Novel platform for identifying rare antigen-specific CD4 T cells in Merkel cell carcinoma patients
Natalie Vandeven1*, Yuexin Xu2, Lance U’Ren3, Erika Hayes1,3, Junbao Yang4, Jackie Stilwell3, Eric Kaldjian3, Edus Warren1,2, William Kwok4, Paul Nghiem1
University of Washington1, Fred Hutchinson Cancer Research Center2, RareCyte, Inc.3, Benaroya Research Institute4
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.
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