Research ArticleCancer

TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance

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Science Translational Medicine  22 Nov 2017:
Vol. 9, Issue 417, eaag1209
DOI: 10.1126/scitranslmed.aag1209
  • Fig. 1. CAR4 and CAR8 cells demonstrate in vitro and in vivo antileukemic efficacy.

    (A) In vitro killing of CD19+ murine ALL after 4 hours of coincubation, as measured by caspase 3/7 expression and loss of viable leukemia cells. E/T ratio, effector-to-target ratio. (B) Cytokine production of three biological replicates of CAR4 and CAR8 cells after 4 hours of coincubation with CD19+ ALL (error, SD; *P < 0.05, **P < 0.01, ****P < 0.001). (C) Survival analysis of syngeneic mice after infusion of 1 × 106 CAR4 or CAR8 or 5 × 105 CAR4 + 5 × 105 CAR8 cells 4 days after CD19+ ALL inoculation (n = 5 per group; P < 0.0001, Mantel-Cox test). (D and E) Persistence of murine CAR4 and CAR8 cells in the bone marrow of syngeneic recipients 14 days after infusion of CAR4, CAR8, or mixed cells [n = 3; **P < 0.01, one-way analysis of variance (ANOVA) performed for all comparisons and log-rank Mantel-Cox test for survival analysis]. ns, not significant.

  • Fig. 2. Exposure of both TCR and CAR antigens diminishes efficacy of CAR8 but not CAR4 cells.

    (A) IL-2 and IFN-γ production by HY-CAR4 and HY-CAR8 cells after 10 hours of incubation with CD19KO female splenocytes (CD19/HY), CD19KO male splenocytes (CD19/HY+), or wild-type (WT) female splenocytes (CD19+/HY) (**P < 0.001, ****P < 0.0001). (B) CD107a expression (red) and isotype control (gray) on HY-CAR T cells after 4 hours of incubation with CD19+ E2aPbx ALL. (C) HY-CAR4 or HY-CAR8 cells were administered on day 4 to E2aPbx-bearing syngeneic male (HY+) and female (HY) recipients. (D) Survival of female (TCR antigen) and male (TCR antigen+) recipients after treatment with 1 × 106 HY-specific CAR4 or CAR8 cells (n = 5 per group; **P < 0.01). (E) Leukemia burden in the bone marrow 7 days after infusion of mice treated with HY-CAR4 or HY-CAR8 cells (****P < 0.001, one-way ANOVA performed for all comparisons and log-rank Mantel-Cox test for survival analysis).

  • Fig. 3. Increased expression of exhaustion markers and apoptosis markers on CAR8 cells in the presence of TCR antigen.

    (A) HY-CAR4 and HY-CAR8 expansion in the bone marrow of leukemia-bearing HY+ or HY mice (n = 5) 7 days after CAR T cell infusion. (B and C) Percent caspase 3/7+ CAR8 and CAR4 cells at 72 hours after infusion. (D and E) Mean fluorescence intensity (MFI) of PD-1 and LAG3 on HY-specific CAR T cells in male (HY+) and female (HY) recipients day +7 after infusion (**P < 0.01, ****P < 0.0001). (F) Overlaid zebra plots of PD-1 and LAG3 expression of endogenous T cells or HY-CAR4 or HY-CAR8 cells in the presence or absence of HY antigen (one-way ANOVA performed for all comparisons).

  • Fig. 4. Restriction of TCR antigen to hematopoietic tissues does not prevent CAR8 exhaustion and failure of leukemia clearance.

    (A) Female/male hematopoietic chimeras were generated by CD3-depleted bone marrow transplantation 21 days before injection of 1 × 106 E2aPbx followed by lymphodepletion on day −1 and treatment with 1 × 106 CD19 CAR T cells or mock-transduced T cells on day 0. (B and C) Survival of HY-CAR8– or HY-CAR4–treated mice. (D and E) Leukemia burden in the bone marrow 7 days after infusion of CAR T cells. (F and G) PD-1 expression and (H and I) LAG3 expression on HY-CAR8 and HY-CAR4 7 days after infusion [for (B) to (I): *P < 0.05, ****P < 0.0001 (relative to the F→F group), one-way ANOVA performed for all comparisons and log-rank Mantel-Cox test for survival analysis].

  • Fig. 5. CAR8 failure in an OT1 TCR transgenic T cell after exposure to OVA.

    (A) CD107a expression on OT1-CAR T cells generated from Rag1−/− OT1 spleens at 4 hours of coincubation with E2aPbx expressing CD19 and/or OVA antigens. Iso cntrl, isotype control. (B) Percent viable E2aPbx cells expressing varying combinations of the CD19 and OVA antigens after 3 days of coculture with OT1-CAR or mock-OT1 T cells. (C) OVA-expressing hematopoietic chimeras were generated by CD3-depleted bone marrow transplant 21 days before inoculation with 1 × 106 E2aPbx, with lymphodepletion and 1 × 106 CD19 OT1-CAR8 cell treatment on day 0. (D) Survival of OVA chimeric mice treated with OT1-CAR8 T cells. (E) Leukemia burden of OT1-CAR8–treated mice 7 days after infusion. BM, bone marrow. (F and G) Analysis of infused OT1-CAR8 cells from the bone marrow 7 days after infusion. (F) PD-1 expression and (G) LAG3 expression on OT1-CAR8 cell [for (E) to (G): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, relative to the WT→WT group, one-way ANOVA performed for all comparisons and log-rank Mantel-Cox test for survival analysis].

  • Fig. 6. Persistence of CAR4 cells is reduced after sustained TCR engagement.

    (A) Percentage of CAR4 cells in the bone marrow 80 days after infusion into leukemia-free (CAR antigen) or leukemia-bearing (CAR antigen+) male (HY+) and female (HY) recipients (n = 5, *P < 0.05). (B) Percentage of caspase 3/7+ CAR4 cells (CD45.2+) in the bone marrow at day 80 compared to endogenous CD4 T cells (CD45.1+) (**P < 0.01, ***P < 0.001). (C and D) PD-1 expression of CAR4 cells and endogenous CD4 T cells in the same recipient at day 80 after infusion into leukemia-free or leukemia-bearing male (HY+) and female (HY) recipients (***P < 0.001, ****P < 0.0001, one-way ANOVA performed for all comparisons).

  • Fig. 7. Stimulation through CAR, TCR, or both receptors induces unique gene expression signatures in CAR4 and CAR8 cells.

    (A) Dendrogram and principal components analysis of global transcriptional profile of three biological replicates of CAR4 and CAR8 cells evaluated 10 hours after stimulation through either TCR, CAR, or both receptors with CD3-depleted splenocytes as detailed in fig. S6. (B) Volcano plots of CAR4 versus CAR8 cells stimulated through both TCR or CAR, respectively [−log P value versus fold change (FC)]. (C) Heat map of z score values of apoptotic, homing and migration, metabolism, effector, activation, and inhibitory receptor gene expression (20) of CAR4 and CAR8 cells stimulated through CAR, TCR, or both receptors.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/417/eaag1209/DC1

    Fig. S1. CAR4 and CAR8 cells demonstrate comparable in vivo efficacy at low doses against murine ALL, E2aPbx.

    Fig. S2. The presence of both TCR and CAR antigens diminishes efficacy of CAR8 but not CAR4 cells at 14 days.

    Fig. S3. TCR stimulation induces down-regulation of CAR expression.

    Fig. S4. Up-regulation of PD-1 and Lag3 expression is dependent on CAR stimulation.

    Fig. S5. CAR8 cells are capable of endogenous B cell clearance despite ineffective leukemia clearance.

    Fig. S6. In vitro CAR4 and CAR8 cell cytokine profiles when stimulated through TCR, CAR, or both receptors.

    Fig. S7. Gene list for pathways/gene sets from CAR4 and CAR8 cells stimulated through CAR, TCR, or both receptors.

    Table S1. Primary data.

  • Supplementary Material for:

    TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance

    Yinmeng Yang, M. Eric Kohler, Christopher D. Chien, Christopher T. Sauter, Elad Jacoby, Chunhua Yan, Ying Hu, Kelsey Wanhainen, Haiying Qin, Terry J. Fry*

    *Corresponding author. Email: fryt{at}mail.nih.gov

    Published 22 November 2017, Sci. Transl. Med. 9, eaag1209 (2017)
    DOI: 10.1126/scitranslmed.aag1209

    This PDF file includes:

    • Fig. S1. CAR4 and CAR8 cells demonstrate comparable in vivo efficacy at low doses against murine ALL, E2aPbx.
    • Fig. S2. The presence of both TCR and CAR antigens diminishes efficacy of CAR8 but not CAR4 cells at 14 days.
    • Fig. S3. TCR stimulation induces down-regulation of CAR expression.
    • Fig. S4. Up-regulation of PD-1 and Lag3 expression is dependent on CAR stimulation.
    • Fig. S5. CAR8 cells are capable of endogenous B cell clearance despite ineffective leukemia clearance.
    • Fig. S6. In vitro CAR4 and CAR8 cell cytokine profiles when stimulated through TCR, CAR, or both receptors.
    • Fig. S7. Gene list for pathways/gene sets from CAR4 and CAR8 cells stimulated through CAR, TCR, or both receptors.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

    [Download Table S1]

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