein arginine methylation plays an essential role in central immune tolerance by maximizing the self-antigen repertoire in medullary thymic epithelial cells (mTECs). Protein arginine methyltransferase-5 (Prmt5) was required for pre-mRNA splicing of certain key genes in tolerance induction, including Aire as well as various genes encoding TRAs. Mice lacking Prmt5 specifically in thymic epithelial cells exhibited an altered thymic T cell selection, leading to the breakdown of immune tolerance accompanied by both autoimmune responses and enhanced antitumor immunity. Thus, arginine methylation and transcript splicing are essential for establishing immune tolerance and may serve as a therapeutic target in autoimmune diseases as well as cancer immunotherapy.

Microbiota modulate immune repertories in lung adenocarcinoma via microbiota-immunity interactive network.

While the resident microbiome of tumors has been shown to be associated with the occurrence and progression of non-small cell lung cancer, there remains a significant knowledge gap in understanding the correlation between the microbial spectrum and immunity response to cancer therapy. In the case of lung adenocarcinoma (LUAD), the tumor microenvironment, encompassing a diverse array of microbes and immune cells, plays a crucial role in modulating therapeutic response. Towards comprehending the underlying mechanism, we present the microbe-immunity interactive networks to delineate the microbiota and immunity repertoires for two distinct molecular subtypes in LUAD. We obtained multi-omics data of LUAD patients from the publicly available database. In this study, we conducted a systematic exploration of the microbial and immunological etiology of cancer prognosis, by integrating the microbiome, genome, transcriptome, and clinic data. The mutational signature analysis, transcriptome analysis, gene set enrichment analysis, and microbiota-immunity network analysis were performed. Based on the transcriptome repertories, we classified the patients into two molecular subtypes and observed that the overall survival of molecular subtype 2 (MS2) was notably shortened. We identified the microbial biomarkers in patients that distinguished between these molecular subtypes. The significant up-regulation of γδT and neutrophil in MS2, suggesting the inflammation augmentation and stimulation of γδT activation. What is more, the MS2 are characterized by a correlation network between microbiota biomarkers and γδT cell, which may contribute to suppression of anti-tumor immunity and poor overall survival. Our findings not only display the repertoires of tumor microbiota and immune cells, but also elucidate the potential contribution of the microbiota-immunity correlation network to unfavorable overall survival and therapeutic resistance, thereby exerting profound implications on future LUAD therapy.

Full-length single-cell BCR sequencing paired with RNA sequencing reveals convergent responses to pneumococcal vaccination

Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells, but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited, especially from widely-used 3′-barcoded libraries. Here, we report a method that can recover paired, full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate, full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys, indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination.

A transgenic mouse with a humanised B cell repertoire mounts an antibody response to influenza infection and vaccination.

The development of a universal influenza vaccine likely requires an understanding of previous exposure to influenza virus (through vaccination or infection) and how that shapes the antibody repertoire to vaccination, sometimes called Original Antigenic Sin or antigenic imprinting. Whilst animal models can have a much more defined exposure history, they lack a human B cell repertoire. Transgenic mice with the complete human immunoglobulin locus enable studies of controlled infection history leading to human-like antibody evolution. Here we evaluated responses to influenza in the Intelliselect Transgenic mouse (the Kymouse). We show the Kymouse is susceptible to disease following infection with either H1N1, H3N2 or B/Yam influenza viruses and that it induces a robust binding and neutralising antibody response to all three strains of influenza virus. This study demonstrates that human B cell repertoire mice can be used for influenza virus studies, providing a tool for further interrogation of the antibody response.

A novel method for characterizing cell-cell interactions at single-cell resolution reveals unique signatures in blood T cell-monocyte complexes during infection.

Communication between immune cells through direct contact is a critical feature of immune responses. Here, we developed a novel high-throughput method to study the transcriptome and adaptive immune receptor repertoire of single cells forming complexes without needing bioinformatic deconvolution. We found that T cells and monocytes forming complexes in blood during active tuberculosis (TB) and dengue hold unique transcriptomic signatures indicative of TCR/MCH-II immune synapses. Additionally, T cells in complexes showed enrichment for effector phenotypes, imaging and transcriptomic features of active TCR signaling, and increased immune activity at diagnosis compared to after anti-TB therapy. We also found evidence for bidirectional RNA exchange between T cells and monocytes, since complexes were markedly enriched for "dual-expressing" cells (i.e., co-expressing T cell and monocyte genes). Thus, studying immune cell complexes at a single-cell resolution offers novel perspectives on immune synaptic interactions occurring in blood during infection.

Isolation and characterization of IgG3 glycan-targeting antibodies with exceptional cross-reactivity for diverse viral families.

Broadly reactive antibodies that target sequence-diverse antigens are of interest for vaccine design and monoclonal antibody therapeutic development because they can protect against multiple strains of a virus and provide a barrier to evolution of escape mutants. Using LIBRA-seq (linking B cell receptor to antigen specificity through sequencing) data for the B cell repertoire of an individual chronically infected with human immunodeficiency virus type 1 (HIV-1), we identified a lineage of IgG3 antibodies predicted to bind to HIV-1 Envelope (Env) and influenza A Hemagglutinin (HA). Two lineage members, antibodies 2526 and 546, were confirmed to bind to a large panel of diverse antigens, including several strains of HIV-1 Env, influenza HA, coronavirus (CoV) spike, hepatitis C virus (HCV) E protein, Nipah virus (NiV) F protein, and Langya virus (LayV) F protein. We found that both antibodies bind to complex glycans on the antigenic surfaces. Antibody 2526 targets the stem region of influenza HA and the N-terminal domain (NTD) region of SARS-CoV-2 spike. A crystal structure of 2526 Fab bound to mannose revealed the presence of a glycan-binding pocket on the light chain. Antibody 2526 cross-reacted with antigens from multiple pathogens and displayed no signs of autoreactivity. These features distinguish antibody 2526 from previously described glycan-reactive antibodies. Further study of this antibody class may aid in the selection and engineering of broadly reactive antibody therapeutics and can inform the development of effective vaccines with exceptional breadth of pathogen coverage.

NKG2C and NKG2A coexpression defines a highly functional antiviral NK population in spontaneous HIV control.

Elite controllers (ECs), a unique group of people with HIV (PWH), exhibit remarkable control of viral replication in the absence of antiretroviral therapy. In this study, we comprehensively characterized the NK cell repertoire in ECs after long-term viral control. Phenotypic profiling of NK cells revealed profound differences compared with other PWH, but marked similarities to uninfected individuals, with a distinctive prevalence of NKG2C+CD57+ memory-like NK cells. Functional analyses indicated that ECs had limited production of functional molecules upon NK stimulation and consequently reduced natural cytotoxicity against non-HIV target cells. Importantly, ECs showed an exceptional ability to kill primary HIV-infected cells by the antibody-dependent cell cytotoxicity adaptive mechanism, which was achieved by a specific memory-like NK population expressing CD16, NKG2A, NKG2C, CD57, and CXCR3. In-depth single-cell RNA-seq unveiled a unique transcriptional signature in these NK cells linked to increased cell metabolism, migration, chemotaxis, effector functions, cytokine secretion, and antiviral response. Our findings underscore a pivotal role of NK cells in the immune control of HIV and identify specific NK cells as emerging targets for immunotherapies.

Abstract B042: Dissection of the T cell infiltrate in mouse pancreatic tumors reveals a diverse tumor-reactive T cell repertoire targeting neoantigens and tumor-associated antigens

Abstract Although pancreatic cancer is generally refractory to immune checkpoint blockade (ICB), recent studies of tumor-infiltrating T cells in human tumor samples demonstrated the presence of in vivo expanded, tumor-reactive T cell receptor (TCR) clonotypes. Here, we explored the T cell repertoire in ICB-refractory murine pancreatic cancer models by combining single-cell transcriptomics with functional TCR characterization. Our initial studies in an orthotopic, transplantable model revealed a remarkable diversity of tumor-reactive TCR clonotypes that could be distinguished from bystander T cells based on gene expression signatures. We identified TCRs exclusively reactive against the tumor of origin, TCRs that also react against an independently derived pancreatic tumor line, and TCRs broadly reactive to tumor cells of diverse tissue origin (e.g. MC38), the latter category being predominant. Subsequent studies were performed in an autochthonous mouse model, in which single focal tumors were induced in the pancreas by means of in vivo electroporation of gene constructs encoding mutant Kras and inducing CRISPR/Cas9-mediated inactivation of tumor suppressor genes. We analyzed the T cell repertoire in tumors with different levels of mutational burden by also inactivating DNA damage repair genes. Interestingly, even in mismatch repair-deficient tumors that featured a high mutational load, the anti-tumor T cell response was primarily directed against recurrent tumor-associated antigens (TAAs) rather than ‘private’ mutanome-encoded neoantigens. Several TAA-reactive TCRs also mediated reactivity against non-transformed cells. Immunopeptidome analysis thus far revealed the identity of 5 cognate antigens targeted by the tumor-reactive TCR repertoire: a mutanome-encoded neoantigen, an epitope encoded by an ectopically expressed endogenous retroviral provirus, and three epitopes derived from TAAs: Lon Peptidase 2 (LONP2), ATPase Na+/K+ Transporting Subunit Beta 3 (ATP1B3) and SEC24B, a component of secretory vesicles. Notably, the overexpression of LONP2 and ATP1B3 was found associated with progression of human cancers, whereas silencing was shown to suppress tumor cell growth. Overexpression of SEC24B in cancers has not been reported, but this protein plays an essential role in the secretory pathway of acinar cells. In summary, in-depth analysis of the natural T cell response in murine pancreatic cancer models revealed a diverse tumor-reactive T cell repertoire that is predominantly directed against TAAs, some of which are also expressed in normal somatic cells. These findings underline the importance of uncovering the antigen-specificity of the natural tumor-reactive TCR repertoire, both in mouse models and human tumor samples, to assess its therapeutic potential and safety with regard to personalized immunotherapy. Citation Format: Stefan Zens, Hannes Kehm, Daniel Baumann, Aline Konrad, Zibo Meng, Arnoud H. de Ru, Julian Mochayedi, Francesca Lucato, Angelika B. Riemer, Isabel Poschke, Michael Volkmar, Peter A. van Veelen, Rienk Offringa. Dissection of the T cell infiltrate in mouse pancreatic tumors reveals a diverse tumor-reactive T cell repertoire targeting neoantigens and tumor-associated antigens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B042.

Novel engineered IL-2 Nemvaleukin alfa combined with PD1 checkpoint blockade enhances the systemic anti-tumor responses of radiation therapy

BackgroundCombining interleukin-2 (IL-2) with radiotherapy (RT) and immune checkpoint blockade (ICB) has emerged as a promising approach to address ICB resistance. However, conventional IL-2 cytokine therapy faces constraints owing to its brief half-life and adverse effects. RDB 1462, the mouse ortholog of Nemvaleukin alfa, is an engineered IL-2 with an intermediate affinity that selectively stimulates antitumor CD8 T and NK cells while limiting regulatory T cell expansion. This study aimed to evaluate the antitumor activity and mechanism of action of the combination of RDB 1462, RT, and anti-PD1 in mouse tumor models.MethodsTwo bilateral lung adenocarcinoma murine models were established using 344SQ-Parental and 344SQ anti-PD1-resistant cell lines. Primary tumors were treated with RT, and secondary tumors were observed for evidence of abscopal effects. We performed immune phenotyping by flow cytometry, analyzed 770 immune-related genes using NanoString, and performed T cell receptor (TCR) repertoire analysis. Serum pro-inflammatory cytokine markers were analyzed by 23-plex kit.ResultsCompared to native IL-2 (RDB 1475), RDB 1462 demonstrated superior systemic antitumoral responses, attributable, at least in part, to augmented levels of CD4 and CD8 T cells with the latter. Our findings reveal substantial reductions in primary and secondary tumor volumes compared to monotherapy controls, with some variability observed among different dosing schedules of RDB 1462 combined with RT. Blood and tumor tissue-based flow cytometric phenotyping reveals an increase in effector memory CD8 and CD4 T cells and a decrease in immunosuppressive cells accompanied by a significant increase in IL-2, IFN-γ, and GM-CSF levels in the combination group. Transcriptomic profiling and TCR sequencing reveal favorable gene expression and T cell repertoire patterns with the dual combination. Furthermore, integrating anti-PD1 therapy with RT and RDB 1462 further reduced primary and secondary tumor volumes, prolonged survival, and decreased lung metastasis. Observations of immune cell profiles indicated that RT with escalating doses of RDB 1462 significantly reduced tumor growth and increased tumor-specific immune cell populations.ConclusionThe addition of Nemvaleukin therapy may enhance responses to RT alone and in combination with anti-PD1.Graphical

B cell tolerance and autoimmunity: Lessons from repertoires.

Adaptive immune cell function is regulated by a highly diverse receptor recombined from variable germline-encoded segments that can recognize an almost unlimited array of epitopes. While this diversity enables the recognition of any pathogen, it also poses a risk of self-recognition, leading to autoimmunity. Many layers of regulation are present during both the generation and activation of B cells to prevent this phenomenon, although they are evidently imperfect. In recent years, our ability to analyze immune repertoires at scale has drastically increased, both through advances in sequencing and single-cell analyses. Here, we review the current knowledge on B cell repertoire analyses, focusing on their implication for autoimmunity. These studies demonstrate that a failure of tolerance occurs at multiple independent checkpoints in different autoimmune contexts, particularly during B cell maturation, plasmablast differentiation, and within germinal centers. These failures are marked by distinct repertoire features that may be used to identify disease- or patient-specific therapeutic approaches.

ScRNA-seq reveals the landscape of immune repertoire of PBMNCs in iMCD.

The etiology of idiopathic multicentric Castleman disease (iMCD) is poorly understood, and the identification of targetable disease mediators remains an unmet clinical need. Thus, we firstly employed single-cell RNA sequencing (scRNA-seq) to elucidate the landscape of the immune repertoire of peripheral blood mononuclear cells (PBMNCs) in iMCD and to identify additional driver cytokines/cells/pathways to address IL-6 blockade-refractory cases. We revealed that the inflammatory cytokine storm observed in iMCD was a significant phenomenon pervasive across all immune cells. B-plasma cell subsets was the main source of IL-6. The IL-6 signaling pathway was significantly activated across a spectrum of immune cells. Systemic upregulation of CXCL13 is mainly driven by peripheral helper T (Tph) and regulatory T (Treg) cells. Notably, a significant positive interaction was observed between CXCL13-expressing T cells and IL-6 signaling-activated B cells. This study provides an immune perspective on PBMNCs in iMCD at the single-cell level, unveiling pathways or targets characterized by atypical inflammatory expression that could potentially serve as promising candidates for therapeutic intervention in iMCD.

Nucleos(T)ide Analogue Treatment Has a More Pronounced Impact on Immune Repertoires of CHB Patients Compared to HCC Patients.

Nucleos(t)ide analogues (NAs) as the first-line treatment for chronic hepatitis B (CHB) have been shown to partially restore the antiviral immunity of the patients. However, hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients have a relatively longer duration of HBV infection and lower level of HBV DNA. Whether NAs treatments have a different effect on their immune repertoires between CHB and HCC patients remains to be determined. In this study, 126 CHB patients and 85 HBV-related HCC patients who received or did not receive NAs treatment, as well as 361 healthy individuals were enrolled to analyze the effect of NAs treatment on T cell receptor β chain (TCRβ) and B cell receptor heavy chain (BCRh) repertoires in peripheral blood of the patients. We found that after NAs therapy, the richness and evenness of TCRβ and BCRh repertoires in CHB patients were significantly lower than those in untreated patients and healthy controls, while the diversity of TCRβ and BCRh repertoires was stable in HCC patients. The alanine aminotransferase and HBV DNA levels were not correlated with the TCR or BCR diversity in CHB and HCC patients. The results suggest that NAs therapy could influence the overall T cell and B cell repertoires diversity in CHB patients but has minimal impact on HCC patients, indicating a significant difference in the potential to restore antiviral immunity between CHB and HCC patients by NAs treatment.