• BMB Reports
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• 제52권9호
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• pp.540-547
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• 2019

Immune repertoire is a collection of enormously diverse adaptive immune cells within an individual. As the repertoire shapes and represents immunological conditions, identification of clones and characterization of diversity are critical for understanding how to protect ourselves against various illness such as infectious diseases and cancers. Over the past several years, fast growing technologies for high throughput sequencing have facilitated rapid advancement of repertoire research, enabling us to observe the diversity of repertoire at an unprecedented level. Here, we focus on B cell receptor (BCR) repertoire and review approaches to B cell isolation and sequencing library construction. These experiments should be carefully designed according to BCR regions to be interrogated, such as heavy chain full length, complementarity determining regions, and isotypes. We also highlight preprocessing steps to remove sequencing and PCR errors with unique molecular index and bioinformatics techniques. Due to the nature of massive sequence variation in BCR, caution is warranted when interpreting repertoire diversity from error-prone sequencing data. Furthermore, we provide a summary of statistical frameworks and bioinformatics tools for clonal evolution and diversity. Finally, we discuss limitations of current BCR-seq technologies and future perspectives on advances in repertoire sequencing.

• Molecules and Cells
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• 제46권2호
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• pp.120-129
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• 2023

Recent technical advances have enabled unbiased transcriptomic and epigenetic analysis of each cell, known as "single-cell analysis". Single-cell analysis has a variety of technical approaches to investigate the state of each cell, including mRNA levels (transcriptome), the immune repertoire (immune repertoire analysis), cell surface proteins (surface proteome analysis), chromatin accessibility (epigenome), and accordance with genome variants (eQTLs; expression quantitative trait loci). As an effective tool for investigating robust immune responses in coronavirus disease 2019 (COVID-19), many researchers performed single-cell analysis to capture the diverse, unbiased immune cell activation and differentiation. Despite challenges elucidating the complicated immune microenvironments of chronic inflammatory diseases using existing experimental methods, it is now possible to capture the simultaneous immune features of different cell types across inflamed tissues using various single-cell tools. In this review, we introduce patient-based and experimental mouse model research utilizing single-cell analyses in the field of chronic inflammatory diseases, as well as multi-organ atlas targeting immune cells.

• Animal Bioscience
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• 제37권12호
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• pp.2178-2188
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• 2024

Objective: The liver plays a dual role in regulating temperature and immune responses. Examining the influence of heat stress (HS) on liver T cells contributes significantly to understanding the intricate interplay between the immune system and hepatic tissues under thermal stress. This study focused on investigating the characteristics of the T-cell receptor (TCR) β chain CDR3 repertoire in bovine liver samples under both HS and pairfed (PF) environmental conditions. Methods: Sequencing data from six samples sourced from the GEO database underwent annotation. Utilizing immunarch and VDJtool software, the study conducted comprehensive analyses encompassing basic evaluation, clonality assessment, immune repertoire comparison, diversity estimation, gene usage profiling, VJ gene segment pairing scrutiny, clonal tracking, and Kmers analysis. Results: All four TCR chains, namely α, β, γ, and δ, were detected, with the α chains exhibiting the highest detection frequency, followed closely by the β chains. The prevalence of αβ TCRs in bovine liver samples underscored their crucial role in governing hepatic tissue's physiological functions. The TCR β CDR3 repertoire showcased substantial inter-individual variability, featuring diverse clonotypes exhibiting distinct amino acid lengths. Intriguingly, HS cattle displayed heightened diversity and clonality, suggesting potential peripheral T cell migration into the liver under environmental conditions. Notably, differential VJ gene pairings were observed in HS cattle compared to the PF, despite individual variations in V and J gene utilization. Additionally, while most high-frequency amino acid 5-mers remained consistent between the HS and PF, GELHF, and YDYHF were notably prevalent in the HS group. Across all samples, a prevalent trend of high-frequency 5mers skewed towards polar and hydrophobic amino acids was evident. Conclusion: This study elucidates the characteristics of liver TCR β chain CDR3 repertoire under HS conditions, enhancing our understanding of HS implications.

• Journal of Korean Neurosurgical Society
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• 제64권4호
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• pp.505-513
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• 2021

Objective : The adaptive immune response following subarachnoid hemorrhage (SAH) is not well understood. We evaluated and compared the T cell receptor (TCR) immune repertoire of good-grade and poor-grade SAH patients to elucidate the T cell immunology after ictus. Methods : Peripheral blood from six SAH patients was collected at two different times, admission and at the 7-day follow-up. Composition and variation of the TCR β-chain (TCRB) complimentary determining regions (CDR) 3 repertoire was examined using high-throughput sequencing; the analysis was based on sampling time and disease severity (good vs. poor-grade SAH). Results : Clonality at admission and follow-up were 0.059 (0.037-0.038) and 0.027 (0.014-0.082) (median, 25th-75th percentile). Poor-grade SAH (0.025 [0.011-0.038]) was associated with significantly lower clonality than good-grade SAH (0.095 [0.079-0.101]). Poor-grade SAH patients had higher diversity scores than good-grade SAH patients. CDR length was shorter in good-grade SAH vs. poor-grade SAH. Differences in clonotype distribution were more prominent in TCRBV gene segments than TCRBJ segments. TCRBV19-01/TCRBJ02-04 and TCRBV28-01/TCRBJ02-04 were the most increased and the most decreased V-J pairs in the 7-day follow-up compared to admission in good-grade SAH. The most increased and decreased V-J pairs in poor-grade SAH patients were TCRBV28-01/TCRBJ02-06 and TCRBV30-01/TCRBJ02-04, respectively. Conclusion : The TCRB repertoire is dynamic in nature following SAH. TCRB repertoire may facilitate our understanding of adaptive immune response according to SAH severity.

• IMMUNE NETWORK
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• 제3권3호
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• pp.227-234
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• 2003

Background: Immunoglobulin (Ig) light chain repertoire has been implicated as a critical determinant in regulation of autoreactive B cells and production of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE). We analyzed the impact of Ig ${\lambda}$ chain repertoire on development of autoimmunity in patients with SLE. Methods: We obtained genomic DNA from individual peripheral CD19+ B cells of 3 untreated active SLE patients, and amplified $V{\lambda}$ rearrangements from each single cell by polymerase chain reaction. Results: A total number of 208 $V{\lambda}J{\lambda}$ rearrangements were analyzed. Analyzed sequences included 158 productive rearrangements and 50 nonproductive rearrangements. The differences in $V{\lambda}$ gene usage in the productive and nonproductive repertoire of SLE patients were found compared to the non-autoimmune individuals. $V{\lambda}$ gene, 9A was significantly overrepresented in nonproducative repertoire of SLE patients (P=0.016). In the productive repertoire, $V{\lambda}$ genes, 3L and 1E were found more often in the SLE patients (P=0.001, P=0.043). When the productive and the nonproductive repertoires were compared, 9A was found significantly less in the productive repertoire in the SLE patients (P=0.000). There were no significant differences in the $J{\lambda}$ gene usage between SLE patients and non-autoimmune individuals, but $J{\lambda}2/3$ gene was the most frequently used in SLE, whereas $J{\lambda}7$ gene was the most frequently used in the normal subjects. In the productive SLE $V{\lambda}$ repertoire, 9.4% of the total sequences employed identical CDR3. It was particularly striking to find 7 identical versions of the 1G-$J{\lambda}2/3$ $V{\lambda}J{\lambda}$ rearrangements from one patient and 3 of the same sequence from another patient. Notably, identical $V{\lambda}$ junctions in the SLE patients utilized significantly more homologous joining compared to $V{\lambda}$ junctions of the normal adults (P=0.044). Conclusion: These data demonstrate regulation of ${\lambda}$ light chain expression in the SLE patients by selection of unique $V{\lambda}$ genes. Also, biased selection and clonal expansion of particular $V{\lambda}$ rearrangements are apparent in the SLE ${\lambda}$ repertoire.

• IMMUNE NETWORK
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• 제22권6호
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• pp.50.1-50.19
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• 2022

Autoreactive B cells are not entirely deleted, but some remain as immunocompetent or anergic B cells. Although the persistence of autoreactive B cells as anergic cells has been shown in transgenic mouse models with the expression of B cell receptor (BCR) reactive to engineered self-antigen, the characterization of naturally occurring anergic B cells is important to identify them and understand their contribution to immune regulation or autoimmune diseases. We report here that a low-level expression of CD138 in the splenic B cells marks naturally arising anergic B cells, not plasma cells. The CD138^int B cells consisted of IgM^lowIgD^high follicular (FO) B cells and transitional 3 B cells in homeostatic conditions. The CD138^int FO B cells showed an anergic gene expression profile shared with that of monoclonal anergic B cells expressing engineered BCRs and the gene expression profile was different from those of plasma cells, age-associated B cells, or germinal center B cells. The anergic state of the CD138^int FO B cells was confirmed by attenuated Ca²⁺ response and failure to upregulate CD69 upon BCR engagement with anti-IgM, anti-IgD, anti-Igκ, or anti-IgG. The BCR repertoire of the CD138^int FO B cells was distinct from that of the CD138^- FO B cells and included some class-switched B cells with low-level somatic mutations. These findings demonstrate the presence of polyclonal anergic B cells in the normal mice that are characterized by low-level expression of CD138, IgM downregulation, reduced Ca²⁺ and CD69 responses upon BCR engagement, and distinct BCR repertoire.

• Clinical and Experimental Vaccine Research
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• 제13권1호
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• pp.1-9
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• 2024

Since the 1950s decade, it has been suggested that a naturally produced or induced repertoire of immunoglobulin G (IgG) idiotypes may exert some immunoregulatory functions. In the last decades, some more advanced theories have suggested that the repertoire of IgG idiotypes may influence the development or control of some atopic diseases. In atopic dermatitis (AD), some evidence indicated that the IgG repertoire obtained from these patients could effectively mediate regulatory functions on thymic and peripheral CD4+ and CD8+ T cells. Furthermore, some recent clinical trials have corroborated the hypothesis that IgG from AD patients can exert regulatory functions in vivo. Here, we revised some historical aspects that yield current approaches developed in vitro and in vivo to elucidate a recently proposed theory termed "hooks without bait" that can strengthen the broad spectrum of research about evaluating different sets of IgG idiotypes and determine their immunological effects.

• IMMUNE NETWORK
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• 제22권6호
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• pp.47.1-47.20
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• 2022

In the face of an endlessly expanding repertoire of Ags, vaccines are constantly being tested, each more effective than the last. As viruses and other pathogens evolve to become more infectious, the need for efficient and effective vaccines grows daily, which is especially obvious in an era that is still attempting to remove itself from the clutches of the severe acute respiratory syndrome coronavirus 2, the cause of coronavirus pandemic. To continue evolving alongside these pathogens, it is proving increasingly essential to consider one of the main effector cells of the immune system. As one of the chief orchestrators of the humoral immune response, the B cell and other lymphocytes are essential to not only achieving immunity, but also maintaining it, which is the vital objective of every vaccine.

• 한국수산과학회지
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• 제28권1호
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• pp.114-122
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• 1995

B 세포가 다양화되어 가는 기작을 규명한다는 것은 면역 반응의 조절이 생체 내에서 어떻게 이루어지고 있는 가를 이해하는데 가장 기본이 되는 것이다. 본 연구는 기 확립한 in situ hybridization 기법을 이용하여 항체의 항원 결합 부위 유전자가 B 세포의 발달 과정 중 어떻게 조절이 되고 있으며 이것은 B 세포의 다양화라는 측면과 어떻게 연관이 되어 있는 지를 분석하였다. Gestation 시기가 16일, 18일, 19일, 20일 되었을 때간에 있는 B 세포는 $V_H7183$와 $V_HQ52$두개의 $V_H$ 유전자군을 가장 많이 이용하고 있었으며 이러한 경향은 gestation 기간 전체를 통하여 변화 없이 일정하게 나타났다. 간에 있는 fetal B 세포를 differentiation 단계별로 구분하기 위하여 표면 항체를 갖고 있는 집단과, 갖고 있지 않은 두 집단으로 나눈 후 각 집단이 발현하는 $V_H$ 유전자를 분석하였을 때 뚜렷한 차이를 나타냄이 없이 양쪽 집단 모두 fetus의 특징적 $V_H$ 이용양식을 보여주었다. 또 다른 조혈 기능 임파 기관인 fetal spleen에 있는 B 세포 또한 fetal liver의 B 세포와 동일한 양상의 $V_H$ 유전자 이용 양식을 보여 주어 각 임파 기관별 B 세포의 다양성 차이를 발견 할 수 없었다. 이와 같이 adult의 B 세포에 대비하여 독특한 $V_H$ 유전자 이용 양상을 보이는 fetal B 세포의 전구 세포를 4주 이상 미리 형성시킨 adult 골수 세포와 직접 접촉시키면서 발달, 성숙시킨 후 다시 나타난 B 세포를 분석하여도 여전히 fetal B 세포로서의 $V_H$ 유전자 이용 양상을 보이는 것은 fetal B세포의 전구 세포가 갖고 있는 유전적 잠재력에 의한 것이지 환경이나 B 세포의 differentiation 단계 또는 B 세포가 머무르고 있는 특수 임파 장기의 생리적 환경 등에 좌우되는 것이 아니라는 것이 확인되었다.

• BMB Reports
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• 제47권5호
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• pp.241-248
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• 2014

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to ${\alpha}{\beta}$ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of ${\alpha}{\beta}$ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant $V{\alpha}14-J{\alpha}18$ TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.