In chimeric antigen receptor (CAR)-T cell immunotherapy, T cells are obtained from a patient and genetically modified to express specific receptors (CARs) against tumor antigens. The best studied and successful CAR-T cells target the CD19 antigen on neoplastic B cells. However, this targeted approach gives quite variable results. The secretion by CD19 CAR-T cells of various cell signaling molecules varies between patients and individual CAR-T cells. The question then is how to measure the ability of CD19 CAR-T cells to release signals, after a specific antigenic challenge and correlate that to patient responses.
T cells are known to have the capacity to produce many different cytokines making them polyfunctional with powerful and long-lasting immunity. This polyfunctionality can be observed to some extent in single CD19 CAR-T cells using flow cytometry approaches. However, the spectrum of cytokines that can be studied is limited using this technique. Therefore, Novartis researchers used microfluidics technology to assess many cytokines at the single-cell level.
This eloquent and remarkable approach entails the miniaturization of in vitro experiments in single cells by exploiting laminar flow properties. Researchers used the single-cell barcode chip (SCBC) platform whereby single cells are each confined to a nanoscale-volume chamber containing an antibody array. This miniature microarray is designed to detect multiple cytokines in one cell, and each of thousands of cells can be analyzed in numerous separate microchambers all at once.
To do this, Novartis activated peripheral blood mononuclear cells obtained by healthy donor apheresis. CD19 receptor coding information was introduced into the cells to generate the CD 19 CAR-T cells. The cells were stimulated and loaded on the SCBC microchips. The 16-cytokine assay panel covered those of various T cell functions including stimulatory, inflammatory, regulator, and effector. Using microscopy and imaging techniques, 16 cytokines from thousands of single CAR-T cells were measured simultaneously. High polyfunctionality with a dominant antitumor effector cytokine profile was observed, and each donor’s cells showed different functional clusters. This harnessing of multiplex cytokine detection at the single-cell level opens the door to linking in vitro CD19 CAR-T response profiles to patient response and outcomes.
Xue, Q., Bettini, E., Paczkowski, P., Ng, C., Kaiser, A., & McConnell, T. et al. (2017). Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response. Journal For Immunotherapy Of Cancer, 5(1). doi:10.1186/s40425-017-0293-7