RT Journal Article
SR Electronic
T1 Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response
JF Journal for ImmunoTherapy of Cancer
JO J Immunother Cancer
FD BMJ Publishing Group Ltd
SP 85
DO 10.1186/s40425-017-0293-7
VO 5
IS 1
A1 Qiong Xue
A1 Emily Bettini
A1 Patrick Paczkowski
A1 Colin Ng
A1 Alaina Kaiser
A1 Timothy McConnell
A1 Olja Kodrasi
A1 Máire F. Quigley
A1 James Heath
A1 Rong Fan
A1 Sean Mackay
A1 Mark E. Dudley
A1 Sadik H. Kassim
A1 Jing Zhou
YR 2017
UL http://jitc.bmj.com/content/5/1/85.abstract
AB Background It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies.Methods We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods.Results We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors.Conclusions Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.Abbreviations:B-ALLB-cell precursor acute lymphocytic leukemiaCARChimeric antigen receptorCLLChronic lymphocytic leukemiaCRSCytokine release syndromeDLBCLDiffuse large B-cell lymphomaICS-FCIntracellular staining flow cytometryPAT PCAPolyfunctional activation topology principal component analysisPBMCPeripheral blood mononuclear cellPDMSPolydimethyl siloxanePSIPolyfunctional strength indexSCBCSingle-cell barcode chip