Immune repertoire profiling for disease pathobiology

Abstract

Lymphocytes consist of highly heterogeneous populations, each expressing a specific cell surface receptor corresponding to a particular antigen. Lymphocytes are both the cause and regulator of various diseases, including autoimmune/allergic diseases, lifestyle diseases, neurodegenerative diseases, and cancers. Recently, immune repertoire sequencing has attracted much attention because it helps obtain global profiles of the immune receptor sequences of infiltrating T and B cells in specimens. Immune repertoire sequencing not only helps deepen our understanding of the molecular mechanisms of immune‐related pathology but also assists in discovering novel therapeutic modalities for diseases, thereby shedding colorful light on otherwise tiny monotonous cells when observed under a microscope. In this review article, we introduce and detail the background and methodology of immune repertoire sequencing and summarize recent scientific achievements in association with human diseases. Future perspectives on this genetic technique in the field of histopathological research will also be discussed.

Abbreviations

ALK

anaplastic lymphoma kinase

BCR

B cell receptor

CDR3

complementarity determining region 3

EGFR

epidermal growth factor receptor

GC

gastric cancer

GlialCAM

glial cell adhesion molecule

HLA

human leukocyte antigen

ICI

immune checkpoint inhibitor

Ig

immunoglobulin

MS

multiple sclerosis

NGS

next‐generation sequencing

PD‐1

Programmed death receptor‐1

RA

rheumatoid arthritis

RPL23A

60 S ribosomal protein L23a

SARS‐CoV‐2

severe acute respiratory syndrome coronavirus 2

SHM

somatic hypermutation

TCGA

The Cancer Genome Atlas

TCR

T cell receptor

V/(D)/J recombination

variable/(diversity)/joining recombination

INTRODUCTION

Lymphocytes are composed of T and B cell lineages and are key players in adaptive immune systems in our body. ¹ Dysregulated immunity is known to cause various diseases such as autoimmunity and allergic diseases. In contrast, proper activation of exhausted T cell immunity is utilized in the treatments of cancers in which immune systems are generally in their inhibited states. ^{2 , 3 , 4} Thus, the adaptive immunity has been gaining attention in many aspects, including the elucidation of the causes as well as the therapeutic applicability for human diseases. However, the precise molecular mechanisms of adaptive immune systems in both healthy and pathological states remain elusive, partly due to the paucity of our knowledge regarding the complex and highly diversified systems in the antigen–receptor interactions of lymphocytes.

Each lymphocyte expresses its unique cell surface receptor called immune receptor, that is, T cell receptor (TCR) in T cell, and B cell receptor (BCR) as a membrane‐bound form, and immunoglobulin (Ig) as a secreted form in B/plasma cell. ⁵ These immune receptors are genetically produced by somatic V/(D)/J recombination of the genomes in each T and B cell, consisting of higher than 10¹⁸ and 10¹³ orders of potential diversities in T and B cell compositions, respectively, in our body. ⁵ These substantially high orders of complexity in the immune receptor compositions are called “repertoires” such as TCR repertoires and BCR/Ig repertoires. Specifically, BCRs further diversify their repertoire compositions by class switch recombination and somatic hypermutations (SHMs) in the maturation and development through the immunological stimulations, resulting in a nearly infinite order of repertoire diversity. ⁵ Thus, the wide spectrum of lymphocyte repertoires plays an important role in the recognition of a wide variety of possible antigens covering, for instance, and not limited to, microorganisms, allergic materials, and cancer neo‐antigens.

In this review article, we briefly summarize the mechanisms that produce highly complex repertoire diversities in lymphocytes, and the methods by which one can obtain quantitative and high‐throughput data of immune receptor repertoire sequences using disease specimens. Finally, future perspectives on the utilization of repertoire sequence data are discussed from the perspective of histopathologists.

IMMUNE REPERTOIRE IN ADAPTIVE IMMUNITY

T and B cells acquire a great diversity of surface receptors from a limited source of the human genome sequence in order to react to every antigen in the environment. Such great diversity in the immune receptor repertoires is created by various mechanisms, including genome‐level somatic recombination of the immune receptor gene loci in each lymphocyte. ^{5 , 6 , 7} In this review, we briefly describe the mechanisms by which BCR/Ig diversity is achieved (Figure 1). BCR/Ig is composed of two heavy and light chains. ⁵ The heavy chain gene is located on chromosome 14, and kappa and lambda light chain genes are located on chromosomes 2 and 22, respectively, in the human genome. Each genetic locus consists of a diversity of gene segments called variable (V), diversity (D), and joining (J) segments, where D segments are included only in the heavy chain locus (Figure 1). During the development of B cells, somatic V/(D)/J recombination occurs where the unique V, D, and J segments are selected and recombined on the genome of each lymphocyte. Additionally, various lengths of mostly random nucleotide deletions and insertions occur during the V/(D)/J recombination processes at the boundaries between V/D and D/J in the heavy chain gene and between V/J in the light chain gene (Figure 1). These regions, including the V/(D)/J junctions with high diversity, correspond to the complementarity determining region 3 (CDR3), which is the most important region in antigen–receptor interactions ^{5 , 6 , 7} (Figure 1). At this stage, a great diversity of BCR repertoires in B cells is basically achieved. When encountered with the corresponding antigen of each BCR, further diversification of the BCR gene sequences occurs by acquiring SHMs on the BCR gene loci. ^{5 , 6 , 7} In lymphoid structures, such as germinal centers in lymph nodes, B cells that acquire higher affinity to the antigens through the SHMs are empowered with growth advantages and expand their dominances, ⁵ which is called affinity maturation. In addition, class switch recombination, such as that between IgM and IgG, also diversifies the BCR repertoires. ^{5 , 6 , 7} Eventually, the BCR/Ig repertoires acquire nearly infinite diversity in our body. In our repertoire analysis, almost no overlap of the CDR3 sequences in the BCR/Ig repertoires was observed among 102‐case gastric cancer (GC) microenvironments, confirming the great diversity of the immune repertoires. ^{8 , 9}

Figure 1.

A schematic summary of the immune receptor repertoire sequencing and its downstream analysis. The left upper panel represents schematic genetic structures and the process of somatic recombination of the immunoglobulin heavy and light chain gene loci. Unique combinations of the V/(D)/J segments are selected and recombined in individual lymphocytes, with deletions/insertions of mostly random nucleotides between the V/(D)/J junctions (black boxes). The middle panels show the representative methods for the immune receptor repertoire sequencing using NGS or publicly available data sets. The bottom panels represent the possible downstream investigations using the repertoire sequencing data. By combining histopathological examinations of the distributions of the disease‐related B and T cells and specific antigens, an in‐depth understanding of disease pathology will be achieved. CDR3, complementarity determining region 3; RNA‐ISH, RNA in situ hybridization; IHC, immunohistochemistry; CAR‐T, chimeric antigen receptor T cell; and COVID‐19, coronavirus disease 2019. For more details, refer to Janeway's Immunobiology (9th edition, 2016). ⁵ This figure was created with BioRender.com.

METHODS IN IMMUNE REPERTOIRE PROFILING

With advancements in the next‐generation sequencing (NGS) technologies, it is now feasible to obtain immune receptor repertoire data with great diversity. The representative methodologies are summarized in Figure 1 and Table 1. Basically, recent repertoire sequencing methods can obtain data of all known isotypes of TCRs and BCRs/Ig, including, IgD, IgM, IgA, IgE, IgG, κ, λ, TCRα, TCRβ, TCRγ, and TCRδ. There are three major approaches to obtain repertoire sequencing data. One is repertoire sequencing using bulk tissue samples, wherein the purified total RNA is subjected to multiplex PCR using multiplex primers designed for all possible V and C segments of the immune receptor genes. ^{8 , 10 , 11 , 12} By performing NGS on the amplicons, more than 10⁵–10⁶ heavy/light and alpha/beta chain sequences for B and T cells, respectively, can theoretically be obtained for each sample. Another method is single‐cell repertoire sequencing, wherein the isolated single cells of B/plasma cells and T cells are subjected to single‐cell transcriptome sequencing via enrichment PCR using specific primers for immune receptor genes. ^{13 , 14} Currently, repertoire sequences of at the most 10⁴ single cells can be acquired by a single experiment per sample. The advantage of bulk sequencing is that substantially deeper repertoire information can be obtained as compared to single‐cell sequencing; however, the repertoire sequences of heavy/light chains of BCR/Ig and alpha/beta chains of TCR are obtained separately, and it is generally not possible to reconstruct heavy/light and alpha/beta chain combinations from the data. In previous studies, only top‐ranked dominant BCR clones in each examined tissue could successfully be reconstructed as Ig molecules. ^{8 , 9 , 10} On the other hand, by single‐cell repertoire sequencing, the proper combinations of heavy/light and alpha/beta chains of BCR/Ig and TCR can be defined at single‐cell resolution. Thus, more diverse repertoires can be reconstructed as immunoglobulins as described later, and biochemical and histopathological examinations can be performed. The other methods of repertoire sequencing uses bioinformatics analytical pipelines where the repertoire sequence data are reassembled from public NGS databases of bulk RNA‐sequencing and single‐cell sequencing ^{15 , 16} (Figure 1 and Table 1). In these bioinformatics methods, data mining of the candidate sequence reads for the immune receptor repertories from the whole NGS data sets is initially performed. Such sequence reads are reassembled and aligned onto possible repertoire sequences. ^{15 , 16} It is now theoretically possible to obtain the immune receptor repertoire data without conducting NGS experiments but using publicly available databases.

Table 1.

Representative methods to obtain immune receptor repertoire data

	Methods	Advantages	Disadvantages	URL
Amplicon sequence of heavy/light chains (BCR/Ig), and alpha/beta chains (TCR)
iRepertoire	Multiplexed PCR amplification of mRNA or gDNA of heavy/light chains (BCR/Ig) and alpha/beta chains (TCR)	Deeper acquisition of data (105~106 repertoires)	Combinations of heavy/light (BCR/Ig) and alpha/beta (TCR) chains are uninformative, except for dominant clones.	https://irepertoire.com/
Adaptive biotechnologies				https://www.adaptivebiotech.com/
Repertoire genesis		Frozen tissue archives can be used	SHMs in the 5' V regions may not be obtained.	https://www.repertoire.co.jp/
Single‐cell repertoire sequence
10X Genomics, single cell V(D)J regent	Single‐cell sequencing combined with enrichment PCR of immune repertoire genes	Accurate combinations of heavy/light (BCR/Ig) and alpha/beta (TCR) chains are defined for all acquired cells	Up to 104 cells per sample can be analyzed (at present date)	https://www.10xgenomics.com/
iRepertoire, iPair				https://irepertoire.com/
Re‐assemble of the repertoire data from bulk and single‐cell RNA‐seq data
TRUST4	Bioinformatics for public bulk and single‐cell RNA‐seq data to reconstruct BCR/TCR repertoires	No need to acquire new data sets of NGS	Combinations of heavy/light (BCR/Ig) and alpha/beta (TCR) chains are uninformative, except for dominant clones.	https://github.com/liulab-dfci/TRUST4

Bioinformatics pipelines used in the analyses of the repertoire sequencing data are summarized in Table 2. ^{16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27} After the initial quality control and adaptor trimming of the NGS data, the obtained NGS reads are aligned on the immune receptor gene segments, and the corresponding V/(D)/J and C segments are assigned. Thus, the transcript and amino acid sequences of BCR/Ig and TCR repertoires are defined. Further repertoire analyses such as clonotype identification (i.e., the classification of the CDR3 sequences) and diversity analysis are performed to interpret the repertoire compositions, and, specifically for the BCR/Ig repertories, SHM profiling and phylogenetic inference analysis are also performed to acquire global views of the affinity maturation processes of BCR/Ig clones. Useful public databases have been constructed for repertoire sequencing data, ^{28 , 29 , 30 , 31 , 32} which include global BCR/TCR repertoire data sets of humans and mice, some containing TCR repertoires combined with information on defined antigens. Information on the publicly shared clonotypes (CDR3 sequences) of the immune repertoires can help obtain the baseline compositions of the immune repertoires in the general populations and identify disease‐specific repertoires from originally obtained NGS data sets in each laboratory.

Table 2.

Analytical tools for the immune repertoire sequencing

Software & Database	Aim	Reference
Repertoire data analysis tool
Fastp	general‐purpose FASTQ preprocessing	[17]
IMGT/HighV‐QUEST	V(D)J assignment for TCR/BCR	[18]
		(https://www.imgt.org/HighV-QUEST)
IgBLAST		[27]
		(https://www.ncbi.nlm.nih.gov/igblast/)
Cell Ranger V(D)J	V(D)J assignment and clonotype identification for single‐cell TCR/BCR	https://support.10xgenomics.com/single-cell-vdj/software
MiXCR	V(D)J assignment and clonotype identification for TCR/BCR	[19]
IGoR		[20]
GLIPH2	find TCR clusters predicted to bind the same MHC‐restricted peptides	[21]
Immcantation	TCR/BCR repertoire data analysis including read assembly, clonotype identification, diversity analysis, and mutation profiling	[22, 23]
		(https://immcantation.readthedocs.io)
IgPhyML	Phylogenetic inference for BCR	[24]
TRUST4	TCR/BCR reconstruction from bulk and single‐cell RNA‐seq data	[16]
BASIC	TCR/BCR reconstruction from single‐cell RNA‐seq data	[25]
TraCeR/BraCeR		[26]
Public data set of repertoire data
iReceptor	bulk BCR/TCR repertoire sequence data (human and mouse)	[28]
		(http://ireceptor.irmacs.sfu.ca/)
cAB‐rep	bulk BCR repertoire sequence data (human)	[29]
		(https://cab-rep.c2b2.columbia.edu)
TCR3d	TCR repertoire sequence with experimentaly determined 3D structure	[30]
		(https://tcr3d.ibbr.umd.edu)
huARdb	single cell TCR/BCR repertoire data (human)	[31]
		(https://huarc.net/database)
VDJdb	curated database of TCR repertoires with antigen specificities	[32]
		(https://vdjdb.cdr3.net/)
Structural simulation of BCR/TCR repertoires
RepertoireBuilder	fast/high‐throughput, less accurate for the analysis of H‐CDR3	[33]
		(https://sysimm.org/rep_builder/)
ABodyBuilder		[34]
		(http://opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/abodybuilder/)
LYRA		[35]
		(https://services.healthtech.dtu.dk/service.php?LYRA-1.0)
Rosetta Antibody	slow/low throughput, more accurate for the analysis of H‐CDR3	[36]
		(http://rosie.rosettacommons.org/)

Abbreviations: BCR, B cell receptor; TCR, T cell receptor.

IDENTIFICATION OF CORRESPONDING ANTIGENS FOR BCR/IG AND TCR REPERTOIRES

Identification of the corresponding antigen for a given BCR/Ig clone is important for understanding the function of specific B cell immunity and clarifying the molecular background of the disease pathology (Figure 1). Once the dominant Ig clones in diseased tissues and/or disease‐related Ig clones are identified by repertoire sequencing, it is feasible to identify their corresponding antigens by biochemical experiments. ^{8 , 9 , 10 , 37} Identified Ig clone sequences of heavy and light chains can be constructed directly in expression plasmids. Subsequently, these heavy and light chain plasmids can be co‐transfected into mammalian expression systems such as HEK293 and CHO cells. Usually, IgG1/kappa isoform is used in the reconstruction of discovered antibodies for the purpose of experimental usages. Reconstructed human IgG antibodies are purified from the culture supernatant of the transfected cells. To explore the corresponding antigens, the purified IgG antibody can be used in various experiments, such as immunoprecipitation followed by mass spectrometry and array hybridization formats consisting of human proteins, carbohydrates, microorganisms, and allergic materials ^{8 , 9 , 10} (Figure 1). For immunoprecipitation experiments, various protein lysates are used to explore protein antigens, such as cell lysates of culture cell lines and tissue lysates of human disease tissues. Structural simulation of the variable regions of the reconstructed antibodies would help decipher the molecular dynamics of the antibody‐antigen interactions ^{33 , 34 , 35 , 36} (Table 2). Experiments with which histopathologists are more familiar are also candidate approaches for identifying the corresponding antigens of reconstructed antibodies. For instance, immunohistochemistry of human disease tissues using the reconstructed antibodies will help identify possible cellular or stromal components recognized by the antibodies, which will be a scientific clue to hypothesize the targets of the antibodies and, thus, the disease pathology.

Although challenging, the corresponding antigens of disease‐related TCR repertoires can be identified. Since the TCR‐antigen recognition machinery requires human leukocyte antigen (HLA)‐mediated antigen presentation, ³⁸ the exploration of the corresponding antigen for a given TCR is technically more challenging than that for BCR/Ig. To discover the corresponding antigens complexed with HLA without hypothesized antigen targets, it is usually necessary to construct an HLA‐specific peptide‐HLA library in the first step. ³⁸ The reconstructed TCRs in their solubilized form are used to precipitate TCR‐antigen‐HLA complexes. There are also other methods that do not use a peptide‐HLA library but use specifically constructed reporter T cell lines or engineered mouse models, ^{38 , 39} to identify candidate antigens. However, more complex and specialized experimental conditions are required.

Through repertoire sequencing and the subsequent experimental exploration of disease‐related antigens, it is feasible to find unidentified physiology and pathology exerted by adaptive immunity, thus further deepening our understanding of disease mechanisms.

HISTOPATHOLOGICAL EVALUATION OF IMMUNE REPERTOIRE

Once disease‐related immune receptor repertoires and their corresponding antigens are discovered, histopathological examination using human disease specimens is an important strategy to clarify the disease pathology (Figure 1). Investigating the distribution of antigens by immunohistochemistry can help identify possible disease‐causing and/or disease‐regulatory mechanisms of immune‐related human pathology. Clarifying the cellular and stromal components by which the antigens are expressed is key to deepening our understanding of the mechanisms of the diseases. Second, RNA in situ hybridization targeting specific CDR3 sequences of interest can be used to investigate the distribution of specific B/plasma cell clones in human histopathological specimens. ¹⁰ This experiment can help obtain the spatial information of disease‐related B/plasma cells in relation to the histological features of the diseases. Recently, repertoire sequencing combined with spatial genomics approach has enabled the high‐resolution spatial analysis of the immune repertoires in histopathological tissues. ⁴⁰ Histopathologically combined information on the localization of both the disease‐related antigens and B/plasma cells would help decipher the larger picture of the pathobiology of various diseases.

RECENT RESEARCH ACHIEVEMENTS OF IMMUNE REPERTOIRE SEQUENCING

Immune repertoire sequencing has been applied to many scientific fields recently and paved the way to understand varieties of disease pathobiology. Selected representative recent achievements of this methodology are summarized in Table 3 and introduced below. ^{8 , 9 , 10 , 13 , 37 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48}

Table 3.

Selected representative achievements of repertoire sequencing studies

Disease category	Materials	Method	Findings	Reference
BCR reoertoire
Cancer	tumor tissues	bulk repertoire sequencing of 30 gastric cancers	sulfated glycosaminoglycans are the major and functional humoral tumor antigens	[8]
		bulk repertoire sequencing of 102 gastric cancers	anti‐sulfated glycosaminoglycan Ig clones exhibit low pH‐selective reactivity to cancer cells	[10]
			proteins in focal adhesion RNP complex are frequent protein antigens in tumor environments	[9]
		reconstruction of IgGs from TCGA data set of 1945 solid tumors	en masse identification of IgGs with either broad or tumor‐specific reactivity	[41]
		single cell repertoire sequencing of four ovarian cancers	intratumor antibody‐secreting cells produce tumor‐reactive anti‐MMP14 antibodies	[37]
		10X‐based spatial transcriptome sequencing of two renal cancers	tumor‐associated tertiary lymphoid structures generate anti‐tumor antibodies	[40]
autoimmune disease	B cells in central nervous system and secondary lymphoid tissues	bulk repertoire sequencing for 5 multiple sclerosis patients	the majority of MS‐associated B cells mature outside of CNS in peripheral lymphoid tissues	[44]
	B cells in peripheral blood and cerebrospinal fluid	single cell repertoire sequencing for nine multiple sclerosis patients	clonally expanded B cells in MS are dual‐reactive to EBNA1 and GlialCAM	[45]
COVID‐19	peripheral blood	single cell repertoire sequencing of peripheral blood plasma cells for 16 patients	discovering SARS‐CoV‐2 reactive neutralizing antibodies	[13]
TCR repertoire
Cancer	tumor tissues	re‐assemble of repertoire data from bulk sequencing data of TCGA	the diversity of TCR of tumor‐infiltrating T cell is prognostic in various cancers	[46]
	tumor tissues and peripheral blood	bulk repertoire sequencing of eight patients who underwent anti‐PD‐1 therapy	blood‐ tumor TCR repertoire overlap is predictable for better clinical response to PD‐ 1 blockade	[47]
		bulk repertoire sequencing of 11 patients who underwent anti‐CD4 antibody treatment	beneficial remodeling of TCR repertoire could be achieved following CD4 + cell depletion	[48]
autoimmune disease	CD4 + T cells from arthritic joints of mouse model of rheumatoid arthritis	bulk repertoire sequencing	RPL23A is an autoantigen in RA	[39]
COVID‐19	peripheral blood	bulk repertoire sequencing of 37 COVID‐19 patients	SARS‐CoV‐2‐specific T cell responses were driven by TCR clusters shared between patients	[42]

Abbreviations: BCR, B cell receptor; TCR, T cell receptor.

IDENTIFICATION OF DISEASE‐RELATED ANTIGENS AND THEIR CLINICAL APPLICABILITY

The identification of humoral antigens in tumor microenvironments will help reveal the unknown aspects of tumor‐related humoral immunological reactions and may lead to the discovery of potential therapeutic human antibodies. Zhang P et al. ⁴¹ demonstrated that a series of reconstructed human IgGs, which were based on the reassembled BCR repertoires from the public TCGA data set of 1945 solid tumors, exhibited either broad or tumor‐specific reactivities. This study indicates that the investigation of BCR repertoires of tumor‐infiltrating B/plasma cells enables efficient en masse identification of anti‐tumor human antibodies. Our study of immune repertoire sequencing of tumor‐infiltrating B/plasma cells in 30 GCs found that the BCR/Ig repertoires showed more clonal compositions than those in the normal counterpart stomach tissues and identified tens of GC‐specific and dominant Ig clones. ⁸ Biochemical investigation surprisingly revealed that many of the identified Igs commonly reacted to sulfated glycosaminoglycans. ⁸ Further analysis clarified that such anti‐sulfated glycosaminoglycan Ig clones surprisingly exhibited low pH‐selective reactivity to human cancer cells, and an antibody‐drug conjugate of the Ig clone exerted strong anti‐tumor effects against multiple types of malignancies in vivo. ¹⁰ In addition, our repertoire sequencing analysis of 102‐case GCs revealed that ribonucleoproteins in the focal adhesion complex are major and frequent protein antigens of humoral immunity in GC microenvironments. ⁹ A recent BCR repertoire sequencing study of ovarian cancers identified MMP14 as a humoral tumor antigen, demonstrating the therapeutic applicability of the discovered human antibodies for the treatment of ovarian cancers. ³⁷

These data‐driven and hypothesis‐free discoveries of tumor‐related and potential therapeutic human antibodies do not require the laborious and cost‐consuming processes of pharmaceutical antibody development, such as the discovery of target proteins, immunization of animals, hybridoma selection, and humanization of seed antibodies. Thus, characterization of the Ig repertoires of tumor‐infiltrating B/plasma cells is a rapid and highly successful strategy to discover monoclonal fully human antibodies that can be used for the diagnosis and/or treatment of human malignancies. These antibodies are also directly applicable to therapeutic antibody development, such as chimeric antigen receptor‐T cells and antibody‐drug conjugates for cancer treatments ^{10 , 49 , 50} (Figure 1).

Autoantibody production and dysregulated T cell immunity are the causes of autoimmune diseases. Through repertoire sequencing analysis of the diseased tissues and fluid samples of autoimmune disease patients and mouse models, novel disease‐related autoantigens have been identified. TCR repertoire sequencing of a genetically engineered rheumatoid arthritis (RA) mouse model identified a novel autoantigen, 60 S ribosomal protein L23a (RPL23A), which was also confirmed in human RA patients. ³⁹ A recent study revealed that oligoclonal autoantibodies found via BCR repertoire sequencing in multiple sclerosis (MS) exhibited cross‐reactivity to both EBNA1, an Epstein–Barr virus protein, and human glial cell adhesion molecule (GlialCAM); moreover, EBNA1 immunization was found to exacerbate the disease status in a mouse model of MS. ⁴⁵ Identifying the accurate sequences of self‐reactive TCRs and BCRs/Igs via repertoire sequencing enables the reconstructions of such immune receptors and thus, the discovery of the corresponding autoantigens. This deepens our understanding of the molecular pathogenesis of diseases.

RELATIONSHIPS BETWEEN IMMUNE REPERTOIRES AND CLINICAL OUTCOMES OF PATIENTS

Apart from the identification of specific antigens of disease‐related immune repertoires, immune repertoire data have another clinically important aspect. Based on repertoire sequencing, it is possible to calculate the diversity score of repertoire compositions in each tissue analyzed. For instance, repertoire entropy is a widely used value that represents the global diversity of immune repertoire composition. Higher clonality (lower entropy) of the BCR/Ig repertoire indicates that a small subset of specific Ig clones expands in the analyzed tissues, and lower clonality (higher entropy) indicates that more diversified lymphocytes exist in the analyzed specimens.

In recent studies, it has been revealed that such repertoire diversities are correlated with various clinical outcomes of the patients. ^{46 , 51} For example, our group found that the lower entropy of the BCR/Ig repertoires in GC microenvironments is an indicator of better prognosis in patients with advanced stages of GC but not in patients with early stage GC. ⁹ In skin cutaneous melanoma, increased clonality of BCR repertoires indicated favorable prognosis. ⁵² On the other hand, among the TCGA data sets, it was indicated that higher diversity of tumor‐infiltrating T cell repertoires commonly indicated better prognoses in patients with a wide spectrum of cancers. ⁴⁶

The clinical efficacy of cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been reported to be correlated with the repertoire profiles of patients. It has been shown that more clonal intratumor TCR repertoires and a higher clonality of the peripheral blood CD8 + T cell repertoire were indicative of a better response to PD‐1 blockade therapy in melanoma. ^{53 , 54 , 55} In EGFR/ALK wild‐type non‐small cell lung cancers, responders to the PD‐1 blockade showed lower diversities of peripheral blood TCR and BCR repertoires than non‐responders; moreover, the lower peripheral blood TCR diversity corresponded to better progression‐free survival of the patients. ⁵⁶ The in‐depth correlations between the repertoire profiles and the clinical outcomes of ICI therapies are worth studying with larger cohorts of a variety of malignancies; this would help establish robust prediction markers of cancer immunotherapy. Interestingly, in patients who underwent PD‐1 blockade treatments, a greater overlap of the TCR repertoires between peripheral blood and tumor microenvironments has been reported to be associated with a better response to PD‐1 blockade therapy. ^{43 , 47} Further studies on the systemic distribution patterns of the immune repertoires are warranted to deepen our understanding of the consequences of cancer immunotherapies.

CONTRIBUTION OF THE IMMUNE REPERTOIRE SEQUENCING IN THE FIGHT AGAINST SARS‐COV‐2

The repertoire sequencing technique plays an important role in both research and clinical fields of SARS‐CoV‐2. Peripheral blood BCR repertoire sequencing substantially helped develop highly potent neutralizing antibodies to the receptor‐binding domain of SARS‐CoV‐2. ^{13 , 57} In addition, investigation of both the peripheral blood BCR and TCR repertoires has contributed to the clarification of the immunological kinetics of COVID‐19 patients in relation to the severity and clinical course of the disease. ^{42 , 58 , 59 , 60 , 61 , 62}

It is noteworthy that these studies were published as early as the middle of 2020, and the clinical trials of the engineered neutralizing antibodies also started around the same time, demonstrating the rapid and high‐throughput applicability of the repertoire sequencing method. Humans, with the state‐of‐the‐art technique of repertoire sequencing, have been empowered with efficient tools to fight against life‐threatening infectious diseases, which have repeatedly caused devastating pandemics in the past centuries.

SUMMARY

Immune receptor repertoire profiling is a powerful tool that can be used in clinical investigations as well as for basic science. Using this genetic approach, the overall profile of lymphocyte biology can be obtained from various perspectives. With the aid of histopathological investigations that focus on the spatial distributions of disease‐related antigens and specific B and T cell clones in human disease specimens, more light will be shed on the pathology of various human diseases. Through the accumulation of TCR/BCR repertoire data from more diverse spectra of patients in future studies, along with the defined catalog of the corresponding antigens of disease‐related repertoires, it will be possible to depict the molecular mechanisms of disease pathology with greater precision and develop novel clinical modalities for the diagnosis and treatment of various diseases.

CONFLICT OF INTERESTS

None declared.

Katoh H, Komura D, Furuya G, Ishikawa S. Immune repertoire profiling for disease pathobiology. Pathol Int. 2023;73:1–11. 10.1111/pin.13284