Sustained Id2 regulation of E proteins is required for terminal differentiation of effector CD8+ T cells

Abstract

CD8⁺ T cells responding to infection differentiate into a heterogeneous population composed of progeny that are short-lived and participate in the immediate, acute response and those that provide long-lasting host protection. Although it is appreciated that distinct functional and phenotypic CD8⁺ T cell subsets persist, it is unclear whether there is plasticity among subsets and what mechanisms maintain subset-specific differences. Here, we show that continued Id2 regulation of E-protein activity is required to maintain the KLRG1^hi CD8⁺ T cell population after lymphocytic choriomeningitis virus infection. Induced deletion of Id2 phenotypically and transcriptionally transformed the KLRG1^hi "terminal" effector/effector-memory CD8⁺ T cell population into a KLRG1^lo memory-like population, promoting a gene-expression program that resembled that of central memory T cells. Our results question the idea that KLRG1^hi CD8⁺ T cells are necessarily terminally programmed and suggest that sustained regulation is required to maintain distinct CD8⁺ T cell states.

Figure 1.

Late deletion of Id2 in antigen-specific CD8⁺ T cells results in conversion from a KLRG1^hi to KLRG1^lo population. (A) Schematic of experimental set-up. CD45.1 hosts were given a cotransfer of Id2^f/f-ERCre⁻ (CD45.1.2) and Id2^f/f-ERCre⁺ (CD45.2) P14 CD8⁺ T cells then subsequently infected with LCMV. More than 30 d after infection, host mice were treated for 5 consecutive days with tamoxifen (Tam) to induce Id2 deletion. Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ CD8⁺ T cells are called Id2WT or Id2KO, respectively, after Tam treatment. (B) Flow cytometry of transferred P14 CD8⁺ T cells from host peripheral blood lymphocytes (PBL) or spleen (Sp) and LN 15 or 16 d after the last Tam treatment, respectively. Frequency of Id2WT and Id2KO cells among P14 CD8⁺ T cells (left), KLRG1 and CD127 expression (middle), and CD27 and CD43 expression (right) are shown. Numbers in plots represent the percentage of cells. (C) Quantification of donor cell frequency and number or (D) frequency of populations from B. (E) Flow cytometry of cells from B for CD62L and CD127 expression. (F) Quantification of donor populations from E. (G) Expression of indicated proteins on KLRG1^hi or KLRG1^lo Id2WT or Id2KO donor populations. Data shown are representative of three (B and D–F) independent or cumulative of two or three (C and G) independent experiments; n = 3–5 mice per group. Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed paired Student’s t test).

Figure 2.

Id2 is necessary to maintain the terminal differentiation of antigen-specific CD8⁺ T cells. (A) Schematic of experimental set-up. CD45.1 hosts were given a cotransfer of Id2^f/f-ERCre⁻ (CD45.1.2) and Id2^f/f-ERCre⁺ (CD45.2) P14 CD8⁺ T cells and then subsequently infected with LCMV. At >30 d after infection, KLRG1^hiCD127^lo and KLRG1^loCD127^hi, Id2^f/f-ERCre⁻, and Id2^f/f-ERCre⁺ P14 CD8⁺ T cells were sort purified. Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ KLRG1^hiCD127^lo (KLRG1^hi transfer) or Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ KLRG1^loCD127^hi (KLRG1^lo transfer) populations were co-transferred into naive CD45.1 hosts that were treated with tamoxifen (Tam). Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ CD8⁺ T cells are called Id2WT or Id2KO, respectively, after Tam treatment. (B) Flow cytometry of transferred P14 CD8⁺ T cells from host spleen 6 d (top) or 32 d (bottom) after last Tam treatment. Frequency of Id2WT and Id2KO cells among P14 CD8⁺ T cells (left), KLRG1 and CD127 expression (middle), and CD27 and CD43 expression (right) for the KLRG1^hi and KLRG1^lo transfer are shown. Numbers in plots represent the frequency of cells in that quadrant. (C) Quantification of donor cell frequency and number from KLRG1^hi (Hi) or KLRG1^lo (Lo) transfers from B. (D) Quantification of donor populations from KLRG1^hi (left) and KLRG1^lo (right) transfer. (E) Flow cytometry of transferred P14 CD8⁺ T cells from host spleen 6 d after last Tam treatment. CD62L and CD127 expression for the KLRG1^hi and KLRG1^lo transfers is shown. (F) Quantification of donor populations from E. (G) Expression of indicated transcription factors on KLRG1^hi or KLRG1^lo Id2WT or Id2KO donor populations from hosts receiving KLRG1^hi transferred (left) and KLRG1^lo transferred (right) cells 6 d after last Tam treatment. Data shown are representative (B, E, and G) or cumulative (C, D, and F) of two independent experiments; n = 3–4 mice per group. Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed paired Student’s t test).

Figure 3.

Deletion of Id2 in KLRG1^hi memory CD8⁺ T cells results in the faster initiation of a recall response. Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ KLRG1^hi or KLRG1^lo P14 CD8⁺ T cells were co-transferred into naive CD45.1 hosts, and Id2 deletion was induced as described in Fig. 2 A. Host mice were infected with LCMV 7 d after the last tamoxifen treatment. Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ CD8⁺ T cells are called Id2WT or Id2KO, respectively, following tamoxifen treatment. (A) The frequency of total Id2WT and Id2KO P14 donor cells (top) or Id2WT and Id2KO P14 cells of total CD8⁺ T cells (bottom) in PBL on indicated days after infection is shown. (B) Flow cytometry (left) and quantification (right) of the frequency of Id2WT and Id2KO P14 CD8⁺ T cells from host spleen 9 d after infection for KLRG1^hi and KLRG1^lo transfers. (C) Flow cytometry of KLRG1 and CD127 expression (left) and CD27 and CD43 expression (right) for KLRG1^hi and KLRG1^lo transfers are shown. (D) Quantification of donor populations from C after KLRG1^hi (top) and KLRG1^lo (bottom) transfer. Numbers in plots represent the percentage of cells. Data shown are cumulative (total donor frequency; A, top) or representative (total CD8⁺ population frequency; A, bottom), representative (B), or cumulative (C) of two independent experiments; n = 2–4 mice per group. Data are expressed as mean ± SEM. *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001 (one-tailed paired [A and B] or two-tailed paired [D] Student’s t test).

Figure 4.

Id2 regulation of E2A binding regulates expression of key effector CD8⁺ T cell genes. (A–D) Id2^f/f-ERCre⁻ (CD45.1.2) and Id2^f/f-ERCre⁺ (CD45.2) KLRG1^hi or KLRG1^lo P14 CD8⁺ T cells were co-transferred into naive CD45.1 hosts, and Id2 deletion was induced as described in Fig. 2 A. Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ CD8⁺ T cells are called Id2WT or Id2KO, respectively, after tamoxifen (Tam) treatment. On day 6 after the last Tam treatment, Id2WT and Id2KO cells were sorted for RNA-Seq. (A) Gene expression by cells from the Id2WT KLRG1^hi transfer versus Id2WT KLRG1^lo transfer (left) or from the Id2WT KLRG1^hi transfer versus Id2KO KLRG1^hi transfer (right) for genes differentially expressed by 1.5-fold or more, a coefficient of variation of ≤0.50, and an expression value of ≥10; colors indicate genes up-regulated 1.5-fold or more in the cells from the Id2WT KLRG1^hi transfer relative to their expression in cells from the Id2WT KLRG1^lo transfer (green) or vice versa (purple), or genes up-regulated in cells from the Id2WT KLRG1^hi transfer relative to cells from the Id2KO KLRG1^hi transfer (green) or vice versa (magenta). Labels in plots indicate genes of published relevance to CD8⁺ T cell differentiation and memory formation. (B) Principal component analysis of gene expression in Id2WT and Id2KO P14 CD8⁺ T cells from the KLRG1^hi and KLRG1^lo transfers, in TE and MP P14 CD8⁺ T cell populations at day 7 of LCMV infection, and in T_CM, and T_EM P14 CD8⁺ T cell populations at day 180 of LCMV infection. (C) Expression of TE- or MP-associated genes (top) or T_EM- or T_CM-associated genes (bottom) assessed in cells from the Id2KO KLRG1^hi transfer versus cells from the Id2WT KLRG1^hi transfer and plotted against p-value. Numbers in the corners indicate the total of those genes up-regulated in cells from the Id2KO KLRG1^hi transfer (top left) or from the Id2WT KLRG1^hi transfer (top right). P < 0.001 (χ² test). (D) Relative expression of TE and MP genes (top) and T_EM and T_CM genes (bottom) from C that are putative E2A-target genes identified by ChIP-Seq (bar colors match dot colors in C). (E) Schematic of experimental set-up. CD45.1 host mice infected 1 d before received a cotransfer of Id2^f/f-ERCre⁻ and Id2^f/f-ERCre⁺ P14 CD8⁺ T cells transduced with control shRNA targeting Cd19 or with shTcf3. At 15 d of infection, host mice were treated with tamoxifen (Tam) to induce Id2 deletion. (F) Flow cytometry of transferred cells 9 d after the last Tam treatment for KLRG1 and CD127 expression (left) and CD27 and CD43 expression (right). Numbers in the plots represent the percentage of cells. (G) Quantification of donor populations from highlighted populations in F. Data are representative of two (A–D) or three (F) and cumulative of three (G) independent experiments; n = 3–5 mice per group. Data are expressed as mean ± SEM. **, P < 0.01 (two-tailed paired Student’s t test).