Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells1,2,3,4,5. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells6,7. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies8,9,10,11,12,13,14,15. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT16,17. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.

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We are thankful to M. Sayegh (Brigham and Women's Hospital and Children's Hospital Boston) for ABM mice, R. Negrin (Stanford University) for the A20-TGL cell line, E. Campeau (University of Massachusetts) for pENTR1A, D. Vignali (University of Pittsburgh Medical Center) for mouse TCR OTI-2A.pMIG II, and P. Khavari (Stanford University) for LZRS-Rfa plasmid. We appreciate the help of J. White, LCP, RARC, the flow cytometry core facility, the integrative genomics core, and the computational biology core at MSKCC. We also appreciate the help of H. Poeck in critical reading of the manuscript. This research was supported by National Institutes of Health award numbers R01-HL069929 (M.R.M.v.d.B.), R01-AI101406 (M.R.M.v.d.B.), P01-CA023766 (M.R.M.v.d.B.), and R01-AI100288 (M.R.M.v.d.B.), LLS (M. Sadelain), the Lymphoma Foundation, the Susan and Peter Solomon Divisional Genomics Program, MSKCC Cycle for Survival, and P30-CA008748 MSK Cancer Center Support Grant/Core Grant. A.G. is a fellow of the Lymphoma Research Foundation. M. Smith received funding from an NIH Diversity Supplement (PA-16-288) under R01-AI100288-08S1, the M.J. Lacher Research Fellowship (The Lymphoma Foundation), and the American Society for Blood and Marrow Transplantation New Investigator Award. M. Smith and S.J. are supported under an NIH T32 grant (T32-CA009207). S.J. is also a Young Investigator Awardee from the Conquer Cancer Foundation of ASCO. M.L.D. is supported by an ASH-AMFDP career development award, the Damon Runyon Fund, and K08-CA148821 (NCI). J.L.Z. is supported by LLS TRP grant 6465-15 and K08-CA160659 (NCI). A.Z.T. is supported by a grant from the Imaging and Radiation Sciences (IMRAS) Program of MSKCC. The content is solely the responsibility of the authors and does not represent the official views of the National Cancer Institute or the National Institutes of Health.

Author information

Author notes

    • Arnab Ghosh
    • , Melody Smith
    • , Scott E James
    • , Marco L Davila
    • , Michel Sadelain
    •  & Marcel R M van den Brink

    These authors contributed equally to this work.

    • Marco L Davila
    • , Jarrod A Dudakov
    •  & Ana Carolina F Motta

    Present addresses: Department of Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA (M.L.D.), Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (J.A.D.) and University of São Paulo, São Paulo, Brazil (A.C.F.M).


  1. Department of Medicine and Immunology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Arnab Ghosh
    • , Melody Smith
    • , Scott E James
    • , Enrico Velardi
    • , Kimon V Argyropoulos
    • , Fabiana M Kreines
    • , Emily R Levy
    • , Sophie Lieberman
    • , Hillary V Jay
    • , Andrea Z Tuckett
    • , Lisa Tan
    • , Lauren F Young
    • , Kate Takvorian
    • , Jarrod A Dudakov
    • , Robert R Jenq
    • , Alan M Hanash
    • , Andrea Schietinger
    • , Michel Sadelain
    •  & Marcel R M van den Brink
  2. Center of Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Scott E James
    • , Marco L Davila
    • , Gertrude Gunset
    • , Fabiana Perna
    • , Michel Sadelain
    •  & Marcel R M van den Brink
  3. Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Johannes L Zakrzewski
  4. Program in Dermatopathology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Ana Carolina F Motta
    •  & George F Murphy
  5. Department of Pathology and Laboratory Medicine, Rutgers–Robert Wood Johnson Medical School, New Jersey, USA.

    • Chen Liu


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A.G. and M.L.D. conceived the project. A.G., M. Smith, S.E.J., and M.L.D. contributed to the design of the experiments, supervised and conducted the experiments, generated the manuscript figures, and wrote the manuscript. E.V. and K.V.A. conducted the experiments and generated the manuscript figures. F.P. contributed to the performance of microarray analyses and interpretation of the data. A.C.F.M., G.F.M., and C.L. contributed to the performance of histological analyses and interpretation of the data. G.G., F.M.K., E.R.L., S.L., H.V.J., A.Z.T., L.T., L.F.Y., and K.T. assisted in conducting experiments. J.L.Z., J.A.D., R.R.J., A.M.H., and A.S. contributed to the design of the experiments and their analyses. M. Sadelain and M.R.M.v.d.B. supervised the project, contributed to the design of the experiments and their analyses, and wrote the manuscript.

Competing interests

M. Sadelain is a scientific cofounder of Juno Therapeutics. All other authors declare no competing financial interests.

Corresponding authors

Correspondence to Michel Sadelain or Marcel R M van den Brink.

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