Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb;23(2):242-249.
doi: 10.1038/nm.4258. Epub 2017 Jan 9.

Donor CD19 CAR T cells exert potent graft-versus-lymphoma activity with diminished graft-versus-host activity

Arnab Ghosh  1 , Melody Smith  1 , Scott E James  1   2 , Marco L Davila  2 , Enrico Velardi  1 , Kimon V Argyropoulos  1 , Gertrude Gunset  2 , Fabiana Perna  2 , Fabiana M Kreines  1 , Emily R Levy  1 , Sophie Lieberman  1 , Hillary V Jay  1 , Andrea Z Tuckett  1 , Johannes L Zakrzewski  3 , Lisa Tan  1 , Lauren F Young  1 , Kate Takvorian  1 , Jarrod A Dudakov  1 , Robert R Jenq  1 , Alan M Hanash  1 , Ana Carolina F Motta  4 , George F Murphy  4 , Chen Liu  5 , Andrea Schietinger  1 , Michel Sadelain  1   2 , Marcel R M van den Brink  1   2
Affiliations

Donor CD19 CAR T cells exert potent graft-versus-lymphoma activity with diminished graft-versus-host activity

Arnab Ghosh et al. Nat Med. 2017 Feb.

Abstract

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 cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. 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-HSCT. 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.

PubMed Disclaimer

Conflict of interest statement

Competing Financial Interest

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

Figures

Figure 1
Figure 1. m1928z T cells eliminate CD19-expressing lymphoma while exerting significantly less GVHD activity
(A) CD8L = mouse CD8 leader, CD8TM= mouse CD8 transmembrane region, Gly-Ser = glycine-serine linker. Representation of murine CD19-CAR constructs: m19delta (mouse-specific CAR lacking non-functional zeta-chain); m19z (mouse-specific functional CAR, no costimulation); m1928z (mouse-specific functional CAR, CD28-stimulation); m19BBz (mouse-specific functional CAR, 4-1BB costimulation); hum1928z (human-specific functional CAR, mouse CD28 costimulation); m19delta.GFP and m1928z.GFP (CARs with GFP reporter). (B) In vitro cytotoxicity assay using m19delta and m1928z CAR T cells as effectors and EL4-CD19 or EL4-OVA (control). (C, D) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells and inoculated with A20-TGL lymphoma cells. Designated groups were treated with 1×106 B6 m19delta or m1928z T cells per mouse. Tumor growth was monitored by in vivo bioluminescence and images from one of multiple independent experiments are depicted. The BLI images are depicted from one of two experiments (C). Survival was monitored for up to 100 days. Data are representative of two independent experiments (D). (E) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells and inoculated with A20-TGL lymphoma cells. Designated groups were treated with 0.5×106, 0.25×106, or 0.125×106 B6 m19delta or m1928z T cells per mouse. Survival was monitored. The mice treated with B6 m19delta T cells are depicted in Suppl Figure 3 for simplicity. (F) Lethally irradiated BALB/c recipients (upper panel) and CBF1 recipients (lower panel) were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with 1×106 B6 m19delta or m1928z T cells. Survival and weekly clinical GVHD scores were monitored. Results are pooled from two independent experiments. (G) Skin, liver, small intestine and large intestine were harvested from the recipients on day 14 post-transplant. H&E sections were analyzed for GVHD in a blinded fashion. Results pooled from 2 independent experiments and representative micrographs are shown. Black bar on micrographs indicate a field length of 100 microns. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant.
Figure 2
Figure 2. Allogeneic m1928z T cells mediate persistent B cell hypoplasia
(A–C) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with 1×106 B6 m19delta and m1928z Thy1.1+B6 T cells. (A) Peripheral blood was drawn at day 60 or day 90 and CD45-gated events analyzed for B220+B cells by flow cytometry. (B) CD45+Thy1.1+T cells were detected in peripheral blood. (C) Splenocytes were harvested at day 14 and used as effectors, and labeled EL4-CD19 were used as targets in Cr-51 release in vitro cytotoxicity assay. The proportions of donor T cells in spleens were determined by flow cytometry and the effective effector-to-target ratios are depicted. A representative of two independent experiments with similar results is shown (n=4, with lysis set up in triplicates). (D) B10.BR recipients were conditioned with cyclophosphamide on days −3 and −2 and TBI (7.5 Gy) on day −1 and reconstituted with 10 × 106 lin-depleted BM cells. Designated groups were treated with 1×106 B6 m19delta or m1928z T cells. The size of B220+ splenic follicles were scored after IHC staining. Pooled data from two independent experiments are depicted in the right panel and representative micrographs are shown in the lower panel. Black bar on micrographs indicate a field length of 200 microns. ****P < 0.0001; NS, not significant.
Figure 3
Figure 3. Allogeneic m1928z T cells exhibit decreased alloreactive proliferation and GVHD target organ infiltration
(A) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with luciferase+Thy1.1+B6 1×106 m19delta, or m1928z T cells. Bioluminescence imaging of the transplanted mice was performed weekly and flux was measured. Animals from one representative experiment and flux pooled from two independent experiments are shown. (B–D) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with 1×106 B6 m19delta, or m1928z T cells. Data from two independent experiments are depicted. (B) Tissues were harvested at day 14, homogenized and counted, and single cell suspensions analyzed by flow cytometry. The calculated numbers of donor Thy1.1+T cells in pLN, LPL and liver are shown. Results are representative of one of two independent experiments. (C) Splenocytes were harvested and frequencies of CCR5 and L-PAM determined by flow cytometry. Data from two independent experiments are depicted. (D) Cytokine expression in the serum was assessed at day 7 with Luminex multiplex assay. Results are representative of one of two independent experiments. (E) Lethally irradiated BM12 recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with 2×105 ABM m19delta or m1928z T cells and survival and GVHD were monitored. (F–H) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells and designated groups were treated with B6 1×106 m19delta, m1928z, m19z, m19BBz and hum1928z T cells. Survival and weekly clinical GVHD scores were monitored. Results are pooled from two independent experiments. (I) Lethally irradiated recipients (BALB/c or BALB/c CD19KO) were reconstituted with B6 or B6 CD19KO lin-depleted bone marrow cells. Designated groups were treated with 1×106 B6 m19delta or m1928z T cells. Survival and weekly clinical GVHD scores were monitored. Results are depicted from one of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant.
Figure 4
Figure 4. Alloreactive m1928z T cells are hyperactive, exhausted and undergo deletion
(A) m1928z-OT-1 TCR T cells or untransduced T cells were used as effectors. Left panel: CFSE labeled EL4-cells pulsed with OVA peptide (10 μM SIINFEKL) were used as targets with or without non-labeled EL4-CD19 cells as distractor. Right panel: CFSE labeled EL4-CD19 cells were used as targets with or without non-labeled EL4 cells pulsed with OVA peptide (10 μM SIINFEKL) as a distractor. Lysis at 5 hours was estimated as the percentage of 7AAD+ CSFE+ targets divided by total CSFE+ targets. Combined data from three experiments are shown (n = 9 replicates/data point). Each data point reflects the average + SEM. Two-tailed paired t-tests were performed for selected comparisons as depicted. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. (B, C) Lethally irradiated BALB/c recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with Thy1.1+B6 1×106 m19-delta.GFP or m1928z.GFP T cells. (B) Splenocytes harvested at day 14 post-HSCT were counted and surface expression of exhaustion markers was measured on Thy1.1+GFP+ gated cells. Results are representative of one of two independent experiments. (C) Splenocytes harvested on designated days post-HSCT were harvested and phosphorylation status evaluated on Thy1.1+GFP+ gated cells. (D, E) Lethally irradiated BM12 recipients were reconstituted with B6 lin-depleted bone marrow cells. Designated groups were treated with 1×105 ABM m19delta.GFP or m1928z.GFP T cells. Splenocytes harvested at day 14 post-HSCT. Results are representative of one of two independent experiments. (D) Global transcriptional profiles of FACS sorted GFP+CAR T cells, (E) Splenocytes were counted and surface expression of exhaustion markers measured on GFP+CAR T gated cells. Results are representative of one of two independent experiments.
See this image and copyright information in PMC

Comment in

References

    1. Ho WY, Blattman JN, Dossett ML, Yee C, Greenberg PD. Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction. Cancer cell. 2003;3:431–437. - PubMed
    1. Sadelain M, Riviere I, Brentjens R. Targeting tumours with genetically enhanced T lymphocytes. Nat Rev Cancer. 2003;3:35–45. - PubMed
    1. Gill S, June CH. Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunological reviews. 2015;263:68–89. - PubMed
    1. Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design. Cancer discovery. 2013;3:388–398. - PMC - PubMed
    1. James SE, et al. Antibody-mediated B-cell depletion before adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell receptors facilitates eradication of leukemia in immunocompetent mice. Blood. 2009;114:5454–5463. - PMC - PubMed

MeSH terms