Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T

Thomas Nerreter,Ralph Götz,Sophia Danhof,Sebastian Letschert,Hermann Einsele,Markus Sauer,Michael Hudecek,Sören Doose

doi:10.1038/s41467-019-10948-w

Thomas Nerreter, Ralph Götz + Show 6 more

Open Access

https://doi.org/10.1038/s41467-019-10948-w

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Abstract

Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.

Highlights

Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma
The achievement of CR was attributed to the administration of CD19 CAR-T even though, according to conventional detection by flow cytometry (FC), CD19 was only present on a minute fraction of myeloma cells (0.05% by FC) in this patient[6]
We have demonstrated the capacity of direct stochastic optical reconstruction microscopy to determine absolute copy numbers of molecules on plasma membranes of human cells10,11. dSTORM exhibits singlemolecule sensitivity[12,13], suggesting that it could be used to detect ultra-low expression of CD19 on myeloma cells that is undetectable by FC

Summary

Introduction

Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens. The achievement of CR was attributed to the administration of CD19 CAR-T even though, according to conventional detection by flow cytometry (FC), CD19 was only present on a minute fraction of myeloma cells (0.05% by FC) in this patient[6] This sparked heavy controversy over the antigen-sensitivity of CD19 CAR-T, and the ability of FC to detect ultra-low CD19 expression. Our findings are corroborated by dSTORM analyses of CD20 expression and data showing elimination of CD20-positive myeloma cells by CD20 CAR-T with similar sensitivity

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