T Cell Repertoire: Diversity, Disease, and Lab Methods

T cells play a central role in adaptive immunity, recognizing and responding to pathogens through a highly diverse set of T cell receptors (TCRs). This diversity allows the immune system to detect a vast range of antigens while maintaining tolerance to self. Disruptions in this repertoire contribute to autoimmune diseases, infections, and cancer.

Understanding how T cell diversity is generated, maintained, and assessed provides critical insights into immune function and disease.

TCR Gene Rearrangement

T cell receptor (TCR) diversity arises from TCR gene rearrangement, an orchestrated process in developing T cells within the thymus. This enables the formation of unique TCRs capable of recognizing a broad spectrum of antigens. The rearrangement occurs through somatic recombination of TCR gene segments—variable (V), diversity (D), and joining (J) segments for the TCRβ chain, and V and J segments for the TCRα chain. Recombination-activating genes RAG1 and RAG2 mediate this process by introducing double-strand breaks at recombination signal sequences. The non-homologous end joining (NHEJ) repair pathway then processes and ligates the DNA, forming a functional TCR gene.

Additional sequence variability arises during recombination through nucleotide addition and deletion. Terminal deoxynucleotidyl transferase (TdT) randomly adds non-templated nucleotides at the junctions, while exonucleases trim nucleotides, further increasing diversity. This junctional diversity generates an enormous number of possible TCR sequences, far exceeding the variation from V(D)J recombination alone.

Recombination is tightly regulated to ensure functional TCR expression. The β-chain rearranges first, pairing with a surrogate α-chain to form the pre-TCR complex. This complex signals proliferation and α-chain rearrangement. If a productive β-chain fails to form, the cell attempts rearrangement on the second allele before undergoing apoptosis. The α-chain has multiple rearrangement opportunities due to the presence of numerous V and J segments. This stepwise process ensures that only T cells with functional TCRs progress in development.

Mechanisms of Repertoire Diversity

Several molecular mechanisms shape TCR repertoire variability, ensuring broad antigen recognition. While gene rearrangement establishes foundational diversity, additional modifications refine and expand the repertoire.

Junctional diversity significantly increases the number of unique TCRs beyond combinatorial gene rearrangement. TdT adds non-templated nucleotides at V(D)J junctions, generating novel sequences, while exonucleases trim nucleotides, further altering junctional composition. These modifications result in an estimated 10^15 distinct TCR sequences. However, many junctional modifications create nonfunctional TCRs, leading to a high rate of nonproductive rearrangements. Despite this inefficiency, the sheer number of possible TCR combinations ensures adaptability.

Allelic exclusion and receptor editing further shape TCR diversity. Once a productive TCRβ rearrangement occurs, recombination halts to prevent additional β-chain rearrangements, ensuring each T cell expresses a single functional β-chain. In contrast, TCRα rearrangement remains flexible, allowing multiple attempts on both alleles, increasing the likelihood of generating a functional α-chain. Unlike immunoglobulin genes in B cells, TCR genes do not undergo somatic hypermutation, meaning diversity is established during thymic development rather than antigen-driven selection in peripheral tissues.

Role of Thymic Selection

In the thymus, T cells undergo selection processes ensuring functional yet self-tolerant TCRs. This occurs in two stages—positive and negative selection—each shaping the repertoire.

Positive selection occurs in the thymic cortex, where immature T cells interact with cortical thymic epithelial cells (cTECs) presenting self-peptides on major histocompatibility complex (MHC) molecules. Thymocytes that fail to recognize MHC undergo apoptosis, ensuring only those capable of engaging with MHC receive survival signals. The strength of these interactions influences lineage commitment, with MHC class I recognition leading to CD8+ differentiation and MHC class II recognition leading to CD4+ differentiation.

Negative selection occurs in the thymic medulla, eliminating autoreactive T cells. Medullary thymic epithelial cells (mTECs) and dendritic cells present self-antigens, facilitated by the autoimmune regulator (AIRE) protein. Thymocytes that bind too strongly to self-antigens receive apoptotic signals, preventing the release of autoreactive T cells. While negative selection is highly effective, some self-reactive T cells escape deletion, but regulatory mechanisms, such as regulatory T cells (Tregs), help maintain self-tolerance.

Phenotypic Variations in Different T Cell Subsets

T cell subsets exhibit distinct phenotypic characteristics reflecting their functional roles. CD4+ and CD8+ T cells, the two primary subsets, further differentiate into specialized populations contributing to immune regulation and response.

CD4+ T cells differentiate into distinct subsets based on transcription factors and cytokine secretion. T helper 1 (Th1) cells, driven by T-bet, produce interferon-gamma (IFN-γ) and aid in intracellular pathogen clearance. Th2 cells, regulated by GATA3, secrete interleukin-4 (IL-4) and support responses against extracellular parasites. Th17 cells, governed by RORγt, produce IL-17 and play a role in mucosal immunity and inflammatory disorders. Regulatory T cells (Tregs), identified by FOXP3 expression, suppress excessive immune activation and maintain tolerance.

CD8+ T cells also exhibit phenotypic diversity. Naïve CD8+ T cells express CD62L and CCR7, enabling circulation through secondary lymphoid organs. Upon activation, they differentiate into effector cells, upregulating CD44 and granzyme B, reflecting cytotoxic capabilities. Memory CD8+ T cells further diversify into central memory (TCM) and effector memory (TEM) subsets. TCM cells retain CCR7, enabling lymphoid homing, while TEM cells lack CCR7 but express integrins facilitating migration to peripheral tissues. These adaptations optimize rapid responses upon antigen re-encounter.

Repertoire Changes in Disease States

TCR repertoire composition undergoes significant alterations in various diseases, reflecting immune system adaptations. Changes include reduced diversity, expansion of specific clonotypes, or shifts in T cell subsets. Infections, autoimmune disorders, and malignancies imprint distinct signatures on the TCR landscape.

Chronic infections drive selective expansion of pathogen-specific T cell clones. This is evident in viral infections such as HIV and hepatitis C, where dominant clonotypes narrow repertoire diversity. While this focused response enhances pathogen control, it reduces immunological flexibility. Autoimmune diseases like multiple sclerosis and rheumatoid arthritis show aberrant expansion of self-reactive T cells, often with reduced regulatory T cell function. Patients with these conditions frequently exhibit restricted TCR repertoires in affected tissues, suggesting specific autoreactive clones drive pathology.

In malignancies, tumor-infiltrating lymphocytes (TILs) often display antigen-driven selection but exhibit exhaustion, marked by diminished proliferative capacity and altered cytokine production. Identifying these repertoire changes has guided immunotherapies, such as checkpoint inhibitors and adoptive T cell therapies, which aim to restore functional diversity and enhance anti-tumor immunity.

Current Laboratory Methods for Assessment

Advanced laboratory techniques enable precise characterization of TCR diversity, clonality, and function. These methods have evolved significantly, allowing high-throughput analysis at both bulk and single-cell levels.

High-throughput sequencing, particularly next-generation sequencing (NGS) of TCR repertoires, has revolutionized the field. This approach amplifies and sequences TCRβ or TCRα chains from bulk T cell populations, revealing clonotype frequencies and repertoire breadth. Analyzing complementarity-determining region 3 (CDR3) sequences—the most variable TCR region—allows tracking of clonotypic expansions and immune responses. Single-cell RNA sequencing (scRNA-seq) enhances this by linking TCR sequences to transcriptional profiles, providing insights into individual T cell function. These methods have identified TCR signatures associated with infections, autoimmunity, and cancer, informing targeted immunotherapies.

Flow cytometry and TCR-specific tetramer staining remain critical for functional characterization. Fluorescently labeled MHC-peptide tetramers identify antigen-specific T cells within complex populations, aiding infection and vaccination studies. Single-cell analysis techniques, such as TCR-dependent activation assays, assess T cell reactivity by measuring cytokine production, proliferation, and cytotoxic activity. Integrating these methodologies has significantly advanced TCR repertoire analysis, paving the way for precision immunology in research and clinical applications.