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Single-Cell Cytokine Analysis to Characterize CAR-T Cell Activation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2097))

Abstract

With the increase in the implementation of adoptive transfer anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we introduce a novel platform to study their heterogeneous cytokine response at the single-cell level following activation. Here, we describe an activation platform which incubates single CAR-T cells with single-target CD19 antigen-presenting cells (APCs) and measures the resulting cytokine secretions. This can be compared to the more traditional bulk activation modalities. Both pipelines of activation are measured using our high-throughput, multiplexed single-cell secretomic microchip composed of an antibody barcode array and subnanoliter microchambers, capable of detecting up to 42 unique proteins. This platform has also been adapted to cell lines as well as primary cells.

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References

  1. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371(16):1507–1517

    Article  Google Scholar 

  2. Porter DL, Hwang W-T, Frey NV, Lacey SF, Shaw PA, Loren AW, Bagg A, Marcucci KT, Shen A, Gonzalez V (2015) Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med 7(303):303ra139

    Article  Google Scholar 

  3. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, Braunschweig I, Oluwole OO, Siddiqi T, Lin Y (2017) Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 377(26):2531–2544

    Article  CAS  Google Scholar 

  4. Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK (2012) A deep profiler’s guide to cytometry. Trends Immunol 33(7):323–332

    Article  CAS  Google Scholar 

  5. Xue Q, Bettini E, Paczkowski P, Ng C, Kaiser A, McConnell T, Kodrasi O, Quigley MF, Heath J, Fan R (2017) Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response. J Immunother Cancer 5(1):85

    Article  Google Scholar 

  6. Woehrling E, Hill E, Torr E, Coleman M (2011) Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity. Neurotox Res 19(3):472–483

    Article  CAS  Google Scholar 

  7. Edwards BS, Oprea T, Prossnitz ER, Sklar LA (2004) Flow cytometry for high-throughput, high-content screening. Curr Opin Chem Biol 8(4):392–398

    Article  CAS  Google Scholar 

  8. Rebhahn JA, Bishop C, Divekar AA, Jiminez-Garcia K, Kobie JJ, Lee FE-H, Maupin GM, Snyder-Cappione JE, Zaiss DM, Mosmann TR (2008) Automated analysis of two-and three-color fluorescent Elispot (Fluorospot) assays for cytokine secretion. Comput Methods Prog Biomed 92(1):54–65

    Article  Google Scholar 

  9. Lu Y, Xue Q, Eisele MR, Sulistijo ES, Brower K, Han L, Amir e-AD, Pe’er D, Miller-Jensen K, Fan R (2015) Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands. Proc Natl Acad Sci U S A 112(7):E607–E615

    Article  CAS  Google Scholar 

  10. Lu Y, Chen JJ, Mu L, Xue Q, Wu Y, Wu P-H, Li J, Vortmeyer AO, Miller-Jensen K, Wirtz D (2013) High-throughput secretomic analysis of single cells to assess functional cellular heterogeneity. Anal Chem 85(4):2548–2556

    Article  CAS  Google Scholar 

  11. Chang ZL, Lorenzini MH, Chen X, Tran U, Bangayan NJ, Chen YY (2018) Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat Chem Biol 14(3):317

    Article  CAS  Google Scholar 

  12. Rossi J, Paczkowski P, Shen Y-W, Morse K, Flynn B, Kaiser A, Ng C, Gallatin K, Cain T, Fan R, Mackay S, Heath JR, Rosenberg SA, Kochenderfer JN, Zhou J, Bot A (2017) Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells associate with clinical outcomes in NHL, Blood. https://doi.org/10.1182/blood-2018-01-828343

    Article  CAS  Google Scholar 

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Correspondence to Rong Fan .

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Finck, A., Fan, R. (2020). Single-Cell Cytokine Analysis to Characterize CAR-T Cell Activation. In: Katz, S., Rabinovich, P. (eds) Cell Reprogramming for Immunotherapy. Methods in Molecular Biology, vol 2097. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0203-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0203-4_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0202-7

  • Online ISBN: 978-1-0716-0203-4

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