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Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response

Xue, Qiong and Bettini, Emily and Paczkowski, Patrick and Ng, Colin and Kaiser, Alaina and McConnell, Timothy and Kodrasi, Olja and Quigley, Máire F. and Heath, James and Fan, Rong and Mackay, Sean and Dudley, Mark E. and Kassim, Sadik H. and Zhou, Jing (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). Art. No. 85. ISSN 2051-1426. PMCID PMC5697351.

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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.

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Heath, James0000-0001-5356-4385
Additional Information:© 2017 The Authors. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated. Acknowledgements: Not applicable. Funding: Not applicable. Availability of data and materials: The data presented in this study is available upon reasonable request to the corresponding authors. Authors’ contributions: QX, OK, MFQ manufactured CD19 CAR-T cells; QX, EB, TM, JZ performed CAR-T cell culture, SCBC, flow cytemotry and ELISA experiemnts; CN, AK perpared and validated the 16-plex cytokine panel slides; QX, EB, SM, MED, SHK, JZ designed the project and experiments; QX, EB, PP, CB, AK, TM, JH, RF, SM, SHK, JZ analzyed the data and prepared figures. All authors read and approved the final manuscript. Ethics approval and consent to participate: Peripheral blood mononuclear cells were obtained from HemaCare from consented healthy donors. Consent for publication: Not applicable. Competing interests: J.R.H. & R.F are board members of IsoPlexis, which is a company seeking to commercialize the SCBC technology. Q.X., O.K., M.F.Q. are current employees of Novartis and have competing interests with Novartis. M.E.D., S.H.K. are former employees of Novartis. E.B., P.P., C.N., A.K., T.M., S.M., J.Z are employees of IsoPlexis and have competing interests with IsoPlexis.
Subject Keywords:Single-cell proteomics; CD19 CAR-T cell product; Polyfunctionality; Microfluidic microdevice; Precision profiling
Issue or Number:1
PubMed Central ID:PMC5697351
Record Number:CaltechAUTHORS:20171121-104006296
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:83400
Deposited By: Tony Diaz
Deposited On:21 Nov 2017 19:06
Last Modified:03 Oct 2019 19:06

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