Search

BACKGROUND: The clustering of immune repertoire data is challenging due to the computational cost associated with a very large number of pairwise sequence comparisons. To overcome this limitation, we developed Anchor Clustering, an unsupervised clustering method designed to identify similar sequences from millions of antigen receptor gene sequences. First, a Point Packing algorithm is used to identify a set of maximally spaced anchor sequences. Then, the genetic distance of the remaining sequences to all anchor sequences is calculated and transformed into distance vectors. Finally, distance vectors are clustered using unsupervised clustering. This process is repeated iteratively until the resulting clusters are small enough so that pairwise distance comparisons can be performed. RESULTS: Our results demonstrate that Anchor Clustering is faster than existing pairwise comparison clustering methods while providing similar clustering quality. With its flexible, memory-saving strategy, Anchor Clustering is capable of clustering millions of antigen receptor gene sequences in just a few minutes. CONCLUSIONS: This method enables the meta-analysis of immune-repertoire data from different studies and could contribute to a more comprehensive understanding of the immune repertoire data space.

Psoriasis (Ps) is one of the most common chronic inflammatory skin disorders with its pathogenesis correlated with dysregulated innate and adaptive system. Even though biological agents have advanced the treatment of psoriasis, however, there are huge limitations, like high adverse reactions and relapse rate. Therefore, it is of great interest in searching clinical resolutions with better safety and efficacy. In the current study, we utilized the adipose-derived mesenchymal stem cell (AD-MSCs) to treat moderate/severe cases of psoriasis in a single-arm clinical study. This AD-MSC treatment has proven to be clinically safe and effective. Interestingly, a trend of adaptome improvement, including increased diversity, elevated uCDR3s and decreased large clone after AD-MSC treatment in a short (2 weeks) and long (12 weeks) terms. In conclusion, allogenic AD-MSC treatment has shown a good safety and efficacy in treating Ps and can effectively improve the compromised adaptive immune system of Ps patients. [ABSTRACT FROM AUTHOR]

Diversity is the cornerstone of the adaptive immune system, crucial for its effectiveness against constantly evolving pathogens that pose threats to higher vertebrates. Accurately measuring and interpreting this diversity presents challenges for immunologists, as changes in diversity and clonotype composition can tip the balance between protective immunity and autoimmunity. In this review, we present the current methods commonly used to measure diversity from single‐cell T‐cell receptor and B‐cell receptor sequencing. We also discuss two case studies where single‐cell sequencing and diversity estimations have led to breakthroughs in autoimmune disease discovery and therapeutic innovation, and reflect upon the necessity and importance of accurately defining and measuring lymphocyte diversity in these contexts. [ABSTRACT FROM AUTHOR]

The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications. [ABSTRACT FROM AUTHOR]

Background: In this study, we comprehensively profiled the T‐cell receptor (TCR) repertoire of the tumor and adjacent normal tissue in patients with HBV‐associated hepatocellular carcinoma (HCC) and determined the baseline characteristics and clinical significance of TCR. Methods: High‐throughput sequencing was used to determine the profile of complementarity‐determining region 3 (CDR3) of the TCR‐β chain variable (TRBV) in the tumor and normal tissue samples of 14 HCC patients. At the same time, TRBV diversity and differences in expression between tumor and normal tissues were investigated. The cumulative frequency of top 100 CDR3 (CF100), clonality, and Shannon entropy as indices to evaluate diversity, Results: The diversity of TRBV CDR3 showed no significant difference between tumor and normal tissues. Of the 58 V gene segments in TRBV, TRBV16 and TRBV7‐6 had a significantly higher frequency in the tumor group than in the normal group (p < 0.05). The frequency of 14 J gene segments showed no significant difference between tumor and normal tissues. In contrast, the frequency of 22 TRBVx/BJx combinations was significantly higher in the tumor than in the normal tissue. In addition, the length and type of TRBV CDR3 were similar in tumor and normal tissues, and a Gaussian distribution was observed in both groups. Conclusion: This study provided a large amount of information about the TCR lineage in HBV‐associated HCC, laying the foundation for further research. In addition, the fact that the immune repertoire (TRBV CDR3) hardly differs between tumor and adjacent normal tissue provides a new clue for exploring the mechanism of the liver as an organ with immune privileges. [ABSTRACT FROM AUTHOR]

The γδ T cells are a subpopulation of T cells that are abundantly found in the skin and mucous membranes. Their reactivity to self‐antigens and ability to secrete various cytokines make them a key component in psoriasis development. Although the correlation between the immune repertoire (IR) of γδ T‐cell receptors and the occurrence and severity of psoriasis remains incompletely explored, high‐throughput sequencing of γδ T cells has led to a deeper understanding of IR in psoriasis. This study investigated the differences between γδ T cells in patients with psoriasis and healthy controls. The γδ T cells were identified via immunofluorescence staining and a correlation analysis was performed according to the psoriasis area and severity index (PASI) scores. The IR sequencing method was used to detect IR in the γδ T‐cell receptors. The findings demonstrated more skin γδ T cells in patients with psoriasis, which were positively correlated with the PASI score. There were subtle differences in most variable (V), diversity (D) and joining (J) gene segments and VJ/VDJ combination segments between patients with psoriasis and healthy controls. However, a higher diversity of complementarity‐determining region 3 (CDR3) was observed in patients with psoriasis. In summary, the IR of skin γδ T cells was significantly altered in patients with psoriasis, and the diversity in the cell's CDR3 population is a promising biomarker for assessment of psoriasis severity. [ABSTRACT FROM AUTHOR]

Guojun Shen,1,&ast; Xueying Li,2,3,&ast; Anqi Zheng,2 Mingxing Gong,4 Zhanhui Wang,2 Baolin Liao5 1Hepatology Unit, the Third People′s Hospital of Jiujiang City, Jiujiang, People’s Republic of China; 2Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 3Department of Gastroenterology, Shenzhen Qianhai Taikang Hospital, Shenzhen, People’s Republic of China; 4Department of Infectious Diseases, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou, People’s Republic of China; 5Department of Hepatology, Guangzhou Medical Research Institute of Infectious Diseases, Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Baolin Liao, Department of Hepatology, Guangzhou Medical Research Institute of Infectious Diseases, Guangzhou Eighth People’s Hospital, Guangzhou Medical University, No. 8 Huaying Road, Guangzhou, 510440, People’s Republic of China, Email polinlbl@163.com Zhanhui Wang, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, No. 1838 North Guangzhou Blvd, Guangzhou, 510515, People’s Republic of China, Email wangzhh@smu.edu.cnBackground: 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.Patients and Methods: 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.Results: 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.Conclusion: 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.Keywords: chronic hepatitis B, Nucleos(t)ide analogue, T cell receptor, immune repertoire, hepatocellular carcinoma

Abstract Purpose Immune checkpoint inhibitors (ICIs)-associated myocarditis was a rare yet severe complication observed in individuals undergoing immunotherapy. This study investigated the immune status and characteristics of patients diagnosed with ICIs- associated myocarditis. Methods A total of seven patients diagnosed with ICIs-associated myocarditis were included in the study, while five tumor patients without myocarditis were recruited as reference controls. Additionally, 30 healthy individuals were recruited as blank controls. Biochemical indices, electrocardiogram, and echocardiography measurements were obtained both prior to and following the occurrence of myocarditis. High-throughput sequencing of T cell receptor (TCR) was employed to assess the diversity and distribution characteristics of TCR CDR3 length, as well as the diversity of variable (V) and joining (J) genes of T lymphocytes in peripheral blood. Results In the seven patients with ICIs-associated myocarditis, Troponin T (TNT) levels exhibited a significant increase following myocarditis, while other parameters such as brain natriuretic peptide (BNP), QTc interval, and left ventricular ejection fraction (LVEF) did not show any significant differences. Through sequencing, it was observed that the diversity and uniformity of CDR3 in the ICIs-associated myocarditis patients were significantly diminished. Additionally, the distribution of CDR3 nucleotides deviated from normality, and variations in the utilization of V and J gene segments. Conclusion The reconstitution of the TCR immune repertoire may play a pivotal role in the recognition of antigens in patients with ICIs-associated myocarditis.

Abstract Colorectal cancer (CRC) is one of the top five most common and life-threatening malignancies worldwide. Most CRC develops from advanced colorectal adenoma (ACA), a precancerous stage, through the adenoma-carcinoma sequence. However, its underlying mechanisms, including how the tumor microenvironment changes, remain elusive. Therefore, we conducted an integrative analysis comparing RNA-seq data collected from 40 ACA patients who visited Dongguk University Ilsan Hospital with normal adjacent colons and tumor samples from 18 CRC patients collected from a public database. Differential expression analysis identified 21 and 79 sequentially up- or down-regulated genes across the continuum, respectively. The functional centrality of the continuum genes was assessed through network analysis, identifying 11 up- and 13 down-regulated hub-genes. Subsequently, we validated the prognostic effects of hub-genes using the Kaplan–Meier survival analysis. To estimate the immunological transition of the adenoma-carcinoma sequence, single-cell deconvolution and immune repertoire analyses were conducted. Significant composition changes for innate immunity cells and decreased plasma B-cells with immunoglobulin diversity were observed, along with distinctive immunoglobulin recombination patterns. Taken together, we believe our findings suggest underlying transcriptional and immunological changes during the adenoma-carcinoma sequence, contributing to the further development of pre-diagnostic markers for CRC.

Colorectal cancer (CRC) is one of the top five most common and life-threatening malignancies worldwide. Most CRC develops from advanced colorectal adenoma (ACA), a precancerous stage, through the adenoma-carcinoma sequence. However, its underlying mechanisms, including how the tumor microenvironment changes, remain elusive. Therefore, we conducted an integrative analysis comparing RNA-seq data collected from 40 ACA patients who visited Dongguk University Ilsan Hospital with normal adjacent colons and tumor samples from 18 CRC patients collected from a public database. Differential expression analysis identified 21 and 79 sequentially up- or down-regulated genes across the continuum, respectively. The functional centrality of the continuum genes was assessed through network analysis, identifying 11 up- and 13 down-regulated hub-genes. Subsequently, we validated the prognostic effects of hub-genes using the Kaplan–Meier survival analysis. To estimate the immunological transition of the adenoma-carcinoma sequence, single-cell deconvolution and immune repertoire analyses were conducted. Significant composition changes for innate immunity cells and decreased plasma B-cells with immunoglobulin diversity were observed, along with distinctive immunoglobulin recombination patterns. Taken together, we believe our findings suggest underlying transcriptional and immunological changes during the adenoma-carcinoma sequence, contributing to the further development of pre-diagnostic markers for CRC. [ABSTRACT FROM AUTHOR]

Objective: Using single cell sequencing to analyze the characteristics of the immune repertoire of SARS-CoV-2 patients at different infection stages, and to explore the possible pathogenesis. Methods: Obtaining data in the NCBI database, and healthy control, progression and convalescence groups were set up. Analysis of single cell sequencing data by RStudio, Origin, Hipliot and Excel software. Results: TCR plays an important role in antigen recognition and virus clearance. Characteristics of the three immune repertoires are very different. The number of clones exceeded 26.92%. The length distribution of α chain CDR3 was concentrated on 14 amino acids, while β-chain was concentrated on 15 amino acids. The frequency of gene fragments in V and J regions were different, and the frequency of TRAV13-1, TRAJ20, TRBV20-1 and TRBJ2-1 were statistically significant (P<0.05). Further V-J gene combination analysis showed that there were significant differences in the frequency of single chain V-J between the control group and the progression group, the control group and the rehabilitation group (P<0.05). The highest frequency of αβ double chain V-J in the control group was TRAV19-J34-TRBV4-1-J2-1, the highest frequency of αβ double-stranded V-J in the progression group was TRAV12-1-J30-TRBV19-1-J2-1, and the highest frequency of αβ double chain V-J in the convalescence group was TRAV12-2-J52-TRBV7-9-J1-5. Conclusion: This study analyzes the global characteristics of T cells in the immune repertoire, which is helpful to understand the pathogenesis of SARS-CoV-2 patients, timely and effective treatment of patients in the early stages of infection. [ABSTRACT FROM AUTHOR]

Abstract Background In this study, we comprehensively profiled the T‐cell receptor (TCR) repertoire of the tumor and adjacent normal tissue in patients with HBV‐associated hepatocellular carcinoma (HCC) and determined the baseline characteristics and clinical significance of TCR. Methods High‐throughput sequencing was used to determine the profile of complementarity‐determining region 3 (CDR3) of the TCR‐β chain variable (TRBV) in the tumor and normal tissue samples of 14 HCC patients. At the same time, TRBV diversity and differences in expression between tumor and normal tissues were investigated. The cumulative frequency of top 100 CDR3 (CF100), clonality, and Shannon entropy as indices to evaluate diversity, Results The diversity of TRBV CDR3 showed no significant difference between tumor and normal tissues. Of the 58 V gene segments in TRBV, TRBV16 and TRBV7‐6 had a significantly higher frequency in the tumor group than in the normal group (p

The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications.

Abstract The T-cell receptor (TCR) repertoires exhibits distinct signatures associated with COVID-19 severity. However, the precise identification of vaccine-induced SARS-CoV-2-specific TCRs and T-cell immunity mechanisms are unknown. We developed a machine-learning model that can differentiate COVID-19 patients from healthy individuals based on TCR sequence features with an accuracy of 95.7%. Additionally, we identified SARS-CoV-2-specific T cells and TCR in HLA-A*02 vaccinated individuals by peptide stimulation. The SARS-CoV-2-specific T cells exhibited higher cytotoxicity and prolonged survival when targeting spike-pulsed cells in vitro or in vivo. The top-performing TCR was further tested for its affinity and cytotoxic effect against SARS-CoV-2-associated epitopes. Furthermore, single-cell RNA sequencing (scRNA-seq), immune repertoire sequencing (IR-seq) and flow cytometry were used to access vaccine-induced cellular immunity, which demonstrated that robust T cell responses (T cell activation, tissue-resident memory T cell (Trm) generation, and TCR clonal expansion) could be induced by intranasal vaccination. In summary, we identified the SARS-CoV-2-associated TCR repertoires profile, specific TCRs and T cell responses. This study provides a theoretical basis for developing effective immunization strategies.

ABSTRACTImmune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRβ) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRβ repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRβ repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRβ clonotypes was observed in responding tumours. Machine learning identified TCRβ CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRβ signatures associated with ICT response.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+ CD5+ clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Treatment options for patients range from historical chemoimmunotherapy (CIT) to small molecule inhibitors targeting pro-survival pathways in leukemic B cells, such as the Brutons tyrosine kinase inhibitor ibrutinib (IBR). Using biobanked blood samples obtained pre-therapy and at standard response evaluation timepoints, we performed an in-depth evaluation of the blood innate and adaptive immune compartments between pentostatin-based CIT and IBR and looked for correlations with clinical sequelae. CD4+ conventional T cells and CD8+ cytotoxic T cells responded similarly to CIT and IBR, although exhaustion status differed. Both treatments dramatically increased the prevalence and functional status of monocyte, dendritic cell, and natural killer cell subsets. As expected, both regimens reduced clonal B cell levels however, we observed no substantial recovery of normal B cells. Although improvements in most immune subsets were observed with CIT and IBR at response evaluation, both patient groups remained susceptible to infections and secondary malignancies during the study.

The dog is valued as a companion animal and increasingly recognized as a model for human disorders. Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells can contribute to our understanding of pathogenesis mechanisms and inform the development of new treatment strategies. However, the diversity of canine T cells is still poorly understood mainly due to the lack of species-reactive antibodies for use in flow cytometry. The aim of this study was to generate a detailed atlas of peripheral blood TCRαβ+ T cells of healthy dogs using single-cell RNA-sequencing (scRNAseq) combined with immune repertoire sequencing. A total of 22 TCRαβ+ T cell clusters were identified, which were classified into three major groups: CD4-dominant (11 clusters), CD8A-dominant (8 clusters), and CD4/CD8A-mixed (3 clusters). Based on differential gene expression, distinct differentiation states (naïve, effector, memory, exhausted) and lineages (e.g. CD4 T helper and regulatory T cells) could be distinguished. Importantly, several T cell populations were identified, which have not been described in dogs before. Of particular note, our data provide first evidence for the existence of canine mucosa-associated invariant T cell (MAIT)-like cells, representing one of three newly identified FCER1G+ innate-like CD8A+ T cell populations in the peripheral blood of healthy dogs. In conclusion, using scRNAseq combined with immune repertoire sequencing we were able to resolve canine TCRαβ+ T cell populations at unprecedented resolution. The peripheral blood TCRαβ+ T cell atlas of healthy dogs generated here represents an important reference data set for future studies and is of relevance for identifying new targets for T cell-specific therapies.

The T-cell receptor (TCR) repertoires exhibits distinct signatures associated with COVID-19 severity. However, the precise identification of vaccine-induced SARS-CoV-2-specific TCRs and T-cell immunity mechanisms are unknown. We developed a machine-learning model that can differentiate COVID-19 patients from healthy individuals based on TCR sequence features with an accuracy of 95.7%. Additionally, we identified SARS-CoV-2-specific T cells and TCR in HLA-A*02 vaccinated individuals by peptide stimulation. The SARS-CoV-2-specific T cells exhibited higher cytotoxicity and prolonged survival when targeting spike-pulsed cells in vitro or in vivo. The top-performing TCR was further tested for its affinity and cytotoxic effect against SARS-CoV-2-associated epitopes. Furthermore, single-cell RNA sequencing (scRNA-seq), immune repertoire sequencing (IR-seq) and flow cytometry were used to access vaccine-induced cellular immunity, which demonstrated that robust T cell responses (T cell activation, tissue-resident memory T cell (Trm) generation, and TCR clonal expansion) could be induced by intranasal vaccination. In summary, we identified the SARS-CoV-2-associated TCR repertoires profile, specific TCRs and T cell responses. This study provides a theoretical basis for developing effective immunization strategies. [ABSTRACT FROM AUTHOR]

Systematic immune responses have been identified in patients with acute spontaneous intracerebral hemorrhage (ICH). T cells have been established to participate in central nervous system damage and repair following brain injury. However, their contribution to the prognosis of patients with ICH remains to be elucidated. In this study, peripheral blood mononuclear cells (PBMCs) were collected from 45 patients with acute spontaneous ICH (<24 h from symptom onset). Our results exposed significant negative correlations between hematoma volume/white blood cell (WBC) density and Glasgow Coma Scale (GCS) score. Contrastingly, lymphocyte density was negatively correlated with hematoma volume and positively correlated with GCS score. Moreover, flow cytometry determined that ICH activated T cells despite their proportion being lower in blood. Afterward, immune repertoire sequencing (IR-seq) revealed a significant decrease in VJ, VDJ usage, and TCR clonotypes in ICH patients. Finally, variations in the complementarity-determining region 3 (CDR3) amino acid (aa) were also detected in ICH patients. This study reveals the occurrence of peripheral T-cell diminishment and activation in response to acute hematoma. ICH lesion also alters the T cell receptor (TCR) immune repertoire, which is associated with patient prognosis. [ABSTRACT FROM AUTHOR]

Immune repertoire is defined as the sum of T cells and B cells, which possesses high diversity and enables immune system to respond to various antigen stimuli. With the development of sequencing technique, immune repertoire sequencing can be utilized to deeply understand the changes of lymphocyte clones when rejection occurs at the gene level, and also provide the possibility for the emergence of novel non-invasive diagnostic techniques based on immune repertoire sequencing. In recent years, more and more attempts have been made to apply immune repertoire sequencing in solid organ transplantation, especially in the fields of kidney transplantation, liver transplantation, heart transplantation and post-transplantation infection. In this article, research progresses on the application of immune repertoire sequencing in these fields were reviewed, and current status of immune repertoire sequencing in organ transplantation and its potential as a novel technique for early non-invasive diagnosis of rejection were summarized, aiming to provide reference for subsequent development and clinical application of this technique. [ABSTRACT FROM AUTHOR]

Background:The pathogenesis of juvenile idiopathic arthritis (JIA) is strongly influenced by an impaired immune system. However, the molecular mechanisms underlying its development and progression have not been elucidated. In this study, the computational methods TRUST4 were used to construct a T-cell receptor (TCR) and B-cell receptor (BCR) repertoire from the peripheral blood of JIA patients via bulk RNA-seq data, after which the clonality and diversity of the immune repertoire were analyzed.Results:Our findings revealed significant differences in the frequency of clonotypes between the JIA and healthy control groups in terms of the TCR and BCR repertoires. This work identified specific V genes and J genes in TCRs and BCRs that could be used to expand our understanding of JIA. After single-cell RNA analysis, the relative percentages of CD14 monocytes were significantly greater in the JIA group. Cell-cell communication analysis revealed the significant role of the MIF signaling pathway in JIA.Conclusion:In conclusion, this work describes the immune features of both the TCR and BCR repertoires under JIA conditions and provides novel insight into immunotherapy for JIA.

Abstract Background The clustering of immune repertoire data is challenging due to the computational cost associated with a very large number of pairwise sequence comparisons. To overcome this limitation, we developed Anchor Clustering, an unsupervised clustering method designed to identify similar sequences from millions of antigen receptor gene sequences. First, a Point Packing algorithm is used to identify a set of maximally spaced anchor sequences. Then, the genetic distance of the remaining sequences to all anchor sequences is calculated and transformed into distance vectors. Finally, distance vectors are clustered using unsupervised clustering. This process is repeated iteratively until the resulting clusters are small enough so that pairwise distance comparisons can be performed. Results Our results demonstrate that Anchor Clustering is faster than existing pairwise comparison clustering methods while providing similar clustering quality. With its flexible, memory-saving strategy, Anchor Clustering is capable of clustering millions of antigen receptor gene sequences in just a few minutes. Conclusions This method enables the meta-analysis of immune-repertoire data from different studies and could contribute to a more comprehensive understanding of the immune repertoire data space.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+ CD5+ clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Treatment options for patients range from historical chemoimmunotherapy (CIT) to small molecule inhibitors targeting pro-survival pathways in leukemic B cells, such as the Bruton’s tyrosine kinase inhibitor ibrutinib (IBR). Using biobanked blood samples obtained pre-therapy and at standard response evaluation timepoints, we performed an in-depth evaluation of the blood innate and adaptive immune compartments between pentostatinbased CIT and IBR and looked for correlations with clinical sequelae. CD4+ conventional T cells and CD8+ cytotoxic T cells responded similarly to CIT and IBR, although exhaustion status differed. Both treatments dramatically increased the prevalence and functional status of monocyte, dendritic cell, and natural killer cell subsets. As expected, both regimens reduced clonal B cell levels however, we observed no substantial recovery of normal B cells. Although improvements in most immune subsets were observed with CIT and IBR at response evaluation, both patient groups remained susceptible to infections and secondary malignancies during the study. [ABSTRACT FROM AUTHOR]

Objective: At present, little is known about the immune mechanism of liver transplantation caused by decompensated cirrhosis. Lymphocytes play an essential important role in the immune rejection of liver transplantation. In this study, we aimed to comprehensively analyze changes in complementary determinant 3 (CDR3) repertoire of T cell receptor β chain (TRβ) and immunoglobulin heavy chain (IGH) in liver transplantation patients and healthy controls (HC). Methods: High-throughput sequencing technology was used to study the characteristics of TRβ/IGH CDR3 repertoire, and identify the amino acid sequences of TRβ and IGH associated with liver transplantation patients and HC. Results: We found that some TRβ and IGH CDR3 repertoire characteristics differed between liver transplant patients and HC. The diversity of TRβ CDR3 increased in the liver transplantation group. First and seven days after live transplantation patients showed a lower degree of T cell clone amplification compared to the HC group. The CDR3 repertoire of the TRβ/IGH chain was certainly biased in the use of some V, D, and J gene segments, TRβ/IGH V-J combined frequency was also skewed and TRβ CDR3 clonotypes were shared at a higher degree in the liver transplantation patients. Importantly, one amino acid sequence in the decompensated cirrhosis group was significantly higher than that in the healthy group. It should be noted that the frequency of some CDR3 sequences is closely correlated with the different stages of liver transplantation, and these sequences may play a key role in liver transplantation. Conclusion: Based on the above results, we can better understand the dynamic changes of TCβ/IGH CDR3 repertoire in patients during liver transplantation. [ABSTRACT FROM AUTHOR]

Antibodies are generated with great diversity in nature resulting in a set of molecules, each optimized to bind a specific target. Taking advantage of their diversity and specificity, antibodies make up for a large part of recently developed biologic drugs. For therapeutic use antibodies need to fulfill several criteria to be safe and efficient. Polyspecific antibodies can bind structurally unrelated molecules in addition to their main target, which can lead to side effects and decreased efficacy in a therapeutic setting, for example via reduction of effective drug levels. Therefore, we created a neural-network-based model to predict polyspecificity of antibodies using the heavy chain variable region sequence as input. We devised a strategy for enriching antibodies from an immunization campaign either for antigen-specific or polyspecific binding properties, followed by generation of a large sequencing data set for training and cross-validation of the model. We identified important physico-chemical features influencing polyspecificity by investigating the behaviour of this model. This work is a machine-learning-based approach to polyspecificity prediction and, besides increasing our understanding of polyspecificity, it might contribute to therapeutic antibody development.

ABSTRACTThe emergence and re-emergence of abundant viruses from bats that impact human and animal health have resulted in a resurgence of interest in bat immunology. Characterizing the immune receptor repertoire is critical to understanding how bats coexist with viruses in the absence of disease and developing new therapeutics to target viruses in humans and susceptible livestock. In this study, IGH germline genes of Chiroptera including Rhinolophus ferrumequinum, Phyllostomus discolor, and Pipistrellus pipistrellus were annotated, and we profiled the characteristics of Rhinolophus affinis (RA) IGH CDR3 repertoire. The germline genes of Chiroptera are quite different from those of human, mouse, cow, and dog in evolution, but the three bat species have high homology. The CDR3 repertoire of RA is unique in many aspects including CDR3 subclass, V/J genes access and pairing, CDR3 clones, and somatic high-frequency mutation compared with that of human and mouse, which is an important point in understanding the asymptomatic nature of viral infection in bats. This study unveiled a detailed map of bat IGH germline genes on chromosome level and provided the first immune receptor repertoire of bat, which will stimulate new avenues of research that are directly relevant to human health and disease.IMPORTANCEThe intricate relationship between bats and viruses has been a subject of study since the mid-20th century, with more than 100 viruses identified, including those affecting humans. While preliminary investigations have outlined the innate immune responses of bats, the role of adaptive immunity remains unclear. This study presents a pioneering contribution to bat immunology by unveiling, for the first time, a detailed map of bat IGH germline genes at the chromosome level. This breakthrough not only provides a foundation for B cell receptor research in bats but also contributes to primer design and sequencing of the CDR3 repertoire. Additionally, we offer the first comprehensive immune receptor repertoire of bats, serving as a crucial library for future comparative analyses. In summary, this research significantly advances the understanding of bats’ immune responses, providing essential resources for further investigations into viral tolerance and potential zoonotic threats.

Fansheng Guo,1 Xiaoye Yuan,2 Jinglin Cao,1 Xin Zhao,1 Yang Wang,1 Wenpeng Liu,1 Baowang Liu,1 Qiang Zeng1 1Department of Hepatobiliary Surgery, the Third Hospital of Hebei Medical University, Shijiazhuang, 050000, People’s Republic of China; 2Department of Gerontology, Hebei General Hospital, Shijiazhuang, 050000, People’s Republic of ChinaCorrespondence: Qiang Zeng, Department of Hepatobiliary Surgery, the Third Hospital of Hebei Medical University, No. 139, Ziqiang Road, Shijiazhuang, 050051, People’s Republic of China, Tel +86-15511308373, Email zengqiangtg@163.comBackground: Hepatocellular carcinoma (HCC) relapse is the main reason for the poor prognosis of HCC after Liver transplantation (LT). This study aimed to explore the molecular mechanisms and immune repertoire profiles of HCC relapse.Material and Methods: RNA-seq of blood samples from patients with normal (n=12) and HCC relapse (n=6) after LT was performed to identify differentially expressed genes (DEGs) and key signalling pathways. The DEGs and immune genes were further analyzed by bioinformatics. TRUST4 was used to analyze the differences in the immune repertoire between the two groups. Another 11 blood samples from patients with HCC who had received LT were collected for RT-qPCR verification of key genes.Results: A total of 131 upregulated and 157 downregulated genes were identified using RNA-seq, and GO enrichment analysis revealed that the top 15 pathways were immune-related. The PPI network identified 10 key genes. Immune infiltration analysis revealed a significant difference in the five immune cell types between the two groups. A total of 83 intersecting genes were obtained by intersecting DEGs and immune genes. 6 key genes, including MX1, ISG15, OAS1, PRF1, SPP1, and THBS1 were obtained according to the intersection of DEGs, PPI network top 10 genes and immune intersecting genes. Immune repertoire analysis showed that the usage frequency of variable (V) and joining (J) genes in the normal group was higher than that in the relapse group. RT-qPCR validation showed that the expression levels of key genes were consistent with the RNA-seq results.Conclusion: Our study identified key pathways and genes that could help determine whether transplant recipients are more prone to HCC relapse. Immune repertoire analysis revealed a difference in the usage frequency of VJ genes between the normal and relapse groups, providing a research direction for immunotherapy in patients with HCC relapse after liver transplantation.Keywords: liver transplantation, hepatocellular carcinoma relapse, RNA-sequencing, immune repertoire, variable and joining genes, V and J genes

Abstract Assessment of minimal residual disease in acute lymphoblastic leukemia by immune repertoire NGS requires spiking CDR3 sequences at known quantities into the patient's sample. Recently, the EuroClonality‐NGS group released one of the most comprehensive protocols for this purpose. ARResT/Interrogate is a closed‐source software for processing these NGS libraries, developed by this same group. Vidjil, an open‐source alternative, currently cannot handle libraries prepared using this protocol. Here, we present a Vidjil add‐on to solve this issue. EuroClonality‐NGS prepared samples analyzed with Vidjil and ARResT/Interrogate were highly concordant (r = 0.998) and presented low error (root‐mean‐square error, RMSE = 0.112).

In this cross-sectional and longitudinal analysis of mapping the T-cell repertoire in kidney transplant recipients, we have investigated and validated T-cell clonality, immune repertoire chronology at rejection, and contemporaneous allograft biopsy quantitative tissue injury, to better understand the pathobiology of acute T-cell fraction, T-cell repertoire and antibody-mediated kidney transplant rejection. To follow the dynamic evolution of T-cell repertoire changes before and after engraftment and during biopsy-confirmed acute rejection, we sequenced 323 peripheral blood samples from 200 unique kidney transplant recipients, with (n=100) and without (n=100) biopsy-confirmed acute rejection. We report that patients who develop acute allograft rejection, have lower (p=0.01) T-cell fraction even before transplantation, followed by its rise after transplantation and at the time of acute rejection accompanied by high TCR repertoire turnover (p=0.004). Acute rejection episodes occurring after the first 6 months post-transplantation, and those with a component of antibody-mediated rejection, had the highest turnover; p=0.0016) of their T-cell repertoire. In conclusion, we validated that detecting repertoire changes in kidney transplantation correlates with post-transplant rejection episodes suggesting that T-cell receptor sequencing may provide recipient pre-transplant and post-transplant predictors of rejection risk.

Tumor-infiltrating B cells can play an important role in anti-tumor responses but their presence is not well understood. In this study, we extracted the B cell receptor repertoires from 9522 tumor and adjacent non-tumor samples across 28 tumor types in the Cancer Genome Atlas project and performed diversity and network analysis. We identified differences in diversity and network statistics across tumor types and subtypes and observed a trend towards increased clonality in primary tumors compared to adjacent non-tumor tissues. We also found significant associations between the repertoire features and mutation load, tumor stage, and age. Our V-gene usage analysis identified similar V-gene usage patterns in colorectal and endometrial cancers. Lastly, we evaluated the prognostic value of the repertoire features and identified significant associations with survival in seven tumor types. This study warrants further research into better understanding the role of tumor-infiltrating B cells across a wide range of tumor types.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+ CD5+ clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Treatment options for patients range from historical chemoimmunotherapy (CIT) to small molecule inhibitors targeting pro-survival pathways in leukemic B cells, such as the Bruton’s tyrosine kinase inhibitor ibrutinib (IBR). Using biobanked blood samples obtained pre-therapy and at standard response evaluation timepoints, we performed an in-depth evaluation of the blood innate and adaptive immune compartments between pentostatin-based CIT and IBR and looked for correlations with clinical sequelae. CD4+ conventional T cells and CD8+ cytotoxic T cells responded similarly to CIT and IBR, although exhaustion status differed. Both treatments dramatically increased the prevalence and functional status of monocyte, dendritic cell, and natural killer cell subsets. As expected, both regimens reduced clonal B cell levels however, we observed no substantial recovery of normal B cells. Although improvements in most immune subsets were observed with CIT and IBR at response evaluation, both patient groups remained susceptible to infections and secondary malignancies during the study.

With the development of immune repertoire sequencing technology, it is hoped that this technology can be used to deeply explore the correlation between postmenopausal osteoporosis and bone immunity from the perspective of immune repertoire. Aided by immune repertoire sequencing technology, we may understand the pathogenesis of postmenopausal osteoporosis, obtain the corresponding biomarkers, and make the diagnosis and treatment of postmenopausal osteoporosis more accurately. This article aims to summarize the research progress in this field. [ABSTRACT FROM AUTHOR]

Background: The tumor microenvironment in hepatocellular carcinoma (HCC) is complicated. Tumor-infiltrating T and B cells play a pivotal role in anti-tumor immunity. T cell receptor (TCR) and B cell receptor (BCR) features may reflect the disease-associated antigen response. Methods: By combining bulk TCR/BCR-sequencing, RNA-sequencing, whole exome-sequencing, and human leukocyte antigen-sequencing, we examined the immune repertoire (IR) features of tumor and adjacent non-tumor tissues obtained from 64 HCC patients. Results: High IR heterogeneity with weak similarity was discovered between tumor and non-tumor tissues. Higher BCR diversity, richness, and somatic hypermutation (SHM) were found in non-tumor tissues, while TCRa and TCRb diversity and richness were comparable or higher in tumor. Additionally, lower immune infiltration was found in tumor than non-tumor tissues; the microenvironment in tumor appeared to keep stably inhibited and changed slightly with tumor progression. Moreover, BCR SHM was stronger, whereas TCR/BCR diversity declined with HCC progression. Importantly, we found that higher IR evenness in tumor and lower TCR richness in non-tumor tissues indicated better survival in HCC patients. Collectively, the results revealed that TCR and BCR exhibited distinct features in tumor and non-tumor tissues. Conclusions: We demonstrated that IR features vary between different tissues of HCC. IR features may represent a biomarker for the diagnosis and treatment of HCC patients, providing references for subsequent immunotherapy research and strategy selection. [ABSTRACT FROM AUTHOR]

Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-β1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.

Early screening and detection of non-small cell lung cancer (NSCLC) is crucial due to the significantly low survival rate in advanced stages. Blood-based liquid biopsy is non-invasive test to assistant disease diagnosis, while cell-free RNA is one of the promising biomarkers in blood. However, the disease related signatures have not been explored completely for most cell-free RNA transcriptome sequencing (cfRNA-Seq) datasets. To address this gap, we developed a comprehensive cfRNA-Seq pipeline for data analysis and constructed a machine learning model to facilitate noninvasive early diagnosis of NSCLC. The results of our study have demonstrated the identification of differential mRNA, lncRNAs and miRNAs from cfRNA-Seq, which have exhibited significant association with development and progression of lung cancer. The classifier based on gene expression signatures achieved an impressive area under the curve (AUC) of up to 0.9, indicating high specificity and sensitivity in both cross-validation and independent test. Furthermore, the analysis of T cell and B cell immune repertoire extracted from cfRNA-Seq have provided insights into the immune status of cancer patients, while the microbiome analysis has revealed distinct bacterial and viral profiles between NSCLC and normal samples. In our future work, we aim to validate the existence of cancer associated T cell receptors (TCR)/B cell receptors (BCR) and microorganisms, and subsequently integrate all identified signatures into diagnostic model to improve the prediction accuracy. This study not only provided a comprehensive analysis pipeline for cfRNA-Seq dataset but also highlights the potential of cfRNAs as promising biomarkers and models for early NSCLC diagnosis, emphasizing their importance in clinical settings.

Abstract Background Clear cell renal cell cancer (ccRCC) is accompanied by T-cell infiltration. In this study, we sought to determine the difference in T-cell infiltration and the T-cell receptor (TCR) immune repertoire between ccRCC and peritumour tissue. Methods T-cell infiltration was examined using immunohistochemistry (IHC) and haematoxylin and eosin (HE) staining. The chi-squared test and Pearson correlation analysis were applied to evaluate the relationship between clinical traits and CD3, CD4, and CD8 expression. Immune repertoire sequencing (IR-Seq) was used to describe the profile of the TCR repertoire. Results The adjacent tissue showed increased expression of CD3, CD4 and CD8 compared with ccRCC tissue (P CD3 = 0.033; P CD4 = 0.014; P CD8 = 0.004). Indicated CD3+ T-cell density in ccRCC tissue was positively correlated with that in peritumour tissue (P = 0.010, r = 0.514), which implied the T cells in peritumour tissue directly infect the number of cells infiltrating in ccRCC tissue. Moreover, there was a positive correlation between Vimentin expression and indicated positive T-cell marker in ccRCC tissue (P CD3 = 0.035; P CD4 = 0.020; P CD8 = 0.027). Advanced stage revealed less CD4+ T-cell infiltration in ccRCC tissue (P CD4 = 0.023). The results from IR-Seq revealed an obvious increase in VJ and VDJ segment usage, as well as higher complementarity-determining region 3 (CDR3) amino acid (aa) clonotypes in ccRCC. The matched antigen recognized by the TCR of ccRCC may be potential targets. Conclusions The current study collectively demonstrates diminished T-cell infiltration and increased CDR3 aa diversity in ccRCC, which may be associated with immunotherapeutic targets for ccRCC patients.

Introduction: Oral lichen planus (OLP) is a common chronic inflammatory disorder of the oral mucosa with an unclear etiology. Several types of immune cells are involved in the pathogenesis of OLP. Methods: We used single-cell RNA sequencing and immune repertoire sequencing to characterize the mucosal immune microenvironment of OLP. The presence of tissue-resident memory CD8+ T cells are validated by multiplex immunofluorescence. Results: We generated a transcriptome atlas from four OLP biopsy samples and their paired peripheral blood mononuclear cells (PBMCs), and compared them with two healthy tissues and three healthy PBMCs samples. Our analysis revealed activated tissue-resident memory CD8+ T cells in OLP tissues. T cell receptor repertoires displayed apperant clonal expansion and preferrential gene pairing in OLP patients. Additionally, obvious BCR clonal expansion was observed in OLP lesions. Plasmacytoid dendritic cells, a subtype that can promote dendritic cell maturation and enhance lymphocyte cytotoxicity, were identified in OLP. Conventional dendritic cells and macrophages are also found to exhibit proinflammatory activity in OLP. Cell-cell communication analysis reveals that fibroblasts might promote the recruitment and extravasation of immune cells into connective tissue. Discussion: Our study provides insights into the immune ecosystem of OLP, serving as a valuable resource for precision diagnosis and therapy of OLP. [ABSTRACT FROM AUTHOR]

The adaptive immune receptor repertoire (AIRR), consisting of T- and B-cell receptors, is the core component of the immune system. The AIRR sequencing is commonly used in cancer immunotherapy and minimal residual disease (MRD) detection of leukemia and lymphoma. The AIRR is captured by primers and sequenced to yield paired-end (PE) reads. The PE reads could be merged into one sequence by the overlapped region between them. However, the wide range of AIRR data raises the difficulty, so a special tool is required. We developed a software package for IMmune PE reads merger of sequencing data, named IMperm. We used the k -mer-and-vote strategy to pin down the overlapped region rapidly. IMperm could handle all types of PE reads, eliminate adapter contamination and successfully merge low-quality and minor/non-overlapping reads. Compared with existing tools, IMperm performed better in both simulated and sequencing data. Notably, IMperm was well suited to processing the data of MRD detection in leukemia and lymphoma and detected 19 novel MRD clones in 14 patients with leukemia from previously published data. Additionally, IMperm can handle PE reads from other sources, and we demonstrated its effectiveness on two genomic and one cell-free deoxyribonucleic acid datasets. IMperm is implemented in the C programming language and consumes little runtime and memory. It is freely available at https://github.com/zhangwei2015/IMperm. [ABSTRACT FROM AUTHOR]

Background: Despite the introduction of trastuzumab, pathologic complete response (pCR) is not attained in approximately 30–40% of Human epithelial growth factor receptor-2-positive breast cancer. Tumor-infiltrating lymphocytes (TIL) have been suggested as a predictive marker of treatment response, albeit not always effective. We investigated the relationship between trastuzumab, docetaxel, carboplatin, and pertuzumab (TCHP) treatment and immune repertoire as a treatment response predictor. Design: In all, 35 cases were divided into two experimental groups: 10 and 25 cases in the preliminary and main experiments, respectively. In the preliminary experiment, the biopsy tissues before TCHP treatment and the surgical tissues after TCHP treatment were compared. In the main experiment, the biopsy tissues before TCHP treatment were compared according to the TCHP treatment response. Methods: The T-cell repertoire for TRA, TRB, TRG, and TRD, and B-cell repertoire for immunoglobulin heavy, immunoglobulin kappa, and immunoglobulin lambda were evaluated. Whole transcriptome sequencing was also performed. Results: In the preliminary experiment, the density and richness of the T-cell receptor (TCR) and B-cell receptor (BCR) repertoires decreased after treatment, regardless of TCHP response. In the main experiment, the Shannon's entropy index, density, and length of CDR3 of the TCR and BCR repertoires did not differ significantly in patients who did and did not achieve pCR. The pCR and non-pCR subgroups according to the level of TILs revealed that the non-pCR/lowTIL group had a higher proportion of low-frequency clones than the pCR/lowTIL group in TRA (non-pCR/lowTIL versus pCR/lowTIL, 0.01–0.1%, 63% versus 45.3%; <0.01%, 32.9% versus 51.8%, p < 0.001) and TRB (non-pCR/lowTIL versus pCR/lowTIL, 0.01–0.1%, 26.5% versus 14.7%; <0.01%, 72.0% versus 84.1%, p < 0.001). Conclusions: The role of the diversity, richness, and density of the TCR and BCR repertoires as predictive markers for TCHP response was not identified. Compositions of low-frequency clones could be candidates for predictive factors of TCHP response; however, validation studies and further research are necessary. [ABSTRACT FROM AUTHOR]

The dog is valued as a companion animal and increasingly recognized as a model for human disorders. Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells can contribute to our understanding of pathogenesis mechanisms and inform the development of new treatment strategies. However, the diversity of canine T cells is still poorly understood mainly due to the lack of species-reactive antibodies for use in flow cytometry. The aim of this study was to generate a detailed atlas of peripheral blood TCRαβ+ T cells of healthy dogs using single-cell RNA-sequencing (scRNAseq) combined with immune repertoire sequencing. A total of 22 TCRαβ+ T cell clusters were identified, which were classified into three major groups: CD4- dominant (11 clusters), CD8A-dominant (8 clusters), and CD4/CD8A-mixed (3 clusters). Based on differential gene expression, distinct differentiation states (naïve, effector, memory, exhausted) and lineages (e.g. CD4 T helper and regulatory T cells) could be distinguished. Importantly, several T cell populations were identified, which have not been described in dogs before. Of particular note, our data provide first evidence for the existence of canine mucosa-associated invariant T cell (MAIT)-like cells, representing one of three newly identified FCER1G+ innate-like CD8A+ T cell populations in the peripheral blood of healthy dogs. In conclusion, using scRNAseq combined with immune repertoire sequencing we were able to resolve canine TCRαβ+ T cell populations at unprecedented resolution. The peripheral blood TCRαβ+ T cell atlas of healthy dogs generated here represents an important reference data set for future studies and is of relevance for identifying new targets for T cell-specific therapies. [ABSTRACT FROM AUTHOR]

The dog is valued as a companion animal and increasingly recognized as a model for human disorders. Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells can contribute to our understanding of pathogenesis mechanisms and inform the development of new treatment strategies. However, the diversity of canine T cells is still poorly understood mainly due to the lack of species-reactive antibodies for use in flow cytometry. The aim of this study was to generate a detailed atlas of peripheral blood TCRαβ+ T cells of healthy dogs using single-cell RNA-sequencing (scRNAseq) combined with immune repertoire sequencing. A total of 22 TCRαβ+ T cell clusters were identified, which were classified into three major groups: CD4-dominant (11 clusters), CD8A-dominant (8 clusters), and CD4/CD8A-mixed (3 clusters). Based on differential gene expression, distinct differentiation states (naïve, effector, memory, exhausted) and lineages (e.g. CD4 T helper and regulatory T cells) could be distinguished. Importantly, several T cell populations were identified, which have not been described in dogs before. Of particular note, our data provide first evidence for the existence of canine mucosa-associated invariant T cell (MAIT)-like cells, representing one of three newly identified FCER1G+ innate-like CD8A+ T cell populations in the peripheral blood of healthy dogs. In conclusion, using scRNAseq combined with immune repertoire sequencing we were able to resolve canine TCRαβ+ T cell populations at unprecedented resolution. The peripheral blood TCRαβ+ T cell atlas of healthy dogs generated here represents an important reference data set for future studies and is of relevance for identifying new targets for T cell-specific therapies.

Immune cells, including T and B cells, are key factors in the success of liver transplantation. And the repertoire of T cells and B cells plays an essential function in mechanism of the immune response associated with organ transplantation. An exploration of their expression and distribution in donor organs could contribute to a better understanding of the altered immune microenvironment in grafts. In this study, using single-cell 5’ RNA sequence and single-cell T cell receptor (TCR)/B cell receptor (BCR) repertoire sequence, we profiled immune cells and TCR/BCR repertoire in three pairs of donor livers pre- and post-transplantation. By annotating different immune cell types, we investigated the functional properties of monocytes/Kupffer cells, T cells and B cells in grafts. Bioinformatic characterization of differentially expressed genes (DEGs) between the transcriptomes of these cell subclusters were performed to explore the role of immune cells in inflammatory response or rejection. In addition, we also observed shifts in TCR/BCR repertoire after transplantation. In conclusion, we profiled the immune cell transcriptomics and TCR/BCR immune repertoire of liver grafts during transplantation, which may offer novel strategies for monitoring recipient immune function and treatment of rejection after liver transplantation.

The immune memory repertoire encodes the history of present and past infections and immunological attributes of the individual. As such, multiple methods were proposed to use T-cell receptor (TCR) repertoires to detect disease history. We here show that the counting method outperforms two leading algorithms. We then show that the counting can be further improved using a novel attention model to weigh the different TCRs. The attention model is based on the projection of TCRs using a Variational AutoEncoder (VAE). Both counting and attention algorithms predict better than current leading algorithms whether the host had CMV and its HLA alleles. As an intermediate solution between the complex attention model and the very simple counting model, we propose a new Graph Convolutional Network approach that obtains the accuracy of the attention model and the simplicity of the counting model. [ABSTRACT FROM AUTHOR]

Important roles of humoral tumor immunity are often pointed out; however, precise profiles of dominant antigens and developmental mechanisms remain elusive. We systematically investigated the humoral antigens of dominant intratumor immunoglobulin clones found in human cancers. We found that approximately half of the corresponding antigens were restricted to strongly and densely negatively charged polymers, resulting in simultaneous reactivities of the antibodies to both densely sulfated glycosaminoglycans (dsGAGs) and nucleic acids (NAs). These anti‐dsGAG/NA antibodies matured and expanded via intratumoral immunological driving force of innate immunity via NAs. These human cancer–derived antibodies exhibited acidic pH–selective affinity across both antigens and showed specific reactivity to diverse spectrums of human tumor cells. The antibody‐drug conjugate exerted therapeutic effects against multiple cancers in vivo by targeting cell surface dsGAG antigens. This study reveals that intratumoral immunological reactions propagate tumor‐oriented immunoglobulin clones and demonstrates a new therapeutic modality for the universal treatment of human malignancies. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) impairs the adaptive immune system during acute infection. Still, it remains largely unclear whether the frequency and functions of T and B cells return to normal after the recovery of Coronavirus Disease 2019 (COVID‐19). Here, we analyzed immune repertoires and SARS‐CoV‐2‐specific neutralization antibodies in a prospective cohort of 40 COVID‐19 survivors with a 6‐month follow‐up after hospital discharge. Immune repertoire sequencing revealed abnormal T‐ and B‐cell expression and function with large T cell receptor/B cell receptor clones, decreased diversity, abnormal class‐switch recombination, and somatic hypermutation. A decreased number of B cells but an increased proportion of CD19+CD138+ B cells were found in COVID‐19 survivors. The proportion of CD4+ T cells, especially circulating follicular helper T (cTfh) cells, was increased, whereas the frequency of CD3+CD4− T cells was decreased. SARS‐CoV‐2‐specific neutralization IgG and IgM antibodies were identified in all survivors, especially those recorded with severe COVID‐19 who showed a higher inhibition rate of neutralization antibodies. All severe cases complained of more than one COVID‐19 sequelae after 6 months of recovery. Overall, our findings indicate that SARS‐CoV‐2‐specific antibodies remain detectable even after 6 months of recovery. Because of their abnormal adaptive immune system with a low number of CD3+CD4− T cells and high susceptibility to infections, COVID‐19 patients might need more time and medical care to fully recover from immune abnormalities and tissue damage. [ABSTRACT FROM AUTHOR]

Background: Clear cell renal cell cancer (ccRCC) is accompanied by T-cell infiltration. In this study, we sought to determine the difference in T-cell infiltration and the T-cell receptor (TCR) immune repertoire between ccRCC and peritumour tissue.Methods: T-cell infiltration was examined using immunohistochemistry (IHC) and haematoxylin and eosin (HE) staining. The chi-squared test and Pearson correlation analysis were applied to evaluate the relationship between clinical traits and CD3, CD4, and CD8 expression. Immune repertoire sequencing (IR-Seq) was used to describe the profile of the TCR repertoire.Results: The adjacent tissue showed increased expression of CD3, CD4 and CD8 compared with ccRCC tissue (PCD3 = 0.033; PCD4 = 0.014; PCD8 = 0.004). Indicated CD3+ T-cell density in ccRCC tissue was positively correlated with that in peritumour tissue (P = 0.010, r = 0.514), which implied the T cells in peritumour tissue directly infect the number of cells infiltrating in ccRCC tissue. Moreover, there was a positive correlation between Vimentin expression and indicated positive T-cell marker in ccRCC tissue (PCD3 = 0.035; PCD4 = 0.020; PCD8 = 0.027). Advanced stage revealed less CD4+ T-cell infiltration in ccRCC tissue (PCD4 = 0.023). The results from IR-Seq revealed an obvious increase in VJ and VDJ segment usage, as well as higher complementarity-determining region 3 (CDR3) amino acid (aa) clonotypes in ccRCC. The matched antigen recognized by the TCR of ccRCC may be potential targets.Conclusions: The current study collectively demonstrates diminished T-cell infiltration and increased CDR3 aa diversity in ccRCC, which may be associated with immunotherapeutic targets for ccRCC patients. [ABSTRACT FROM AUTHOR]