Basic Procedures for Detection and Cytotoxicity of Chimeric Antigen Receptors

  • Keichiro MiharaEmail author
  • Tetsumi Yoshida
  • Joyeeta Bhattacharyya
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)


Chimeric antigen receptors against CD19 (anti-CD19-CAR) are widely recognized and used by not only researchers associated with immunology, molecular biology, and cell biology but also physicians to treat B-cell malignancies. Anti-CD19-CAR is currently clinically available as one of the therapeutic modalities for refractory acute B-cell-typed lymphoblastic leukemia (B-ALL) patients. However, to detect CAR on the cell surface and investigate the efficacy of CAR-T cells, there are numerous experimental modalities including flow cytometry, the Cr-releasing assay, immunoblot, and immunostaining. We have chosen several techniques, which are necessary and sufficient as well as reliable and reproducible to detect and assess the killing effect of CAR-T cells. Here, we describe protocols for basic experiments and procedures for the detection of CAR on transduced cells and in in vitro coculture experiments to assess cytotoxicity using CAR-T cells.

Key words

CD19 CD38 Flow cytometry Autolysis CAR Antibody capping 



This work was supported by a Japanese Grant-in-Aid for Science Research.


  1. 1.
    Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger TL et al (2004) Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 18:676–684CrossRefGoogle Scholar
  2. 2.
    Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR et al (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368:1509–1518CrossRefGoogle Scholar
  3. 3.
    Porter DL, Levine BL, Kalos M, Bagg A, June CH (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365:725–733CrossRefGoogle Scholar
  4. 4.
    Mihara K, Yanagihara K, Takigahira M, Imai C, Kitanaka A, Takihara Y et al (2009) Activated T-cell-mediated immunotherapy with a chimeric receptor against CD38 in B-cell non-Hodgkin lymphoma. J Immunother 32:737–743CrossRefGoogle Scholar
  5. 5.
    Mihara K, Yanagihara K, Takigahira M, Kitanaka A, Imai C, Bhattacharyya J et al (2010) Synergistic and persistent effect of T-cell immunotherapy with anti-CD19 or anti-CD38 chimeric receptor in conjunction with rituximab on B-cell non-Hodgkin lymphoma. Br J Haematol 151:37–46CrossRefGoogle Scholar
  6. 6.
    Mihara K, Bhattacharyya J, Kitanaka A, Yanagihara K, Kubo T, Takei Y et al (2012) T-cell immunotherapy with a chimeric receptor against CD38 is effective in eliminating myeloma cells. Leukemia 26:365–367CrossRefGoogle Scholar
  7. 7.
    Bhattacharyya J, Mihara K, Kitanaka A, Yanagihara K, Kubo T, Takei Y et al (2012) T-cell immunotherapy with a chimeric receptor against CD38 is effective in eradicating chemotherapy-resistant B-cell lymphoma cells overexpressing survivin induced by BMI-1. Blood Cancer J 2:e75CrossRefGoogle Scholar
  8. 8.
    Mihara K, Yoshida T, Ishida S, Takei Y, Kitanaka A, Shimoda K et al (2016) All-trans retinoic acid and interferon-α increase CD38 expression on adult T-cell leukemia cells and sensitize them to T cells bearing anti-CD38 chimeric antigen receptors. Blood Cancer J 6:e421CrossRefGoogle Scholar
  9. 9.
    Yoshida T, Mihara K, Takei Y, Yanagihara K, Kubo T, Bhattacharyya J et al (2017) All-trans retinoic acid enhances cytotoxic effect of T cells with an anti-CD38 chimeric antigen receptor in acute myeloid leukemia. Clin Transl Immunology 5:e116CrossRefGoogle Scholar
  10. 10.
    Mihara K, Yoshida T, Takei Y, Sasaki N, Takihara Y, Kuroda J et al (2017) T cells bearing anti-CD19 and/or anti-CD38 chimeric antigen receptors effectively abrogate primary double-hit lymphoma cells. J Hematol Oncol 10:116CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Keichiro Mihara
    • 1
    Email author
  • Tetsumi Yoshida
    • 1
  • Joyeeta Bhattacharyya
    • 2
  1. 1.Department of Hematology and Oncology, Research Institute for Radiation Biology and MedicineHiroshima UniversityHiroshimaJapan
  2. 2.Department of Cardiac ResearchCumballa Hill Hospital and Heart InstituteMumbaiIndia

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