Regulation of CD8 T Cell Differentiation by the RNA-Binding Protein DDX5

Abstract

The RNA-binding protein DEAD-box protein 5 (DDX5) is a polyfunctional regulator of gene expression, but its role in CD8+ T cell biology has not been extensively investigated. In this study, we demonstrate that deletion of DDX5 in murine CD8+ T cells reduced the differentiation of terminal effector, effector memory T, and terminal effector memory cells while increasing the generation of central memory T cells, whereas forced expression of DDX5 elicited the opposite phenotype. DDX5-deficient CD8+ T cells exhibited increased expression of genes that promote central memory T cell differentiation, including Tcf7 and Eomes. Taken together, these findings reveal a role for DDX5 in regulating the differentiation of effector and memory CD8+ T cell subsets in response to microbial infection.

FIGURE 1.

DDX5 may regulate CD8⁺ T cell proliferation and apoptosis. (A) Experimental set-up. Congenically distinct Ddx5^fl/fl Cd4-Cre⁺ (Ddx5^cKO) cells and Ddx5^fl/fl CD4-Cre⁻ (WT) CD8⁺ P14 T cells were mixed at a 1:1 ratio and adoptively transferred i.v. into CD45.2⁺ hosts. One day after transfer, recipient mice were infected with LCMV, sacrificed at days 7 and 30 p.i., and donor cells analyzed by flow cytometry. (B) Representative flow cytometry plot showing the proportions of WT vs Ddx5^cKO cells (left) and ratio of the absolute numbers of Ddx5^cKO to WT CD8⁺ P14 cells at days 7 and 30 p.i., normalized to the input ratio (right). (C) Absolute numbers of WT vs. Ddx5^cKO CD8⁺ P14 cells at days 7, 14, 30 and 60 post-infection. (D) Ratio of Ddx5^cKO to WT CD8⁺ P14 cells at days 7, 14, 30 and 60 post-infection, normalized to the input ratio. (E-F) Representative flow cytometry plots and histograms showing the frequency, absolute numbers, and MFI of Ki67⁺ WT vs, Ddx5^cKO CD8⁺ P14 cells at day 7 post-infection. Gates were determined by Ki67 fluorescence minus one (FMO). (G-H) CFSE proliferation analysis of WT and Ddx5^cKO CD8⁺ T cells at 48 hours p.i., represented as (G) flow cytometry plots (left), histograms (right). (H) Frequency and absolute numbers of CFSE-labeled undivided, Division 1 (Div 1), Division 2 (Div 2), Division 3 (Div 3), and CFSE⁻ WT vs. Ddx5^cKO CD8⁺ P14 cells. (I-J) Representative flow plots and histograms and showing the frequency, absolute numbers, and MFI of Annexin V⁺ WT and Ddx5^cKO CD8⁺ P14 cells at 48 hours post-infection. All data are from one representative experiment out of two independent experiments with n = 5 to 10 per group: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s paired two-tailed t test). Graphs indicate mean ± SEM, symbols represent individual mice.

FIGURE 2.

DDX5 regulates CD8⁺ TE, T_EM, and t-T_EM cell differentiation. (A) Representative flow cytometry plots (left) and quantification (right) of WT and Ddx5^cKO TE- and MP-phenotype cells. (B) Representative flow cytometry plots (left) and quantification (right) of WT and Ddx5^cKO T_CM, T_EM, t-T_EM cells at day 30 p.i. (C) Ratio of Ddx5^cKO and WT T_CM-, T_EM-, t-T_EM-phenotype CD8⁺ P14 cells at day 30 post-infection. (D) Representative flow cytometry plots (bottom) and quantification (top) of TNF⁺IFNy⁺ WT and Ddx5^cKO cells isolated at day 30 p.i. and restimulated ex vivo in the presence of GP_33–41 peptide for 3 hours. (E) Representative flow plots (left) and quantification (right) of WT and Ddx5^cKO IL-2⁺ CD8⁺ T cells after ex vivo restimulation at day 30 p.i. (F) Experimental set-up. Congenically distinct CD8⁺ T cells were activated and transduced with empty vector (EV) or forced Ddx5 expression (Ddx5^OE) retroviral constructs. Transduced cells were mixed at a 1:1 ratio and adoptively transferred into CD45.2⁺ recipients. Recipient mice were infected with LCMV and sacrificed at days 7 and 30 p.i. (G) Representative flow cytometry plots (left) and quantification (right) of EV and Ddx5^OE TE- and MP-phenotype cells at day 7 p.i. (H) Representative flow cytometry plots (left) and quantification (right) of EV and Ddx5^OE T_CM, T_EM, t-T_EM cells at day 30 p.i. (I) Ratio of Ddx5^OE and WT T_CM-, T_EM-, t-T_EM-phenotype CD8⁺ P14 cells at day 30 post-infection. (J) Representative flow plots (left) and quantification (right) of EV and Ddx5^OE IFN-γ⁺ and TNF⁺ CD8⁺ T cells after ex vivo restimulation at day 30 p.i. (I) Representative flow plots (left) and quantification (right) of EV and Ddx5^OE IL-2⁺ CD8⁺ T cells after ex vivo restimulation at day 30 p.i. All data are from one representative experiment out of two independent experiments with n = 5–10 per group; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; student’s paired two-tailed t test (A,B,D,E,G,H,J,K), one sample t test (C and I). Graphs indicate mean ± SEM, symbols represent individual mice.

FIGURE 3.

DDX5 regulates the maintenance of established CD8⁺ t-T_EM cells. (A) Experimental set-up. Congenically distinct Ddx5^fl/fl Ert2-Cre⁻ (WT ) and Ddx5^fl/fl Ert2-Cre⁺ (Ddx5^iKO ) CD8⁺ P14 T cells were mixed at a 1:1 ratio and adoptively transferred into CD45.2⁺ recipients intravenously. One day post-transfer, recipient mice were infected with LCMV. At days 30–33 p.i., mice received 1μg tamoxifen i.p. to induce the deletion of Ddx5. Recipient mice were sacrificed at day 40 p.i. and donor cells were analyzed by flow cytometry. (B) Representative flow cytometry plots (left) and quantification (right) of WT and Ddx5^iKO CD8⁺ P14 cells at day 40 p.i. (C) Representative flow cytometry plots (left) and quantification (right) of WT and Ddx5^iKO T_CM, T_EM, and t-T_EM cells at day 40 p.i. (D) Representative flow plots (left) and quantification (right) of WT and Ddx5^iKO IFN-γ⁺ and TNF⁺ CD8⁺ P14 cells after ex vivo restimulation at day 30 p.i. (E-F) IFNγ and TNF median fluorescence intensity in WT and Ddx5^iKO T_CM, T_EM, and t-T_EM cells at day 40 p.i. (G) Representative flow plots (left) and quantification (right) of WT and Ddx5^iKO IL-2⁺ CD8⁺ T cells after ex vivo restimulation at day 40 p.i. (H) IL-2 median fluorescence intensity of WT and Ddx5^iKO T_CM, T_EM, and t-T_EM cells at day 40 p.i. All data are from one representative experiment out of two independent experiments with n = 5 to 10 per group; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s paired two-tailed t test). Graphs indicate mean ± SEM, symbols represent individual mice.

FIGURE 4.

Genes associated with T_CM cell differentiation are upregulated in the absence of DDX5. (A) Experimental set-up. Congenically distinct Ddx5^fl/fl Cd4-Cre⁻ (WT) and Ddx5^fl/fl Cd4-Cre⁺ (Ddx5^cKO) CD8⁺ P14 cells were mixed and adoptively transferred into CD45.2⁺ recipients. One day after transfer, recipient mice were infected with LCMV. Mice were sacrificed at days 4, 7, and 30 p.i. and stained with a TotalSeq-A antibody panel (Supplemental Table 1) before FACS-sorting and processing for CITE-seq. (B) UMAP analyses of WT and Ddx5^cKO CD8⁺ T cells on days 4, 7, and 30 p.i. (C) Proportions of WT (red) and Ddx5^cKO (blue) cells within each cluster. (D-F) T_CM, T_EM, and t-T_EM gene signature scores applied to Day 30 clusters, represented as violin plots. (G-I) T_CM, T_EM, and t-T_EM gene signature scores applied to WT and Ddx5^cKO cells within each Day 30 cluster and represented as violin plots. (J) Heatmap representing the expression of selected genes among cells from each of the Day 30 clusters. (K) Quantification of expression level of selected genes among cells from each of the Day 30 clusters, represented as violin plots. (L) Quantification of selected proteins by Day 30 cells, represented as relative expression plots superimposed on individual cells in the UMAP.

FIGURE 5.

DDX5-deficient t-TEM cells exhibit increased TCF1 and Eomes protein expression. (A) Representative flow cytometry plots (left) and quantification (right) of proportions of WT and Ddx5^cKO TE and MP CD8⁺ T cells expressing TCF1 at day 7 p.i. (B) Representative histograms (left) and median fluorescence intensity (right) of TCF1 expression in WT and Ddx5^cKO TE and MP cells at day 7 p.i. (C) Representative flow cytometry plots (left) and quantification (right) of proportions of WT and Ddx5^OE TE and MP CD8⁺ T cells expressing TCF1 at day 7 p.i. (D) Representative histograms (left) and median fluorescence intensity (right) of TCF1 expression in WT and Ddx5^OE TE and MP cells at day 7 p.i. (E) Representative flow cytometry plots (left) and histograms (middle) and quantification (right) of TCF1 MFI in WT and Ddx5^cKO CD8⁺ P14 cells at day 30 p.i. (F) Representative histograms (left) and quantification (right) of proportions of WT and Ddx5^cKO T_CM, T_EM, t-T_EM cells expressing TCF1. (G) Representative flow cytometry plots (left), histograms (middle), and quantification (right) of TCF1 MFI in WT and Ddx5^OE CD8⁺ P14 cells at day 30 p.i. (H) Representative histograms (left) and quantification (right) of proportions of WT and Ddx5^OE T_CM, T_EM, t-T_EM cells expressing TCF1. (I) Representative flow cytometry plots (left) and histograms (middle) and quantification (right) of Eomes MFI in WT and Ddx5^cKO CD8⁺ P14 cells at day 30 p.i. (J) Representative histograms (left) and quantification (right) of proportions of WT and Ddx5^cKO T_CM, T_EM, t-T_EM cells expressing Eomes.(K) Representative flow cytometry plots (left), histograms (middle), and quantification (right) of Eomes MFI in WT and Ddx5^OE CD8⁺ P14 cells at day 30 p.i. (L) Representative histograms (left) and quantification (right) of proportions of WT and Ddx5^OE T_CM, T_EM, t-T_EM cells expressing Eomes. All data are from one representative experiment out of two independent experiments with n = 5 to 10 per group; ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s paired two-tailed t test). Graphs indicate mean ± SEM, symbols represent individual mice.