Research ArticleCANCER IMMUNOLOGY

Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory phenotype

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Science Immunology  02 Nov 2018:
Vol. 3, Issue 29, eaat7061
DOI: 10.1126/sciimmunol.aat7061
  • Fig. 1 Anti-GITR and anti–PD-1 combination synergistically rejects established murine tumors and reinvigorates intratumoral dysfunctional T cells.

    (A) Survival curve of MC38 tumor–bearing mice treated with anti-GITR and/or anti–PD-1 Ab treatment (days 6 and 13). Results depict cumulative survival curves with indicated treatment (n = 16 to 17 mice per group). (B) CD8+ T cell–dependent long-term tumor protection mediated by combination treatment. C57BL/6 mice were treated with anti-CD8–, anti-CD4–, or anti-CD25–depleting Ab before and during therapy with anti-GITR and anti–PD-1 Ab or control Ab. Data shown are average tumor growth curve upon treatment with different depletion Abs (n = 6 mice per group). (C and D) Combination treatment increases intratumoral TEff/Treg ratio. (C) Representative FACS plots showing tumor T cell subsets on day 11 (FoxP3+ versus CD8+, cells pregated on live/single cells/CD45+/CD3+). FITC, fluorescein isothiocyanate. (D) Summary FACS result of intratumoral CD8+ T cell/Treg and CD4+ TEff/Treg ratio on days 8 and 11 after tumor challenge. Data are representative of three independent experiments (n = 7 mice per group). (E to G) Combination treatment reinvigorates intratumoral dysfunctional T cells. Tumors were harvested on days 11 and 12 after implantation, dissociated into single-cell suspension restimulated with phorbol 12-myristate 13-acetate/ionomycin with the presence of brefeldin A. Cells were fixed and permeabilized, followed by intracellular staining with Ki67 (E), granzyme A (F), and granzyme B (G). Data shown are percentages of positive cells (n = 8 to 9 mice per group). All error bars in figures show SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, log-rank test (A) and one-way ANOVA with Tukey’s test (C to G).

  • Fig. 2 Single-cell RNA-seq analysis of intratumoral CD8+ T cells reveals unique gene profiles upon combination treatment.

    (A) Schematic of tumor immunotherapy and single-cell sorting study design. (B) Heat map of significantly changed genes in clonal expanded intratumoral CD8+ T cells upon different treatment. Numbers indicate unique gene clusters (see table S3 for gene list). Color gradient indicates the P value. (C) Distribution of genes significantly affected by each treatment on the dysfunctional/activation plot. (D) KS plot of the values of the gene signatures on the dysfunction ↔ activation axis (KS, P < 0.05).

  • Fig. 3 Identification of combination treatment–responsive dysfunctional tumor-infiltrating CD8+ T cell population.

    (A) Density viSNE plots of OVA-specific intratumoral CD8+ T cells from each treatment group days 9 and 12 after tumor challenge. (B) viSNE plots of tumor-infiltrating T cells overlaid with the expression of indicated markers. (C) viSNE plot of intratumoral OVA-specific CD8+ T cells overlaid with color-coded T cell clusters identified by SPADE. (D) Frequency of selected T cell clusters displayed on a per-mouse basis with means ± SEM. Indicated cluster was highlighted on viSNE plot below the bar graph. (E) Histogram displaying the expression level of indicated markers on T cell clusters. (F) CITRUS cluster result overlaid with indicated markers. Cluster that was significantly affected with Ab treatment is circled (C1). (G) Frequency of cluster C1 is shown with means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test.

  • Fig. 4 Identification of combination treatment–responsive effector/memory tumor-infiltrating CD8+ T cell population.

    (A) viSNE plots of tumor-infiltrating T cells overlaid with the expression of selected markers. (B) Density viSNE plots of OVA-specific tumor-infiltrating CD8+ T cells from each treatment group days 9 and 12 after tumor challenge. (C) viSNE plot of MC38 infiltrating OVA-specific CD8+ T cells overlaid with color-coded T cell clusters identified by SPADE. (D) Frequency of selected T cell clusters (means ± SEM). Indicated cluster was highlighted on viSNE plot below the bar graph. (E) FACS plots of CD44 and CD62L expression on cluster 11 compared with spleen T cell subsets. Tcm central memory T cells. (F) Histogram displaying the expression level of selected markers on T cell clusters. (G) Frequency of cytokine production of CD8+ T cells upon restimulation in vitro with OVA peptide. GzmB, granzyme B. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s test).

  • Fig. 5 CD226 signaling pathway is essential in mediating antitumor response induced by combination treatment in MC38 tumor model.

    (A) Cumulative distribution function plots show the expression of CD226 in total, clonal expanded, or nonexpanded CD8+ T cells. Fold changes of CD226 expression level are indicated for each subset. RPKM, reads per kilobase million. (B) FACS analysis of CD226 expression [mean fluorescence intensity (MFI)] on spleen/tumor CD8+ T cell populations, gating strategy indicated in FACS plots. Data show one representative experiment of two independent experiments (n = 10 mice per group). (C) Schematic shows LUVs. (D) Western blot shows phosphorylation status of ICOS, CD28, and CD226 with increasing PD-1 concentrations. (E) Western blot shows phosphorylation status of ICOS and CD226 from intratumoral OVA-specific CD8+ T cells purified from mice treated with isotype or anti–PD-1 Ab. (F) MC38 tumor–bearing mice were treated with CD226-blocking Ab or isotype IgG before immunotherapy with anti-GITR and anti–PD-1 or isotype IgGs. The percentages of survival are shown here. Data show one representative experiment of three independent experiments (n = 5 mice per group). (G) CD226 KO mice or WT littermates were challenged with MC38 tumor cells and treated with either anti-GITR and anti–PD-1 Ab or isotype Abs on days 6 and 13 after tumor implantation. Data show one representative experiment of two independent experiments (n = 7 to 8 mice per group). (H) CITRUS cluster result overlaid with indicated markers. Cluster C1 that was significantly affected with Ab treatment is circled. (I) Frequency of T cell cluster C1 displayed on a per-mouse basis with means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s test.

  • Fig. 6 CD226 signaling pathway is essential in mediating antitumor response induced by combination treatment in RENCA tumor model.

    (A) RENCA tumor–bearing mice were treated with either CD226-blocking Ab or isotype IgG before immunotherapy with anti-GITR and anti–PD-1 or isotype IgGs. The percentages of survival are shown here (n = 8 mice per group). ns, not significant. (B) Effect of Ab treatment on intratumoral Treg frequency (n = 7 to 8 mice per group). (C) Frequency of tumor and spleen gp70-specific CD8+ T cells from tumor-bearing mice treated with anti-GITR and/or anti–PD-1 Ab (n = 8 mice per group). (D) Frequency of CD226+Ki67+ cell in gp70-specific CD8+ T cell population (n = 7 to 8 mice per group). (E) viSNE plot of tumor-infiltrating T cells overlaid with the expression of selected markers. (F) Density viSNE plot of gp70-specific tumor-infiltrating CD8+ T cells from each treatment group day 11 after tumor challenge. (G) viSNE plot of RENCA infiltrating gp70-specific CD8+ T cells overlaid with color-coded T cell clusters identified by SPADE. (H) Frequency of selected T cell clusters (means ± SEM). Indicated cluster was highlighted on viSNE plot below the bar graph. (I) RENCA tumor–bearing mice were treated with CD226-blocking Ab or isotype IgG before immunotherapy with anti-GITR and anti–PD-1 or isotype IgGs. The percentages of survival are shown here (n = 10 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s test.

  • Fig. 7 Proposed model for anti–PD-1 and anti-GITR Ab combination therapy.

    pMHC, peptide–major histocompatibility complex; APC, antigen-presenting cells.

  • Table 1 Pathways specifically up-regulated in clonal expanded CD8+ T cells with Ab treatment (day 11).

    Genes specifically up-regulated in monotherapy or combination therapy compared with isotype control were analyzed using Illumina Gene Ontology Engine. Jak, Janus kinase; STAT, signal transducers and activators of transcription.

    TreatmentPathwayP value
    Anti–PD-1 + Anti-GITRAdaptive immune response1.50 × 10−08
    Cell cycle5.80 × 10−07
    Metabolism of lipids and lipoproteins3.00 × 10−04
    Anti–PD-1Lymphocyte activation2.30 × 10−05
    Gene targets for miR-124u (T cell activation)1.80 × 10−05
    Jak-STAT signaling pathway3.00 × 10−04
    Anti-GITRGlucose metabolism8.20 × 10−10
    Protein metabolism7.50 × 10−08
    The citric acid cycle and respiratory electron transport3.90 × 10−06

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/29/eaat7061/DC1

    Fig. S1. T cell depletion with Abs.

    Fig. S2. Bioinformatic pipeline rpsTCR.

    Fig. S3. Combination therapy expands intratumoral high-frequency tumor-reactive CD8+ T cell clones.

    Fig. S4. Combination treatment expands tumor antigen–specific CD8+ T cells with effector function.

    Fig. S5. Mean fluorescence intensity of markers for dysfunctional cell clusters identified in Fig. 2.

    Fig. S6. Mean fluorescence intensity of markers for effector/memory cell clusters identified in Fig. 3.

    Fig. S7. TIGIT expression at single-cell RNA level and FACS analysis of TIGIT/CD226 expression level on different T cell subsets.

    Fig. S8. GITR and PD-1 combination treatment significantly reduced highly activated Treg subsets.

    Fig. S9. GITR and PD-1 combination treatment induced intratumoral CD8+ T cell subsets distinct from CD25 and PD-1 combination therapy.

    Fig. S10. CD226−/− mice show normal T cell development and homeostatic function.

    Fig. S11. Effectiveness of combination treatment does not rely on CD28, OX40, and 4-1BB pathway.

    Fig. S12. Expression level of CD155.

    Table S1. Negative controls for rpsTCR.

    Table S2. Comparison of TCR detection rate.

    Table S3. Selected genes differentially regulated by each treatment.

    Table S4. Abs for flow cytometry.

    Table S5. Primers for TCRα/β repertoire sequencing.

    Table S6. Raw data.

    References (4852)

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. T cell depletion with Abs.
    • Fig. S2. Bioinformatic pipeline rpsTCR.
    • Fig. S3. Combination therapy expands intratumoral high-frequency tumor-reactive CD8+ T cell clones.
    • Fig. S4. Combination treatment expands tumor antigen–specific CD8+ T cells with effector function.
    • Fig. S5. Mean fluorescence intensity of markers for dysfunctional cell clusters identified in Fig. 2.
    • Fig. S6. Mean fluorescence intensity of markers for effector/memory cell clusters identified in Fig. 3.
    • Fig. S7. TIGIT expression at single-cell RNA level and FACS analysis of TIGIT/CD226 expression level on different T cell subsets.
    • Fig. S8. GITR and PD-1 combination treatment significantly reduced highly activated Treg subsets.
    • Fig. S9. GITR and PD-1 combination treatment induced intratumoral CD8+ T cell subsets distinct from CD25 and PD-1 combination therapy.
    • Fig. S10. CD226−/− mice show normal T cell development and homeostatic function.
    • Fig. S11. Effectiveness of combination treatment does not rely on CD28, OX40, and 4-1BB pathway.
    • Fig. S12. Expression level of CD155.
    • Table S1. Negative controls for rpsTCR.
    • Table S2. Comparison of TCR detection rate.
    • Table S3. Selected genes differentially regulated by each treatment.
    • Table S4. Abs for flow cytometry.
    • Table S5. Primers for TCRα/β repertoire sequencing.
    • References (4852)

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