Eomesodermin is required for antitumor immunity mediated by 4-1BB-agonist immunotherapy

Abstract

CD8+ T cells in progressing tumors frequently fail to mount an effective antitumor response often in association with the expression of inhibitory receptors, including programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (Lag3). Using a lymphoma tumor model, we demonstrate that tumor-infiltrating CD8+ T cells from growing tumors co-express inhibitory receptors and co-stimulatory receptors, including 4-1BB (TNFRSF9) as well as high levels of 2 transcription factors, Eomesodermin (Eomes) and T-bet (Tbx21), critical determinants of CD8+ T cell fate. Immunotherapy with an agonistic anti-4-1-BB antibody altered the ratio of Eomes to T-bet expression in tumor-infiltrating CD8+ T cells by increasing Eomes and decreasing T-bet expression. 4-1BB-agonist immunotherapy was also associated with downregulated expression of the inhibitory receptors PD-1 and Lag3 on tumor-infiltrating CD8+ T cells, a molecular phenotype associated with subsequent attenuation of tumor growth. Furthermore, 4-1BB-agonist immunotherapy failed to effect tumor progression in mice with Eomes deficient T cells. However, upon resumption of tumor growth, tumor-infiltrating CD8+ T cells from treated animals continued to express high levels of Eomes as well as elevated levels of the inhibitory receptors PD-1 and Lag3. Our data suggest that tumor-infiltrating CD8+ T cells are poised between activation and inhibition as dictated by expression of both co-stimulatory receptors and inhibitory receptors and demonstrate that T cell expression of Eomes is necessary, but not sufficient, for efficacious 4-1BB-agonist-mediated immunotherapy.

Keywords: 4-1BB; Lag3; PD-1; T cells; eomesodermin; tumor immunotherapy.

Figures

Figure 1. Co-expression of Eomes, T-bet, PD-1, Lag3, 4-1BB, and OX40 by CD8+ CD44hi infiltrating lymphocytes in EG7 tumors. (A–C) Cytofluorometric analysis of tumor-infiltrating lymphocytes (TILs) present in harvested EG7 tumors 21 d after tumor cell s.c. inoculation into the flank of C57BL/6 host mice. Tumors were enzymatically dissociated to a single cell suspension and cells were stained with fluorophore-conjugated antibodies against the indicated markers. Flow cytometry was performed by gating on CD8+ cells. (A) Dot plot showing the expression of T-bet and Eomes in CD8+ CD44hi cells among TILs vs. splenic controls. (B) Histograms of the expression of the indicated proteins in either CD8+ CD44lo or CD8+ CD44hi tumor or spleen control cells, as indicated. Data in (A) and (B) are representative of at least 3 independent experiments. (C) 5 × 104 OVA-specific CD8+ T cells magnetically enriched from spleen preparations from CD45.1 OT-1 donor mice were adoptively transferred to each mouse by i.v. injection on the same day as EG7 flank tumor inoculation. TILs were harvested 21 d later and analyzed by flow cytometry, as above. Dot plots show expression of the indicated marker in CD45.1+ (donor derived) and CD45.1− (host derived) subsets of CD8+ CD44hi cells. Data are representative of three independent experiments. 4-1BB/TNFRSF9, tumor necrosis factor receptor superfamily member 9; Eomes, eomesodermin; CTLA4, cytotoxic T-lymphocyte antigen 4; Lag3, lymphocyte-activation gene 3; PD-1, programmed cell death-1; T-bet/Tbx21, T-box 21.

Figure 2. Diminished Lag3 expression on Eomes-deficient CD8+ CD44hi TILs. (A–C) 1 × 106 EG7 cells were subcutaneously injected into the flank of 6–12 wk old C57BL/6 wild-type (WT), Eomes KO (EKO), T-bet KO (TKO) or double KO (DKO) recipient mice. Data are representative of at least 3 independent experiments. (A) Cytofluorometric analysis of immunofluorescence stained, dissociated tumor cells 21 d after tumor cell inoculation. Histograms of the expression level of the indicated marker in CD8+ CD44hi splenocytes or EG7 tumor-infiltrating lymphocytes (TILs). (B) Growth curves for EG7 tumors in C57BL/6 controls vs. EKO, TKO, and DKO mice. Each point represents the mean ± SEM of tumor measurements (n = 8 per group). Statistical analyses were performed by Student t test with no significant differences between WT and EKO, TKO, or DKO at any time point (P > 0.05). (C) Quantitation of CD8+ CD44hi TILs in EG7 tumors from mice in (B), 21 d post tumor inoculation as determined by flow cytometry. Bars represent the mean ± SEM of the log CD8+ CD44hi TILs per gram tumor (n = 8 per group). Statistical analyses were performed by Student t test with no significant differences found between WT and EKO, TKO, or DKO (P > 0.05). 4-1BB/ TNFRSF9, tumor necrosis factor receptor superfamily member 9; Eomes, eomesodermin; KO, knockout; Lag3, lymphocyte-activation gene 3; PD-1, programmed cell death-1; Tbet, T-bet/Tbx21, T-box 21;

Figure 3. Enhanced Eomes expression and diminished T-bet expression in CD8+ CD44hi TILs after in vivo administration of α4-1BB. (A–C) C57BL/6 mice bearing EG7 tumors were administered 100 μg α4-1BB antibody or control rat IgG on day 7 and day 10 after tumor cell inoculation. (A) Tumor growth curves. Each data point represents the mean ± SEM of tumor measurements (n = 8 per group). Data are from one of four independent experiments). Statistical analyses were performed by Student t test; *P < 0.05 and **P < 0.01. (B–C) Cytofluorometric analysis of immunofluorescence stained, dissociated tumor cells 13 d after tumor cell inoculation and 3 d after the second dose of α4-1BB antibody or IgG control. Data are representative of three independent experiments. (B) Histograms of the expression level of T-bet (Tbx21, T-box 21) and Eomes (eomesodermin) in CD8+ CD44hi gated tumor-infiltrating lymphocytes (TILs) in EG7 tumors from α4-1BB (red line in B) or rat IgG (blue line in B) treated animals. Isotype control staining of CD8+ CD44hi gated TILs from IgG-treated animal is shown for comparison (black dashed line in B). (C) Dot plot showing change in distribution of Eomes, T-bet and KLRG1 expression in CD8+ CD44hi TILs in response to the indicated treatment.

Figure 4. Eomes is required for α4-1BB mediated delay in tumor progression. (A) C57BL/6 (WT) or Eomes KO (EKO) mice bearing EG7 tumors were administered 100 μg α4-1BB or control rat IgG on d7 and d10 after tumor cell inoculation. Tumor growth curves are shown, with each point representing the mean ± SEM of the tumor area (n = 8 per group). Data are from one of three independent experiments. Statistical analyses were performed by Student t test; IgG vs. α4-1BB in WT mice *P < 0.05 and **P < 0.01 whereas in EKO mice no significant differences were detected between treatment groups. (B) Tumor growth curves for s.c. flank EG7 tumors in WT or EKO mice immunized by s.c. injection of phosphate-buffered saline (PBS) or CpG oligodeoxynucleotide and ovalbumin (CPG-OVA) 14d prior to tumor inoculation and on the day of tumor cell inoculation. Each point represents the mean ± SEM of the tumor area (n = 4 per group). Data are from one of two independent experiments. Statistical analyses were performed by Student t test for differences between PBS and CPG-OVA groups in WT mice (*P < 0.05 and **P < 0.01) or EKO mice (†P < 0.05 and ††P < 0.01). (C) Cytofluorometric analysis of immunofluorescence stained, dissociated tumor cells for KLRG1, PD-1 and Lag3 in CD8+ CD44hi gated TILs from EG7 tumors 13d after tumor inoculation and 3d after the second dose of α4-1BB antibody (red line) or rat IgG (blue line) treatment in vivo. Isotype control staining of CD8+ CD44hi gated TILs from IgG-treated animal is shown for comparison (black dashed line). Data are representative of 3 independent experiments. (D) Quantitation of lung nodules from WT or EKO mice 14 d after i.v. injection of B16 cells followed by administration of 100 μg α4-1BB or control rat IgG on d6 and d9 after B16 injection. Bars represent mean ± SEM of lung nodule counts (n = 5 per group). Data are from 1 of 2 independent experiments. Statistical analyses were performed by Student t test; IgG vs. α4-1BB groups *P < 0.05 in WT mice whereas no significant differences were detected between treatment groups in EKO mice. (E) Photo of representative lungs from each of the four groups from (D).

Figure 5. Analysis of Eomes, PD-1 and Lag3 expression in CD8+ CD44hi TILs from tumors progressing after α4-1BB immunotherapy. Immunofluorescent staining and cytofluorometric analysis for expression of Eomes, KLRG1, PD-1, or Lag3 expression in CD8+ CD44hi gated tumor-infiltrating lymphocytes (TILs) derived from EG7 tumors during α4-1BB antibody mediated tumor stasis/regression (d13 post tumor inoculation and d3 after the second dose of α4-1BB or control rat IgG) and upon resumption of tumor growth (d22 post tumor inoculation and d12 after the second dose of α4-1BB). All data are representative of at least three experiments.