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

Abstract

BackgroundIt 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.MethodsWe 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.ResultsWe 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.ConclusionsSingle-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.Electronic supplementary materialThe online version of this article (10.1186/s40425-017-0293-7) contains supplementary material, which is available to authorized users.

CD29 marks superior cytotoxic human T cells

“…Previous studies showed that the cytokine production profile of CAR T cells was highly diverse (2). As culturing T cells with different cytokines did not alter the relative distribution of CD29 + and CD29 -T cells, we propose to select for CD29 expression for the generation of highly toxic T cell products, in particular when donors have a low percentage of CD29 + T cells.…”

Cell therapy products: focus on issues with manufacturing and quality control of chimeric antigen receptor T‐cell therapies

“…Availability of multiplexed, single-cell approaches will greatly assist pre-infusion assessments of cellular immunotherapies, and these are now emerging. 100 The relevancy of readouts based on short-term effector functions to overall CAR-T potency is also questionable. Kunkele wileyonlinelibrary.com/jctb et al 107 demonstrated that CAR-T constructs that generated the highest activity in assays measuring specific lysis and cytokine secretion exhibited attenuated anti-tumour potency in vivo.…”

Single-cell integrative analysis of CAR-T cell activation reveals a predominantly TH1/TH2 mixed response independent of differentiation

“…In contrast, the cytokine profiles from single CAR-T cells that did not kill tumor cells showed no distinguishable grouping among these cytokines ( Figure 1g). A diverse landscape of antigen-specific response was observed in CD19-BB-3z CAR-T cells 13 . Their dominant polyfunctional cells also produce GranzymeB, GM-CSF, IL8, TNFa, IL13, and IFNg, in concordance with our observation in the third-generation CAR-T cells.…”

“…Intra-cellular cytokine staining for flow cytometric analysis is not a true secretion assay and often leads to over-estimation of cytokine-secreting cells. Recently, Xue et al used single-cell multiplex cytokine profiling to measure the cytokine output of CD19-BB-3z CAR-T cells using CD19coated beads and revealed a diverse polyfunctional response upon activation 13 . However, the cytokine profile of a CAR-T cell is yet to be directly correlated to cytotoxicity, subtype and signaling in order to elucidate the underlying mechanisms.…”