DNA-barcode labelled MHCII multimers for detection of antigen-specific CD4 T cells across large libraries of epitopes

Authors

Basavaraju, Y., Dijkstra, S., Tamhane, T., Skadborg, S. K., Lu, L., Kwok, W. W., Stern, L. J., Lauer, G. M., Hadrup, S. R.

Abstract

The role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 25 Jun 2026.

Stats

1-terrible, 9-excellent. How would you rate this preprint? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.