Cutting edge: The transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche

Abstract

CD8(+) T cells responding to intracellular infection give rise to cellular progeny that become terminally differentiated effector cells and self-renewing memory cells. T-bet and eomesodermin (Eomes) are key transcription factors of cytotoxic lymphocyte lineages. We show in this study that CD8(+) T cells lacking Eomes compete poorly in contributing to the pool of Ag-specific central memory cells. Eomes-deficient CD8(+) T cells undergo primary clonal expansion but are defective in long-term survival, populating the bone marrow niche and re-expanding postrechallenge. The phenotype of Eomes-deficient CD8(+) T cells supports the hypothesis that T-bet and Eomes can act redundantly to induce effector functions, but can also act to reciprocally promote terminal differentiation versus self-renewal of Ag-specific memory cells.

Figure 1. Diminished central-memory CD8⁺ T cell compartment in the absence of Eomes

(A) Eomes expression in effector (8 days post LCMV infection, GP33⁺) and memory (90 days post LCMV infection, GP33⁺) CD8⁺ T cells relative to naïve (CD44^lo GP33^-) CD8⁺ T cells from spleens of C57BL/6 mice. (B) Quantification of CD8⁺ GP33⁺T cells harvested from spleens of wild-type and Eomes KO mice 60 days after infection with LCMV. Data in (A) and (B) are representative of three independent experiments. (C) GP33-specific central-memory CD8⁺ T cells in spleens of wild-type versus Eomes KO mice 60 days after infection with LCMV. Plots show CD8⁺CD44 ⁺GP33 ⁺ cells. Numbers within plots refer to percentage of cells in the upper right quadrant. Graph shows mean ^+/− SEM from six mice of each genotype. (D) Central-memory (CD62L^hi CD44^hi), effector-memory (CD62L^loCD44 ^hi) and naïve (CD62L^hi CD44^lo) CD8⁺ T cell compartments in spleens of 6–12 month old naïve, non-infected wild-type, T-bet KO, or Eomes KO mice. Numbers within plots refer to percentage of cells in the respective quadrant. Data are representative of three independent experiments. (E) Listeria monocytogenes-GP33 (LMgp33) burden in liver or spleen 3 days after infection with 5×10⁵ LMgp33 organisms in non-immune mice (non-immune WT), immunized (by LCMV infection 100 days prior) wild-type mice (immune WT), and immunized Eomes KO mice (immune KO) with three mice per group. Data is representative of two independent experiments.

Figure 2. Defective memory CD8⁺ T cell persistence and re-expansion in the absence of Eomes in competitive models

(A) Serial flow cytometric analysis of peripheral blood from Eomes KO/WT bone marrow chimeras, showing CD8⁺GP33 ⁺ and CD8⁺ NP396⁺ cells derived from Eomes KO bone marrow or WT bone marrow, as labeled, versus time in relation to initial LCMV infection. Data are representative of three independent experiments with four chimeric mice each. (B) Ratio of wild-type to Eomes KO CD8⁺ GP33⁺ T cells (top plot) and IFN-γ⁺ CD8⁺ T cells (bottom plot) in the spleen of a Eomes KO/WT bone marrow chimera spleen five days after rechallenge with LCMV (65 days after primary infection). Numbers within quadrants represent percentage of cells within that quadrant. Data are representative of five independent experiments. (C) Relative number of Eomes KO (CD8⁺ Thy1.2⁺ CD45.1⁻) and wild-type (CD8⁺ Thy1.2⁻ CD45.1⁻) P14 cells in the blood of recipients 8 days or 45 days after primary LCMV infection. Data are mean ^+/− SEM and are derived from four chimeric mice in each of three independent experiments. (D) Ratio of WT versus Eomes KO CD8⁺ GP33⁺ cells in Eomes KO/WT bone marrow chimeras 60 days after LCMV infection. Data presented as mean and SEM from four individual chimeras. * P < .01 vs. blood, P < .05 vs. lymph node or spleen; Student’s two-tailed T test (E) Ratio of P14 cells in spleen relative to bone marrow 8 days after LCMV infection for both wild-type and Eomes KO populations in a competitive adoptive transfer setting. Data are representative of 7 co-transfer recipients over two independent experiments. (F) Relative number of Eomes KO (CD8⁺ Thy1.2⁺ CD45.1⁻) and wild-type (CD8⁺ Thy1.2⁻ CD45.1⁻) P14 cells in the blood of recipients on the day of LCMV re-challenge (45 days after primary infection) and five days after re-challenge. Data are mean ^+/− SEM and are representative of three independent experiments with four mice per experiment. (G) Relative number of GP33⁺ or NP396⁺ CD8⁺ cells in the blood of Eomes KO or Wild-type mice at least 90 days after initial LCMV infection on the day of LCMV re-challenge and five days after re-challenge. Data are mean ^+/− SEM with four mice per group. (H) Eomes expression in short-lived effector (KLRG1^hi CD127^lo CD8⁺ GP33⁺) and memory-precursor (KRLG1^lo CD127^hi CD8⁺ GP33⁺) populations in spleens from mice 8 days after LCMV infection. Data is representative of four independent experiments. (I) KLRG1^hi CD127^lo cells (number in left upper quadrant) and KLRG1^lo CD127^hi cells (number in right lower quadrant) in wild-type (CD8⁺ Thy1.2⁻ CD45.1⁻) and Eomes KO (CD8⁺ Thy1.2⁺ CD45.1⁻) P14 cells in a competitive adoptive transfer setting 8 days after LCMV infection. Data is representative of 2 independent experiments with a total of 7 co-transfer recipients. (J) Eomes expression in naïve (CD44^lo CD62L^hi GP33⁻), central-memory (CD44^hi CD62L^hi GP33⁺) and effector-memory (CD44^hi CD62L^lo GP33⁺) CD8⁺ T cell populations from the spleen of a mouse 240 days after LCMV infection. Data are representative of two independent experiments.

Figure 3. Defective bone marrow localization and proliferation Eomes KO memory CD8⁺ T cells

(A) CXCR4, integrin α4, and integrin β1 expression on CD8⁺ GP33⁺T cells from spleens of Eomes KO/WT bone marrow chimeras 60 days after LCMV infection. Plots are representative results from three chimeric mice. (B) Quantitative RT-PCR of CXCR4 or CXCR3 mRNA from wild-type versus Eomes KO central-memory (CD44^hi CD62L^hi) CD8⁺ cells sorted from an individual Eomes KO/WT bone marrow chimera 60 days after infection with LCMV. Data are representative of two independent experiments. (C) BrdU uptake in Eomes KO versus wild-type CD8⁺ GP33⁺ T cells from the bone marrow of Eomes KO/WT bone marrow chimeras 60 days after LCMV infection. Data are derived from four chimeric animals. (D) Quantitative RT-PCR of Bcl-2 mRNA as in (B). (E) CD122, CD127, and CD27 expression on CD8⁺ GP33⁺ T cells from spleens of wild-type and Eomes KO mice 60 days after infection with LCMV. Data are representative of three independent experiments.