Loss of E protein transcription factors E2A and HEB delays memory-precursor formation during the CD8+ T-cell immune response

Abstract

The transcription factors E2A and HEB (members of the E protein family) have been shown to play essential roles in lymphocyte development, while their negative regulators, the Id proteins, have been implicated in both lymphocyte development and in the CD8(+) T-cell immune response. Here, we show that E proteins also influence CD8(+) T cells responding to infection. E protein expression was upregulated by CD8(+) T cells during the early stages of infection and increased E protein DNA-binding activity could be detected upon TCR stimulation. Deficiency in the E proteins, E2A and HEB, led to increased frequency of terminally differentiated effector KLRG1(hi) CD8(+) T cells in mice during infection, and decreased generation of longer-lived memory-precursor cells during the immune response. These data suggest a model whereby E protein transcription factor activity favors rapid memory-precursor T-cell formation while their negative regulators, Id2 and Id3, are both required for robust effector CD8(+) T-cell response during infection.

Figure 1

E2A-GFP expression is upregulated upon CD8⁺ T-cell activation. Flow cytometric analysis of E2A-GFP expression by CD8⁺ splenocytes (A) after activation in vitro or (B) after infection. (A) Flow cytometric analysis of CD8⁺ T cells stimulated in vitro with anti-CD3 and anti-CD28 and analyzed at indicated time points. Gray-line histogram indicates WT control CD8⁺ T cells and black-line histogram indicates E2A-GFP-expressing CD8⁺ T cells. (B) WT control mice and E2A-GFP-reporter mice were infected with Lm-OVA and CD8⁺ Lm-OVA-specific T cells in spleen were analyzed by flow cytometry on indicated days after infection. K^b-OVAp tetramer⁻ CD8⁺ T cells are shown as controls. Gray-line histogram indicates WT control CD8⁺ T cells and black-line histogram indicates E2A-GFP-expressing CD8⁺ T cells. Data shown are representative of two independent experiments performed with a total of four mice.

Figure 2

E proteins show increased DNA-binding activity in activated CD8⁺ T cells in vivo. Electrophoretic mobility-shift assay of nuclear extracts prepared from OT-I⁺ T cells stimulated during infection. (A) OT-I⁺ T cells were transferred to recipient mice and the mice subsequently infected with VSV-OVA. One day 4 and day 6 after infection OT-I⁺ T cells were harvested from the spleens and lymph nodes of infected mice and nuclear extracts were isolated. Nuclear extracts were incubated with a radiolabeled probe containing an E-box site for detection of E protein DNA-binding (µE5) or a control probe containing an octamer-binding site (Oct) for confirmation of the quality of the nuclear extracts. E47 blocking antibody was used to show E47-specific binding to radiolabeled E-box site-specific probe. (B) WT and Id2-deficient OT-I⁺ T cells were transferred to recipient mice that were then subsequently infected with VSV-OVA. On day 6 after infection OT-I⁺ T cells were harvested from the spleens and lymph nodes of infected mice and nuclear extracts treated as in (A). Data are representative of two independent experiments.

Figure 3

E protein-deficient CD8⁺ cells develop a KLRG1^hi CD127^lo effector phenotype during Listeria infection. CD45.1⁺-recipient mice received either 1 × 10⁴ OT-I WT (CD45.2) or OT-I E protein-deficient (CD45.2) cells 1 day before infection with Lm-OVA. (A) Percent population expansion of OT-I WT, OT-I E2A-deficient (E2AKO), OT-I HEB-deficient (HEBKO) or OT-I E2A/HEB double-deficient (DKO) T cells over time in peripheral blood is shown. (B) Flow cytometric analysis of KLRG1 and CD127 expression by OT-I WT and OT-I DKO cells on indicated days after infection in peripheral blood. Numbers in quadrants indicate percentage of cells. (C) Percent population expansion of KLRG1^hi OT-I WT or OT-I DKO cells and KLRG1^lo OT-I WT or OT-I DKO in peripheral blood was measured over time during Lm-OVA infection. Histograms indicate KLRG1^hi and KLRG1^lo OT-I cells on day 7 after infection. (D) Flow cytometric analysis of KLRG1 and CD127 expression by OT-I WT and OT-I DKO cells at day 77 (memory) after infection in peripheral blood. Numbers in quadrants indicate percentage of cells. Bar graphs indicate percent population expansion of OT-I WT or DKO cells, KLRG1^lo OT-I WT or DKO cells and KLRG1^hi OT-I WT or DKO cells in peripheral blood on day 77 (memory) after infection. Data shown are average (+ SEM) with n = 2–7 mice per group. Statistical significance was determined using unpaired two-tailed t-test where **p < 0.005, **p < 0.0005. Data are representative of two independent experiments.

Figure 4

Splenic E protein-deficient CD8⁺ cells develop a KLRG1^hi CD127^lo effector phenotype during Listeria infection. CD45.1⁺-recipient mice received either 1 ×10⁴ OT-I WT (CD45.2) or OT-I E protein-deficient (CD45.2) cells 1 day before infection with Lm-OVA. (A) Flow cytometry of KLRG1 and CD127 expression by OT-I WT and OT-I DKO cells in the spleen on indicated days after infection. Numbers in quadrants indicate percentage of cells. (B) Percent population expansion of KLRG1^hi OT-I WT or OT-I DKO cells and absolute cell number of KLRG1^hi OT-I WT or OT-I DKO in the spleen on indicated days after infection. Data are shown as mean + SEM of 2–3 mice per group and are pooled from three independent experiments. *p < 0.05, **p < 0.005 (unpaired two-tailed t-test).

Figure 5

E protein-deficient CD8⁺ cells develop an effector-memory phenotype during early stages of infection. CD45.1.2⁺-recipient mice received a mixture of 0.25 × 10⁶ OT-I WT (CD45.1⁺) and OT-I DKO (CD45.2⁺) cells 1 day before infection with Lm-OVA. (A) Flow cytometric analysis of OT-I T cells in spleen. Numbers beside gated area indicate percent in each. Bar graph indicates percent population expansion of OT-I WT and OT-I DKO cells in spleen from 2 to 3 different mice per group per time point. (B) Infected mice received 1 mg BrdU 4 h before analysis of splenocytes. Bar graph indicates percent BrdU incorporation by OT-I cells from day 3 to day 6 after infection. (C) Viability of OT-I WT and OT-I DKO cells assessed by ex vivo staining with Annexin V from day 3 to day 6 postinfection. (D) Flow cytometry of IFN-γ and TNF-α production by splenocytes restimulated for 6 h in vitro with OVAp or incubated in media alone as unstimulated control on day 6 after infection. Lower flow cytometry plots indicate gated OT-I cells IFN-γ production after in vitro restimulation with OVAp. (E) Bar graphs indicating KLRG1 expression by OT-I WT and OT-I DKO splenocytes from day 3 to day 6 postinfection. Data are shown as mean + SEM of 2–3 mice per group and are representative of three independent experiments. *p < 0.05, **p < 0.005, ***p < 0.0005 (unpaired two-tailed t-test).

Figure 6

Gene-expression profile of WT versus E protein-deficient T cells stimulated in vitro. Affymetrix microarray analysis of mRNA from CD8⁺ T cells from WT and DKO mice, stimulated in vitro with anti-CD3 and anti-CD28 for 16 h. (A) Normalized expression values for WT versus DKO CD8⁺ T cells. Numbers in corners indicate genes with a difference in expression of 1.7-fold or more in DKO CD8⁺ T cells versus WT cells (upregulation, red dots; downregulation, blue dots). Data are representative of one experiment with two data sets per group. (B) Gene-expression values of genes of interest with a difference in expression 1.7-fold or more in DKO CD8⁺ T cells versus WT cells (upregulation, red; downregulation, blue). (C) Selected genes involved in immune effector processes that are activated or repressed in DKO CD8⁺ T cells. (D) Selected cytokine and chemokine genes that are activated or repressed in DKO CD8⁺ T cells. (E) E2A DNA-binding sites as identified using ChIP-Seq. Representative examples of Cd28 and Id2 are shown. UCSC Genome Browser was used to visualize binding patterns. Blue pattern denotes E2A DNA binding. Green pattern denotes H3K4 mono-methylation. Red arrow denotes the transcription start site. The Table indicates selected genes up- or downregulated in the microarray that display E2A occupancy as identified by ChIP-Seq.