I. Applications of Single-Cell Immune Repertoire Technologies and Highlighted Case Studies

Single-cell immune repertoire technologies have been widely adopted across diverse areas of immunological research:

Tumor Immunology

Characterizing clonal expansion, exhaustion signatures, and functional reprogramming of tumor-infiltrating T cells during cancer immunotherapy, including checkpoint blockade and adoptive cell transfer.

Case Study 1: Clonal Dynamics in Cancer Immunotherapy

Clonal analysis of tumor-infiltrating T cells in lung cancer patients undergoing immunotherapy revealed that expanded Th1 clones were significantly enriched in treatment responders compared to non-responders. Integration with published basal cell carcinoma datasets further validated the critical role of clonally expanded Th1 cells in mediating responses to immune checkpoint blockade across cancer types¹.

Infection and Vaccine Studies

Investigating the formation, clonal selection, and long-term maintenance of antigen-specific T and B cell responses following infection or vaccination.

Case Study 2: Multi-omics Immune Profiling in Omicron Breakthrough Infection

Integrated multi-omics profiling of blood samples from SARS-CoV-2 Omicron patients revealed dynamic immune and platelet responses across disease stages. Single-cell and immune repertoire analyses identified enhanced interferon-driven platelet activity and extensive platelet–leukocyte interactions that modulate immune function. Notably, re-positive patients exhibited reduced B cell receptor clonality, impaired antibody production, and decreased neutralizing capacity. These findings highlight the power of multi-omics and single-cell TCR/BCR sequencing to characterize immune dysregulation and predict disease outcomes in viral infections².

Autoimmune Diseases

Identifying pathogenic clonally expanded populations and dissecting their functional states to elucidate disease mechanisms and potential therapeutic targets.

Case Study 3: B Cell–Mediated Tolerance in Neuromyelitis Optica

Analysis of immune tolerance mechanisms revealed that B cells can intrinsically express and present the autoantigen AQP4 upon CD40 activation, enabling the deletion of AQP4-specific T cell clones in the thymus. Thymic B cells were shown to play a critical role in shaping the TCR repertoire by mediating central tolerance, and loss of AQP4 expression in B cells led to the escape of autoreactive T cells and enhanced autoantibody production. These findings highlight a noncanonical role of B cells in immune tolerance and provide a framework for studying autoreactive clonal selection using single-cell TCR/BCR sequencing³.

Immune Cell Therapy Research

Enabling high-resolution clonal tracking and functional profiling in TCR-engineered T cell (TCR-T) and chimeric antigen receptor T cell (CAR-T) therapies to assess clonal dominance, persistence, and therapeutic efficacy.

Case Study 4: Targeting Pro-inflammatory T Cells in Atherosclerosis

Single-cell multi-omics analysis of atherosclerotic plaques identified a population of activated, pro-inflammatory PD-1⁺ T cells contributing to disease progression. Clinical cohort studies revealed that anti–PD-1 monoclonal antibodies with FcγR-binding capability significantly reduced plaque size by interacting with myeloid Fcγ receptors and functionally suppressing PD-1⁺ T cells in ligand-deficient environments. These findings highlight a novel immunomodulatory mechanism and support T cell–targeted therapies as a potential strategy for resolving atherosclerosis, which can be further dissected using single-cell TCR profiling to track pathogenic clonal populations⁴.

II. Technical Challenges and Limitations

Despite its transformative capabilities, single-cell TCR sequencing faces several technical and analytical challenges:

3. Lack of Standardized Clonotype Definitions

Variability in clonotype calling criteria (e.g., CDR3 sequence identity thresholds, gene usage requirements) across studies hinders cross-study comparisons and meta-analyses.

4. Unknown Antigen Specificity

TCR sequences alone do not reveal antigen specificity, requiring orthogonal functional validation approaches such as peptide-MHC multimer staining or high-throughput screening.

5. Cost and Scalability Constraints

Single-cell methods remain significantly more expensive and lower throughput compared to bulk repertoire sequencing, limiting their application in large-scale population studies.

III. Future Directions and Emerging Technologies

Ongoing developments are expanding the scope and impact of single-cell immune repertoire profiling:

1. Multi-Omics Integration

Coupling immune repertoire data with epigenomic profiling (e.g., ATAC-seq, DNA methylation), surface proteomics (e.g., CITE-seq), and spatial transcriptomics to achieve comprehensive, spatially resolved characterization of clonal states.

2. Machine Learning–Based Antigen Specificity Prediction

Developing computational models that leverage TCR/BCR sequence features and structural information to predict antigen specificity, reducing reliance on experimental validation and enabling large-scale epitope mapping.

Reference

1. Liu, B., Hu, X., Feng, K. et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nat Cancer 3, 108–121 (2022).
2. Wang H, Liu C, Xie X., et al. Multi-omics blood atlas reveals unique features of immune and platelet responses to SARS-CoV-2 Omicron breakthrough infection. Immunity, 2023; 56, 1410-1428.e8
3. Afzali, A.M., Nirschl, L., Sie, C. et al. B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4. Nature 627, 407–415 (2024).
4. Fan, L., Liu, J., Hu, W. et al. Targeting pro-inflammatory T cells as a novel therapeutic approach to potentially resolve atherosclerosis in humans. Cell Res 34, 407–427 (2024).