Role of nuclear localization in the regulation and function of T-bet and Eomes in exhausted CD8 T cells

Abstract

The transcription factors T-bet and Eomesodermin (Eomes) regulate CD8 T cell exhaustion through undefined mechanisms. Here, we show that the subcellular localization of T-bet and Eomes dictate their regulatory activity in exhausted T cells (T_EXs). T_EXs had a higher ratio of nuclear Eomes:T-bet than memory T cells (T_MEMs) during chronic lymphocytic choriomeningitis virus (LCMV) infection in preclinical cancer models and in human tumors. Biochemically, T-bet and Eomes compete for the same DNA sequences, including the Pdcd1 T-box. High nuclear T-bet strongly represses Pdcd1 transcription in T_MEM, whereas low nuclear T-bet in T_EX leads to a dominant effect of Eomes that acts as a weaker repressor of Pdcd1. Blocking PD-1 signaling in T_EXs increases nuclear T-bet, restoring stronger repression of Pdcd1, and driving T-bet-associated gene expression programs of chemotaxis, homing, and activation. These data identify a mechanism whereby the T-bet-Eomes axis regulates exhaustion through their nuclear localization, providing insights into how these transcription factors regulate T_EX biology.

Keywords: Eomes; PD-1; T cell exhaustion; T-bet; cancer; checkpoint blockade; chronic infection; exhausted T cell reinvigoration.

Figure 1.. Exhausted CD8 T cells have a high ratio of nuclear Eomes to T-bet that correlates with PD-1 expression during LCMV infection

ImageStream analysis was performed on CD8⁺ T cells from Armstrong-immune (T_MEM, blue)- or clone 13 (T_EX, red)-infected mice at day 30 p.i. (A) Representative cell images acquired in IDEAS software from an Armstrong-immune mouse (T_MEM, left) or chronic clone 13 mouse (T_EX, right) are shown. Splenocytes were permeabilized and stained with T-bet (yellow). The location of the nucleus is indicated by DAPI (cyan). (B) Representative ImageStream flow plots displaying T-bet localization in T_MEMs or T_EXs are shown (left). Bar graphs display the frequency and median fluorescence intensity (MFI) of nuclear T-bet in LCMV-specific H-2D^b gp276⁺ CD8⁺ T cells (right). (C) Representative cell images acquired in IDEAS software from an Armstrong-immune mouse (T_MEM, left) or chronic clone 13 mouse (T_EX, right) are shown. Splenocytes were permeabilized and stained with Eomes (magenta). The location of the nucleus is indicated by DAPI (cyan). (D) Representative ImageStream flow plots displaying Eomes localization are shown (left). Bar graphs show the frequency and MFI of nuclear Eomes in LCMV-specific H-2D^b gp276⁺ CD8⁺ T cells (right). (E) The ratio of the MFI of nuclear Eomes:T-bet in LCMV-specific H-2D^b gp276⁺ T-bet⁺ Eomes⁺ CD8 T cells is shown. (F) A correlation plot displaying the ratio of the MFI of nuclear Eomes:T-bet versus MFI of PD-1 in LCMV-specific T-bet⁺ Eomes⁺ CD8⁺ T cells from T_EXs is shown. A Pearson coefficient (r value) is displayed on the graph. All data are represented as mean ± SEM; p values were determined using the Student’s t test (***p < 0.0001, **p < 0.001, *p < 0.05). Each graph is representative of 3 or 4 independent experiments with 4–5 mice per experimental condition.

Figure 2.. The ratio of nuclear Eomes:T-bet correlates with severe exhaustion in TIL from metastatic melanoma patients

Peripheral blood and tumor-infiltrating lymphocytes were isolated from stage IV human metastatic melanoma patients and analyzed on the ImageStream. (A) Representative images of CD8⁺ (orange) TIL from two patients are shown. Cells were stained with PD-1 (blue), T-bet (yellow), and Eomes (magenta), and nuclei are indicated by DAPI (cyan). (B) The bar graphs show the MFI of nuclear T-bet (left) and the MFI of nuclear Eomes (right) in non-naive CD8⁺ PBMC (gray) and TIL (light purple). (C) The ratio of the MFI of nuclear Eomes:T-bet in non-naive CD8⁺ T-bet⁺ Eomes⁺ PBMC and TIL is displayed. Data are represented as mean ± SEM; p values were determined using the unpaired Mann-Whitney Student’s t test (***p < 0.0001; **p < 0.001). Bar graphs were generated from 6 blood samples and 9 TIL samples.

Figure 3.. Blocking the PD-1 pathway re-balances the nuclear Eomes:T-bet ratio during chronic LCMV infection

(A) LCMV-specific H-2D^b gp276⁺ CD8 T cells from B6 control PBS (gray) or αPD-L1-treated mice (green) infected with LCMV clone 13. Representative flow plots show the co-expression of T-bet and Eomes in LCMV-specific CD8 T cells. The frequency of the T-bet⁺Eomes⁺ population is shown. (B) Representative ImageStream flow plots displaying the subcellular distribution of T-bet (top) or Eomes (bottom) in LCMV-specific T-bet⁺ Eomes⁺ CD8 T cells from PBS-treated (gray) or αPD-L1-treated (green) mice are shown. (C) The ratio of the MFI of nuclear Eomes:T-bet in LCMV-specific T-bet⁺ Eomes⁺ CD8 T cells is shown. (D) The mean viral load of mice pre-PBS (day 15) and post-PBS (day 35) or αPD-L1 therapy is shown. Data are represented as mean ± SEM; p values were determined using the unpaired Mann-Whitney Student’s t test (***p < 0.0001). Bar graphs were generated from a single experiment with 4–5 mice per experimental condition. Each bar graph is representative of 3 independent experiments.

Figure 4.. Blocking the PD-1 pathway in CT26 tumors re-balances the nuclear Eomes-to-T-bet ratio and correlates with tumor progression

(A) Experimental design for CT26 tumor studies.BALB/C mice were injected with CT26 tumor cells, and beginning at day 10 post-injection, mice were treated 4 times with control PBS or αPD-L1 every 3 days. Mice were sacrificed at day 20 post-tumor injection. (B) Bar graph displaying the weight of CT26 tumors at day 20 post-tumor cell injection is shown for control PBS-treated (gray) or αPD-L1-treated (blue) mice. (C) The frequency of CD8⁺ TIL that contain nuclear T-bet is shown. (D) Correlation plots displaying the frequency ofCT26 TIL with nuclear T-bet compared to tumor weight are shown. (E) A bar graph displaying the ratio of nuclear Eomes:T-bet in CT26 CD8⁺ TIL is shown (left). A correlation plot highlighting the ratio of the MFI of nuclear Eomes:T-bet in CD8⁺ TIL versus tumor weight in control PBS-treated mice (gray) compared to αPD-L1-treated mice (blue) is shown (right). Data are represented as mean ± SEM; p values were determined using the unpaired Mann-Whitney Student’s t test (**p < 0.001). Bar graphs were generated from a single experiment with 20–30 mice per experimental condition. For correlation graphs, a Pearson correlation analysis was performed, and p values are displayed on the graph.

Figure 5.. Blocking the PD-1 pathway re-engages T-bet transcriptional circuits during chronic LCMV infection

(A) Heatmaps display the raw expression values of chronic-only T-bet (left) or Eomes (right) gene neighbors from clone-13-infected mice following PBS (T_EX) or αPD-L1 treatment. Genes were clustered by K-means. (B) Gene Ontology pathway analysis was performed on the gene clusters from T-bet neighbors (top, cluster 3) or Eomes neighbors (bottom, cluster 4). (C) Histograms from a representative clone-13-infected control (shaded gray) or αPD-L1-treated (green line) mouse show the MFI of candidate proteins in CD44⁺ PD-1⁺ CD8 T cells. Expression of candidate proteins in naive CD8 T cells are shown (dashed line). Histograms are representative of 5 mice per experimental condition.

Figure 6.. Forcing T-bet to the nucleus engages an effector-like program in T_EXs

(A) Naive CD45.2⁺ recipient mice were infected with LCMV clone 13 on day 0. Concurrently, naive CD45.1⁺ CD45.2⁺ P14 cells were isolated, activated using anti-CD3/CD28/IL-2 for 24 h, and transduced with a MSCV-T-bet-ER-GFP retroviral vector. Transduced cells were then transferred into LCMV-clone-13-infected mice on day 1. Beginning on day 22, recipient mice were treated with tamoxifen or PBS (control) every day for 5 days. Splenocytes were harvested on day 32 post-infection and analyzed by flow cytometry. (B) The frequency (left) and total cell number (right) of GFP⁺ non-naive (CD44⁺) P14 cells from tamoxifen-treated or control PBS-treated mice are shown. (C) The frequency (left) and total cell number (right) of KLRG1⁺CD39⁺ effector-like cells within the GFP⁺ P14 splenocytes are shown. (D) A representative histogram of CD69 expression within GFP⁺ P14 cells from control (black) or tamoxifen-treated (red) mice is shown (left). The frequency of CD69⁺ cells within the GFP⁺ P14 population is plotted (right). Data are represented as mean ± SEM; p values were determined using the unpaired Mann-Whitney Student’s t test (**p < 0.001). Bar graphs were generated from a single experiment with 4–5 mice per experimental condition and are representative of 2 independent experiments. Histograms are representative of 5 mice per experimental condition.

Figure 7.. T-bet and Eomes compete for binding to and repressing Pdcd1 transcription

(A) Immobilized T-bet bound to a T-box consensus sequence was competed with recombinant Eomes, and binding of both T-bet and Eomes was determined using an ELISA-based binding assay (described in STAR Methods and Figure S2B). The bar graph displays the fold change in binding of T-bet or Eomes following competition with Eomes⁺ lysate relative to each uncompleted protein. (B) Immobilized Eomes bound to a T-box consensus sequence was competed with recombinant T-bet, and binding of both T-bet and Eomes was determined using an ELISA-based binding assay (described in STAR Methods and Figure S2B). The bar graph displays the fold change in binding of Eomes or T-bet following competition with T-bet⁺ lysate relative to each uncompleted protein. (C) T-bet or Eomes binding to a consensus T-box sequence was competed with oligos corresponding to a canonical T-box half site sequence or the T-box sequences found in Pdcd1 or Il2. The bar graph displays the fold change in binding of Eomes or T-bet following competition with various T-box-containing oligos relative to each uncompleted protein. (D) PCR amplification of immunoprecipitated DNA sequences in EL4 cells expressing exogenous T-bet or Eomes is shown. Ifng(positive) and Il4 (negative) regions were used as controls. (E) Dual-luciferase reporter assays were performed using a reporter plasmid containing two conserved regions within the Pdcd1 promoter region (CR-B+C). A bar graph shows the luciferase activity observed in PMA/ionomycin-stimulated EL4 cells nucleofected with wild-type T-bet, Eomes, or co-nucleofected T-bet and Eomes plasmids. Data were normalized to EL4 cells containing the firefly luciferase reporter only. (F) Endogenous PD-1 expression in unstimulated (left) or PMA/ionomycin-stimulated (right) EL4 cells is shown in T-bet⁺ (green), Eomes⁺ (blue), or T-bet⁺ Eomes⁺ (pink) cells relative to T-bet⁻, Eomes⁻, or T-bet⁻Eomes⁻ cells (gray shaded). Representative histograms from one experiment are shown. Data in bar graphs are represented as mean ± SEM; p values were determined using a one-way ANOVA test (****p < 0.0001; ***p < 0.001, *p < 0.05).