Integrated Antigen Discovery and TCR-pMHC Interaction Validation

Why Connect Antigen Discovery with T Cell Response Analysis?

In modern cellular immunity research, no single technology fully explains antigen-specific T cell responses. While advanced mass spectrometry can identify thousands of naturally presented peptides on the cell surface, it provides no information about which of these peptides actually trigger a T cell response. Conversely, T cell receptor (TCR) sequencing expertly profiles clonal expansion and repertoire diversity, but a TCR sequence alone does not directly reveal its target antigen identity.

To build a stronger case for antigen-specific T cell immunity, researchers need an integrated workflow that connects antigen presentation, clonotype dynamics, and downstream validation. This integrated approach reduces reliance on purely predictive assumptions and helps prioritize biologically relevant candidates for downstream development.

Immunopeptidomics for Naturally Presented Antigen Discovery

The foundation of the workflow is our immunopeptidomics service, which directly sequences the peptides physically eluted from HLA molecules. By utilizing HLA peptidomics analysis, we provide direct, physical proof of which antigens are naturally processed and presented on the cell surface. To accommodate diverse sample types and research scales, we utilize multiple state-of-the-art mass spectrometry platforms.

Antigen Discovery Mass Spectrometry Platforms

TCR Sequencing for Immune Repertoire Profiling and Clonotype Tracking

To understand the immune effector side, TCR sequencing profiles the diversity and expansion of T cells. We offer flexible options supporting both bulk TCR repertoire profiling (profiling alpha and beta chains across a population) and single-cell immune profiling (scTCR-seq combined with scRNA-seq). This tracks which specific T cell clones are expanding in response to a disease state, vaccination, or immunotherapy treatment.

TCR-pMHC Interaction Validation via SPR and BLI Binding Assays

Identifying a peptide and a reactive T cell clone is powerful, but confirming their exact physical interaction requires downstream validation. We rely primarily on Surface Plasmon Resonance (SPR) to deliver high-precision kinetic data (K_D, k_on, k_off) for highly selected "lead" TCR-pMHC pairs. While SPR is our gold standard for absolute kinetic quantification, we can also supplement with Bio-Layer Interferometry (BLI) for broader immunopeptide affinity screening.

Because TCR-pMHC interactions are often weak and technically challenging, SPR/BLI is typically reserved for highly prioritized candidates with suitable reagent quality and assay feasibility. Where appropriate, orthogonal validation strategies such as peptide-HLA stabilization assays, tetramer staining, or cell-based functional assays may also be recommended.

Key Applications in Immuno-Oncology, Neoantigen, and Vaccine Research

This integrated solution is designed with a balanced approach, providing equal utility for both immuno-oncology and infectious disease research:

Tumor Antigen Studies

Accelerate tumor neoantigen discovery and validation services by confirming that mutant peptides are not only presented by tumor cells but are also recognized by circulating or tumor-infiltrating lymphocytes (TILs).

Vaccine Epitope Discovery

Map the presented viral or bacterial epitopes and confirm they engage specific T cell clonotypes, providing a rational basis for next-generation vaccine design.

Autoimmune Disease Research

Track autoreactive T cell clones and validate their cross-reactivity or specific binding affinity to self-antigens.

T Cell Therapy Development

Identify the exact antigen targets of orphan TCRs discovered in patient repertoires, enabling the engineering of targeted TCR-T cell therapies.

End-to-End Workflow: From Antigen Discovery to TCR-pMHC Validation

Bridging multiple complex omics platforms requires sophisticated coordination. Our integrated workflow seamlessly moves your project from initial discovery to final confirmation.

Our Integrated Antigen Discovery and TCR Profiling Services

Mass Spectrometry and SPR Platforms for Immune Profiling

By managing the entire pipeline under one roof, we eliminate the variability and data-loss risks associated with using disjointed vendors for mass spectrometry, genomics, and structural biology.

• Comprehensive Bioinformatic Integration: We do not simply hand over isolated datasets. We perform comprehensive integration using public databases (e.g., VDJdb, IEDB), advanced structural predictions, and clonal frequency metrics to rank probable TCR-peptide associations.
• End-to-End Reagent Sourcing: For the physical validation phase, you do not need to source complex reagents. We provide end-to-end synthesis and expression, generating the required high-purity peptides, recombinant MHC complexes, and soluble TCRs entirely in-house.
• Targeted High-Precision Validation: Rather than wasting resources on massive, low-confidence screening, we strategically filter the data to select highly promising "lead" pairs for low-throughput, high-precision kinetic validation.

Multi-Omics Workflow vs. Single-Platform Analysis

Sample Requirements for Immunopeptidomics and TCR Sequencing

Example Data for Peptide Discovery and TCR Validation

Our final deliverables provide publication-ready visualizations that clearly communicate the journey from multi-omics discovery to biophysical validation.

Deliverables for the Integrated Antigen and TCR Workflow

Because this is an integrated service, you will receive staged data packages culminating in a comprehensive final report:

• Immunopeptidomics Report: Complete list of naturally presented peptides with confidence scores, source protein annotations, and HLA restriction motifs.
• TCR Repertoire Report: Diversity metrics, V(D)J usage, and identification of significantly expanded clonotypes.
• Bioinformatic Integration Report: Cross-referencing of peptide and TCR data utilizing public databases and structural predictions to prioritize top interaction candidates.
• Interaction Validation Data: If the downstream module is selected, raw SPR sensorgrams and calculated kinetic constants (K_D, k_on, k_off) for the selected lead TCR-pMHC pairs.
• Executive Summary: A synthesized research conclusion linking the discovery, tracking, and validation phases into actionable insights for your therapeutic or vaccine pipeline.

Frequently Asked Questions

Summary

Using MHC-associated peptide proteomics (MAPPs), researchers identified naturally presented AAV9 capsid peptides and then evaluated whether these peptides could drive measurable T cell responses in human donor PBMCs. Rather than relying only on overlapping peptide libraries, the study focused on peptides that were actually presented on HLA molecules, creating a more biologically relevant route for epitope discovery. By combining immunopeptidomics-derived peptide selection with downstream immune stimulation assays, the authors identified multiple immunodominant CD8 T cell epitopes of the AAV9 capsid, including several previously unreported epitopes. This example study illustrates how naturally presented peptides can guide downstream immune response mapping and candidate validation.

Methods

This study used an integrated antigen-to-response workflow centered on naturally presented peptide discovery.

Researchers first used MAPPs to define the HLA-presented peptide repertoire of the AAV9 capsid. They then compared two peptide strategies for downstream immune testing: conventional overlapping peptide pools and MAPPs-derived peptide pools. Because direct ex vivo detection of capsid-specific T cells in healthy donor PBMCs showed very low sensitivity, the team introduced an in vitro PBMC expansion step before FluoroSpot restimulation assays.

To identify immunodominant CD8 epitopes in greater detail, the HLA Class I MAPPs-derived peptides were further deconvoluted and tested across a donor cohort enriched for predicted binding alleles. Binding assays were then used to confirm whether selected novel epitopes bound stably to their predicted HLA alleles.

Key Technical Features

Sample types: Healthy donor PBMCs, including seropositive and seronegative donors for AAV9

Antigen discovery: MHC-associated peptide proteomics (MAPPs) to identify naturally presented AAV9 capsid peptides

Immune follow-up: PBMC expansion followed by IFN-γ FluoroSpot restimulation assays

Comparison strategy: Overlapping peptide pools versus MAPPs-derived peptide pools

Epitope deconvolution: Screening of 41 HLA Class I MAPPs peptides in matrix pools across 24 healthy donors

Validation: Binding assays to confirm stable interaction of selected epitopes with predicted HLA alleles

Biological insight: Naturally presented MAPPs peptides and overlapping peptides expanded largely distinct T cell populations

Creative Proteomics can offer integrated discovery and follow-up services that support similar research pipelines, including:

• HLA immunopeptidomics for naturally presented antigen discovery
• Peptide pool design and epitope deconvolution strategies
• T cell response profiling using donor PBMC-based functional assays
• TCR repertoire tracking to monitor responsive clonotype expansion
• Peptide–HLA binding validation and downstream interaction analysis
• Integrated interpretation linking presented peptides to immune response prioritization

These services support viral epitope discovery, gene therapy immunogenicity assessment, vaccine target screening, and broader antigen-specific T cell research.

Results

Naturally Presented Peptides Outperformed Generic Peptide Assumptions
The study showed that conventional overlapping peptides and MAPPs-derived peptides did not stimulate the same T cell populations. PBMCs expanded with overlapping peptides responded poorly to MAPPs-derived peptides, while PBMCs expanded with MAPPs-derived peptides responded preferentially to the MAPPs pools. This indicated that naturally presented peptides can reveal antigen-specific responses that are not captured well by standard overlapping peptide designs.

MAPPs-Derived Responses Were Dominated by CD8 T Cells
When donor PBMCs were expanded with the MAPPs-derived capsid peptides, the immune response was dominated by HLA Class I-derived peptides, consistent with a capsid-specific CD8 T cell response. The authors further confirmed this trend through CD4 depletion experiments, supporting the use of naturally presented class I peptides for identifying cytotoxic T cell-relevant targets.

Novel Immunodominant Epitopes Were Identified
By screening 41 HLA Class I MAPPs peptides in 13 matrix pools across 24 healthy donors, the researchers identified 10 epitopes that elicited IFN-γ release in at least one donor. Notably, 9 of these 10 epitopes were novel, and the identified peptides ranged from 9 to 13 amino acids in length.

Binding Validation Strengthened Candidate Confidence
The study reported that the newly identified epitopes displayed strong binding to their predicted HLA alleles, and binding assays confirmed stable interaction for several of the novel candidates. This added an extra validation layer beyond peptide discovery and T cell activation alone.

Matrix-based screening of HLA class I peptides derived from immunopeptidomics reveals immunodominant AAV9 capsid epitopes capable of inducing IFN-γ responses in donor PBMCs.