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Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional
Qiong Xue1,2, Emily Bettini3, Patrick Paczkowski3
1Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. chelsea.xue@novartis.com.
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.
Insights
This study developed a new single-cell method to analyze chimeric antigen receptor (CAR)-T cells, revealing diverse immune responses and providing insights into CAR-T cell therapy efficacy and safety.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Characterizing the functional attributes (potency, immunotoxicity) of CD19 CAR-T cells ex vivo is challenging.
- Despite challenges, CAR-T cell therapy shows promising in vivo efficacy for B cell malignancies.
Purpose of the Study:
- To develop and apply a novel single-cell, multiplexed cytokine profiling method for CD19 CAR-T cells.
- To evaluate the functional heterogeneity and polyfunctional cytokine secretion profiles of CD19 CAR-T products.
Main Methods:
- Utilized a single-cell, 16-plex cytokine microfluidics device for antigen-specific stimulation of CD19 CAR-T cells.
- Manufactured CAR-T cells from human PBMCs and expanded them using lentiviral transfection and bead-based expansion.
- Analyzed protein secretions from single cells using a microfluidic chip, antibody array, and proprietary bioinformatics tools.
Main Results:
- Demonstrated marked heterogeneity in cytokine secretions and polyfunctional subsets of CD19 CAR-T cells upon stimulation.
- Identified a broad range of effector, stimulatory, regulatory, and inflammatory cytokine responses.
- Developed new bioinformatics tools for enhanced visualization and comparison of polyfunctional immune responses.
Conclusions:
- Single-cell proteomic profiling reveals a diverse immune effector landscape in CD19 CAR-T products.
- The new platform provides sensitive, comprehensive functional assessment of CAR-T pre-infusion products.
- This approach may offer insights into the safety and efficacy of CAR-T cell therapy.
