Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45ROCD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

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We thank the patients for their participation in the clinical trials from which research samples were obtained. We also acknowledge A. Fesnak, A. Lamontagne, A. Malykhin, C. Corl, Y. Ohayon and other members of the Clinical Cell and Vaccine Production Facility for cell manufacturing and testing. In addition, we are grateful to V. Gonzalez, J. Finklestein, F. Nazimuddin, J.-M. Navenot, M. Bogush, Y. Tanner, N. Kengle, K. Marcucci, A. Chew, C. Pletcher, P. Hallberg and R. Schretzenmair for contributions to correlative studies and/or other research support. D. Campana, C. Imai and others at St. Jude Children’s Research Hospital designed, developed and provided, under material-transfer agreements, the CAR used in this study. B. Jena and L. Cooper (MD Anderson Cancer Center) are acknowledged for providing the CAR anti-idiotype detection reagent. The functional anti-idiotypic antibody that was used for in vitro CAR stimulation experiments was a kind gift from Novartis Pharmaceutical Corporation. This work was supported by funding from NCI T32CA009140 (J.A.F.) R01CA165206 (D.L.P. and C.H.J.), P01CA214278 (C.H.J), a Stand Up to Cancer Phillip A. Sharp Innovation in Collaboration Award (C.H.J) and Novartis.

Author information

Author notes

  1. These authors contributed equally: Simon F. Lacey and Elena J. Orlando.


  1. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Joseph A. Fraietta
    • , Simon F. Lacey
    • , F. Brad Johnson
    • , Megan M. Davis
    • , Bruce L. Levine
    • , Donald L. Siegel
    • , David L. Porter
    • , Carl H. June
    •  & J. Joseph Melenhorst
  2. Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA

    • Joseph A. Fraietta
    • , Simon F. Lacey
    • , Mercy Gohil
    • , Stefan Lundh
    • , Alina C. Boesteanu
    • , Yan Wang
    • , Roddy S. O’Connor
    • , Edward Pequignot
    • , David E. Ambrose
    • , Changfeng Zhang
    • , Nicholas Wilcox
    • , Felipe Bedoya
    • , Corin Dorfmeier
    • , Fang Chen
    • , Lifeng Tian
    • , Harit Parakandi
    • , Minnal Gupta
    • , Regina M. Young
    • , Irina Kulikovskaya
    • , Li Liu
    • , Jun Xu
    • , Megan M. Davis
    • , Bruce L. Levine
    • , Noelle V. Frey
    • , Donald L. Siegel
    • , Carl H. June
    •  & J. Joseph Melenhorst
  3. Parker Institute for Cancer Immunotherapy at University of Pennsylvania, Philadelphia, PA, USA

    • Joseph A. Fraietta
    • , Simon F. Lacey
    • , Alexander C. Huang
    • , E. John Wherry
    • , Carl H. June
    •  & J. Joseph Melenhorst
  4. Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    • Elena J. Orlando
    • , Iulian Pruteanu-Malinici
    • , Sadik H. Kassim
    • , Hans Bitter
    •  & Jennifer L. Brogdon
  5. Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA, USA

    • Wei-Ting Hwang
  6. Division of Hematology-Oncology, Department of Internal Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Noelle V. Frey
    •  & David L. Porter
  7. Division of Transfusion Medicine and Therapeutic Pathology, University of Pennsylvania, Philadelphia, PA, USA

    • Donald L. Siegel
  8. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Alexander C. Huang
    •  & E. John Wherry


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J.A.F., S.F.L., F.B.J., R.M.Y., N.V.F., B.L.L., D.L.S., E.J.W., J.L.B., D.L.P., C.H.J. and J.J.M. designed the experiments and/or performed analysis. J.A.F., M.Gohil, S.L., A.C.B., Y.W., R.S.O., D.E.A., C.Z., N.W., F.B., C.D., F.C., L.T., H.P., M. Gupta, I.K., L.L., J.X., S.H.K., M.M.D. and A.C.H. performed experiments. E.J.O. and H.B. analyzed RNA-seq. data. I.P.-M. carried out the computational analyses of flow cytometric data. W.-T.H. and E.P. performed statistical analyses. J.A.F., C.H.J. and J.J.M. wrote the paper, and all authors contributed to writing and providing feedback.

Competing interests

J.A.F., S.F.L., F.B., R.M.Y., B.L.L., J.L.B., D.L.P., C.H.J. and J.J.M. hold patents related to CTL019 cell therapy. These authors declare no additional interests. The remaining authors declare no competing interests.

Corresponding author

Correspondence to J. Joseph Melenhorst.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–11 and Supplementary Tables 1, 7, 9, 10

  2. Reporting Summary

  3. Supplementary Table 2

    Treatment and clinical characteristics of responding patients.

  4. Supplementary Table 3

    Genes differentially expressed in pre-infusion CAR T cells between CR/PRTD and PR/NR patients.

  5. Supplementary Table 4

    List of leading edge genes associated with the gene sets of Figure 2c.

  6. Supplementary Table 5

    Transcriptomic profiling of mock-stimulated (control) and CAR-stimulated CTL019 infusion products as well as ex vivo CD3+ T cells (leukapheresis).

  7. Supplementary Table 6

    Phenotypes of leukapheresed CD8+ and CD4+ T cells identified by the flowType analysis that segregate CR from NR patients.

  8. Supplementary Table 8

    Phenotypes of CD8+ T cells in the CAR T cell infusion product identified by the flowType analysis that segregate CR from NR patients.

  9. Supplementary Table 11

    Holm-Sidak adjusted P values and summary statistics for data sets involving multiple comparisons.