Description

Novel platform for identifying rare antigen-specific CD4 T cells in Merkel cell carcinoma patients

Natalie Vandeven¹*, Yuexin Xu², Lance U’Ren³, Erika Hayes^1,3, Junbao Yang⁴, Jackie Stilwell³, Eric Kaldjian³, Edus Warren^1,2, William Kwok⁴, Paul Nghiem¹
University of Washington¹, Fred Hutchinson Cancer Research Center², RareCyte, Inc.³, Benaroya Research Institute⁴
*Corresponding Author

Merkel cell carcinoma (MCC) is an often-lethal skin cancer associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases. CD8 T cells can recognize MCPyV and CD8 intratumoral infiltration is associated with dramatically improved survival. However, most MCC patients lack significant CD8 T cell infiltration and adoptive T cell therapy using MCPyV-specific CD8 T cells has had limited efficacy. One potential explanation for these findings is that MCPyV-specific CD8s are not sufficiently ‘helped’ by CD4 T cells. Antigen-specific CD4 T cells have been challenging to study due to their low frequency (~1 per million T cells). We have therefore developed a platform to detect & phenotype rare antigen-specific CD4 T cells.
MCC patient peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL) were stained and plated onto 2-well cover slides and evaluated on the RareCyte CyteFinder 6-color fluorescent scanning microscope. Single MCPyV-specific CD4 T cells were picked using an integrated computer-controlled retrieval system (CytePicker). Single cell sequencing to identify the alpha and beta chains of the T cell receptor (TCR) as well as phenotypic characterization was performed using RT-PCR.

We consistently identified antigen-specific CD4 T cells with a frequency of ~1 per million total T cells from the peripheral blood without a need for enrichment, a striking improvement on what we could achieve via flow cytometry. We can pair TCR alpha and beta chains from single cells, and investigate the T-helper subtype based upon a panel of 24 genes. Using this approach to identify rare but functionally important MCPyV-specific CD4 T cells will enhance our understanding of the immune response against this virus as well as antigen-specific cells more broadly.