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T-cell Epitope Discovery & Validation

Identify T-cell epitopes using peptide microarray-based HLA-binding assays. Screen thousands of peptides for MHC presentation, validate computational predictions experimentally, and confirm functional T-cell responses with activation assays.
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What T-cell immune profiling enables

From HLA binding to functional T-cell response

T-cell immunity depends on which peptides are presented on major histocompatibility complex (MHC) molecules and the type and extent of responses they trigger. Microarray-based human leukocyte antigen (HLA)-binding assays identify candidate epitopes at scale, while downstream assays confirm which peptides drive biologically relevant responses.

Table of Contents

Screen HLA-binding epitopes

Identify which peptides are capable of binding specific HLA alleles and being presented to T-cells.

Screen thousands of peptides in parallel

Evaluate large peptide libraries against recombinant HLA molecules using peptide microarray-based assays.

Map HLA-restricted binding patterns

Identify allele-specific binding motifs and compare relative binding affinities.

Reduce dependency on PBMCs early on

Perform initial screening without requiring cellular assays or fresh donor material.

Prioritize T-cell relevant candidates

Filter large peptide sets down to sequences most likely to drive T-cell responses before entering functional assays.

Focus on experimentally validated binders

Select peptides with confirmed HLA binding instead of relying solely on prediction.

Reduce assay complexity downstream

Limit functional testing to high-confidence candidates.

Validate or challenge in silico predictions

Compare chip-based binding data with in silico predictions to identify epitopes missed by algorithms or confirm computational hits.

Validate functional T-cell responses

Confirm whether candidate epitopes trigger biologically relevant T-cell activity.

Measure T-cell activation

Use ELISpot or FluoroSpot assays to detect cytokine secretion in response to peptides.

Characterize response type

Apply flow cytometry to distinguish effector, regulatory, or memory T-cell responses.

Assess functional outcomes

Evaluate cytotoxicity or suppression to determine whether responses are immunogenic or tolerogenic.

Research questions answered

T-cell epitope discovery & validation in practice

From HLA-binding screening to functional validation, T-cell profiling workflows help identify which peptides are presented and which drive immune responses. See how you can apply these approaches across infectious disease and immunogenicity studies.

HLA Binding
Which peptides bind my target HLA allele for T-cell presentation?
Screening 4,327 HCMV peptides with recombinant HLA-DRB1*1101 identified 21 viral proteins containing HLA binders. FluoroSpot validation confirmed microarray-identified binders (KPRKTTRPFKVIIKP, FCTPNVQTRRGRVKI) induced IFN-γ production in HLA-matched PBMCs, while non-binders did not, demonstrating predictive accuracy.
Read poster →
Immunogenicity risk
Which therapeutic antibody regions pose T-cell immunogenicity risk?
Chip-based HLA-binding screening of PD-L1 inhibitors with recombinant HLA-DRB1*01:01 identified four T-cell epitope candidates on atezolizumab but none on durvalumab. Functional Treg suppression assays confirmed one atezolizumab epitope failed to suppress T-cell responses, correlating with clinically observed higher ADA incidence.
Read poster →
Coincident epitopes
Can antibody-defined epitopes also trigger T-cell responses?
Peptide microarray screening of EBNA-1 peptides identified antibody-binding epitope FHPVGEADY in EBV-infected donor sera. Functional ELISpot validation showed peptide FHPVGEADYFEYH (containing this motif) also activated T-cells to produce IFN-γ, revealing a coincident B- and T-cell epitope critical for EBV immunity.
Read application note →
How it works​

From HLA-binding screening to T-cell validation

T-cell epitope discovery begins with identifying peptides that bind HLA molecules. HLA-binding assays reveal which sequences can be presented to T-cells. Our chip-based approach enables high-throughput, cell-free screening and functional validation of selected candidates downstream.
Peptide microarrays enable high-throughput HLA-binding screening to identify candidate T-cell epitopes. These peptides can then be synthesized and evaluated using functional assays assessing T-cell activation and effector function (e.g., ELISpot and cytotoxicity assays), alongside phenotypic analyses by flow cytometry.

T-cell Assays

Your T-cell immunity questions answered: a guide to functional assays

HLA-binding identifies peptides capable of T-cell presentation, but functional assays confirm whether they actually elicit immune responses. From activation screening to phenotyping to functional characterization, we offer comprehensive T-cell services for the experimental validation of epitope candidates beyond in silico data.

activation

Do these peptides activate T-cells?

ELISpot / FluoroSpot
  • Detect cytokine secretion (e.g. IFN-γ, IL-2, TNF-α) from activated T-cells
  • Rank immunogenic epitopes across candidate peptides
When to use
  • Validation of HLA-binding candidates
  • Vaccine and immunotherapy epitope screening

Phenotype

What type of T-cell response is triggered?

Flow cytometry-based phenotyping
  • Distinguish CD4+, CD8+, and regulatory T-cell subsets
  • Measure cell activation states and monitor population shifts
When to use
  • Immunogenicity assessment for biologics
  • Mechanistic immune response studies

Function

What functional effect do activated T-cells have?

Cytotoxicity and suppression assays
  • Measure target cell killing by CD8+ T-cells
  • Assess suppressive activity of regulatory T-cells
When to use
  • Validating tumor cell killing
  • Identifying suppressive signals that may impact outcomes

Not sure which solution is best for you? We can help.

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PROJECT WORKFLOW

What does a T-cell immune profiling project look like?

T-cell immune profiling workflows combine chip-based HLA-binding screening with functional validation assays to identify and confirm immunogenic epitopes. The workflow below focuses on T-cell epitope discovery; for more flexible antibody profiling workflows, see our Epitope Mapping or Cancer Immunology application pages.

1

Target definition & library design

~1–2 days

PEPperPRINT

Microarray & study design
Based on your target antigens and research objectives, we design your peptide microarray and source recombinant HLA molecules (currently only available for HLA class II). Microarray production begins once the final study design is approved.

You

Define your question
Provide your research question and antigen sequence (FASTA, UniProt ID or peptide list). We're happy to sign an NDA in the case of proprietary sequence information.
2

Chip-based HLA-binding assay

~5–6 weeks

PEPperPRINT

HLA-binding screening
Once your microarrays and recombinant HLAs are ready, we run the fluorescence-based immunoassays to identify HLA-restricted T-cell epitope candidates based on binding affinity data.
View example scan output

You

Sit tight
Nothing required at this stage. If requested, we can send you the resulting scans at this point, though we usually send everything together with the report.
3

Functional T-cell validation

~6-8 weeks

PEPperPRINT

Run the assay
Synthesize candidate peptides (through our partners) and run ELISpot/FluoroSpot, phenotyping, or cytotoxicity assays. Deliver full report with epitope rankings and functional data.
View sample report

You

Provide PBMCs or we source them
Provide PBMCs (fresh or frozen), or we source HLA-matched PBMCs for validation experiments.
4

Optional: Antibody isolation & TCR/BCR sequencing

~6-8 weeks

PEPperPRINT

Isolate & sequence receptors
Isolate antigen-specific B-cells or T-cells using single-cell sorting. Perform RNA sequencing and deliver receptor sequences of dominant clones.

You

Provide biological samples
Provide PBMCs for T-/B-cell isolation and TCR/BCR sequencing.
1

Target definition & library design

~1–2 days

PEPperPRINT

Microarray & study design
Based on your target antigens and research objectives, we design your peptide microarray and source recombinant HLA molecules (currently only available for HLA class II). Microarray production begins once the final study design is approved.

You

Define your question
Provide your research question and antigen sequence (FASTA, UniProt ID or peptide list). We're happy to sign an NDA in the case of proprietary sequence information.
2

Chip-based HLA-binding assay

~5–6 weeks

PEPperPRINT

HLA-binding screening
Once your microarrays and recombinant HLAs are ready, we run the fluorescence-based immunoassays to identify HLA-restricted T-cell epitope candidates. We analyze the results and deliver a full written report with annotated scans, intensity plots, and a list of peptide candidates based on binding affinity data.
View example output

You

Receive HLA-binding report
Your analysis report is delivered digitally, ready to share with your team or include in a publication. If planning on running additional validation experiments in your lab, we also offer to provide synthetic peptides of your hit sequences through our partners.
View example output
3

Functional T-cell validation

2 days

PEPperPRINT

Provide peptides & protocol support
Optionally synthesize candidate peptides (through our partners) and ship them to your facility for further in-house validation experiments.

You

Run validation assays in your lab
Run functional T-cell assays in your lab. Contact us for protocol support, PBMC sourcing assistance, or data interpretation.
4

Optional: Antibody isolation & TCR/BCR sequencing

~1-2 weeks

PEPperPRINT

Isolate & sequence receptor
Isolate antigen-specific B-cells or T-cells using single-cell sorting. Perform RNA sequencing and deliver receptor sequences of dominant clones.

You

Provide biological samples
Provide PBMCs for T-/B-cell isolation and TCR/BCR sequencing.
Related applications

Where T-cell immune profiling fits

Cellular immune profiling connects to multiple research contexts. Here's where researchers often go next:
Vaccines & Therapeutic Immunogenicity Profiling
Profile immune responses for vaccine optimization, anti-drug antibody detection, and immunotherapy development across preclinical to clinical stages.
Explore application →
Cancer Immunology & Neoantigen Discovery
Discover tumor-specific antigens, profile anti-tumor antibody responses, and develop therapeutic antibodies for precision cancer immunotherapy.
Explore application →
Infectious Disease & Pathogen Profiling
Profile immune responses to viral and bacterial antigens for vaccine development, seroprevalence studies, and pathogen surveillance.
Explore application →

Discover which peptides drive T-cell immunity

Whether you're assessing immunogenicity, characterizing disease pathology, or optimizing therapeutic efficacy, design a T-cell profiling workflow tailored to your targets, HLA alleles, and assay requirements.
Not sure if this approach fits your study design? Let’s talk it through.​
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Frequently Asked Questions

Before you get started

How do peptide-microarray based HLA-binding assays work?
Our chip-based HLA-binding assays use recombinant HLA molecules. Peptide microarrays displaying thousands of peptides are incubated with biotinylated HLA proteins, and binding is detected via fluorescent streptavidin. This cell-free approach eliminates need for fresh PBMCs during epitope discovery, accelerating timelines and reducing sample requirements. HLA typing information helps us select relevant alleles to screen, or we test common alleles representing global populations.
Currently, our chip-based HLA-binding assays focus on MHC class II (HLA-DR) for CD4+ T-cell epitope prediction. Contact us to discuss the best approach for your research question.
Different samples can definitely be tested against the same set of peptides in parallel, but for this kind of study, we would create duplicate microarrays. You could fully outsource both analyses to us, or we could send you the duplicate microarray for performing epitope mapping at your facility while we perfom the HLA-binding assays.

When starting with chip-based HLA-binding assays, you’ll need to provide your target sequence/s and HLA information if known. For T-cell functional assays, we would need PBMCs in addition to your target peptides (both can be either provided by you, or sourced by us). For BCR/TCR seq, we would need PBMCs. Serum would be required for antibody isolation.

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