T cell epitope mapping

The challenge of T cell epitope mapping can be approached from many angles. At ImmuMap we apply state-of-the-art wet lab protocols in combination with world-recognized in silico prediction servers based on artificial neural networks.

In the following case study, performed in the academic setting, we wanted to map new melanoma-associated T cell epitopes as targets for immune therapeutic intervention as well as for monitoring of existing or raised immunity in melanoma patients.

Multiple T cell epitopes restricted to HLA-A02:01 have been identified previously, while the exact T cell recognition in context of other tissue has only been sporadically investigated. Although HLA-A*02:01 is the most common allele in the Western European population, it is only present in approximately half of the patients leaving the remaining half of the patients with suboptimal treatment and monitoring possibilities.

Therefore we aimed at using cell culturing and flow cytometry testing for mapping of new T cell epitopes restricted to HLA-A01:01, -A03:01, -A11:01 and –B07:02 from melanoma-associated proteins. For these four HLA molecules we performed a study with 249 peptides by flow cytometry testing of PBMCs from 39 melanoma patients. We identified a total of 27 T cell responses against novel peptide sequences and were able to confirm three unique peptide sequences as T cell epitopes (Frøsig et al., Cancer Immunology Immunotherapy, 2015).

The above described T cell epitope mapping case study was broken down into individual work elements as described below:

1. Identifying melanoma-associated candidate antigens based on literature search.


2. In silico prediction of short peptides within the sequence of melanoma-associated candidate antigens for binding to relevant HLA molecules


3. Measuring the actual binding affinity of predicted ligands to the relevant HLA molecules (Rodenko et al., Nature Protocols, 2006) to select the confirmed binding ligands.


4. Enumerating the occurrence of spontaneous T cell responses ex vivo in PBMCs from melanoma patients by staining with multiple MHC multimers generated from relevant HLA molecules and selected peptide ligands analyzed by flow cytometry (combinatorial encoding of MHC multimers, Hadrup & Bakker et al., Nature Methods, 2009; Andersen et al., Nature Protocols, 2012 or DNA-barcoding of MHC multimers, Bentzen et al., Nature Biotechnology, 2016).


5. Enriching PBMCs with MHC multimers generated from the relevant HLA molecules and confirmed peptide ligands to increase the frequency of the T cell responses above the detection limit. As readout was used flow cytometry testing after staining with multiple MHC multimers as in IV (T-cell enrichment, Hombrink et al., PloS One, 2011).


6. Confirming processing and presentation of selected T cell epitope candidates by flow cytometry testing after T cell co-culture with cell lines known to produce the relevant proteins intracellularly and following VITAL-FR cytotoxicity assay (Stanke et al., Journal of Immunological Methods, 2010) and/or intracellular flow cytometry staining of cytokines and/or extracellular staining of activation markers CD107a/b ((Betts et al., Journal of Immunological Methods, 2003) and CD137 (Wolfl et al., Blood, 2007)


7. Investigating the clinical relevance of identified T cell epitope candidates by flow cytometry testing of tumor-infiltrating lymphocytes with MHC multimers (combinatorial encoding of MHC multimers, Hadrup & Bakker et al., Nature Methods, 2009; Andersen et al., Nature Protocols, 2012 or DNA-barcoding of MHC multimers, Bentzen et al., Nature Biotechnology, 2016).

Note, several of the above mentioned methods are patented and licenses are required in order to perform these analyses.

ImmuMap offer to perform single, multiple or all of the tasks necessary for T cell epitope mapping in our laboratory or to train or advice you on how to perform your own epitope mapping.