Basal NF-κB controls IL-7 responsiveness of quiescent naïve T cells

Abstract

T cells are essential for immune defenses against pathogens, such that viability of naïve T cells before antigen encounter is critical to preserve a polyclonal repertoire and prevent immunodeficiencies. The viability of naïve T cells before antigen recognition is ensured by IL-7, which drives expression of the prosurvival factor Bcl-2. Quiescent naïve T cells have low basal activity of the transcription factor NF-κB, which was assumed to have no functional consequences. In contrast to this postulate, our data show that basal nuclear NF-κB activity plays an important role in the transcription of IL-7 receptor α-subunit (CD127), enabling responsiveness of naïve T cells to the prosurvival effects of IL-7 and allowing T-cell persistence in vivo. Moreover, we show that this property of basal NF-κB activity is shared by mouse and human naïve T cells. Thus, NF-κB drives a distinct transcriptional program in T cells before antigen encounter by controlling susceptibility to IL-7. Our results reveal an evolutionarily conserved role of NF-κB in T cells before antigenic stimulation and identify a novel molecular pathway that controls T-cell homeostasis.

Keywords: IKKβ; IkBaDN; Il7r enhancer; STAT5.

Fig. 1.

Naïve T cells display basal NF-κB activity. (A) EMSA for NF-κB using nuclear extracts of WT and IκBαΔN FACS cell-sorted CD4⁺CD44^lo and CD8⁺CD44^lo T cells. (B) EMSA supershift for NF-κB subunits RelA and p50 performed with nuclear extracts from WT naïve T cells. Results are representative of at least three independent experiments. ^†Supershifted bands for RelA. ^‡Supershifted band for p50.

Fig. 2.

Basal NF-κB is required for T-cell survival in vivo. (A) Equal numbers of CD45.1/2 WT and CD45.2 WT or IκBα∆N CD4⁺CD44^lo and CD8⁺CD44^lo cells were coadoptively transferred into CD45.1 recipients. Seven days later, ratios of IκBαΔN:WT and WT:WT splenic CD4⁺CD44^lo and CD8⁺CD44^lo T cells were assessed as follows: (% CD45.2_final/% CD45.1/2_final)/(% CD45.2_initial/% CD45.1/2_initial). The graph represents recipients receiving WT:WT (n = 13) and IκBαΔN:WT (n = 14) cells. Data are pooled from five independent experiments and analyzed by Kruskal–Wallis test with Dunn’s posttest. (B) DO11.10/WT and DO11.10/IκBα∆N mice were thymectomized, and presence of CD4⁺TCRVβ8.2⁺ T cells in peripheral blood was assessed weekly by flow cytometry (WT, n = 6; IκΒαΔN, n = 8). Values displayed are percentages of CD4⁺TCRVβ8.2⁺ T cells with respect to the live gate and relative to the value obtained at the time of thymectomy (day 0). Data are representative of two independent experiments.

Fig. 3.

Basal NF-κB activity is required for IL-7–mediated survival. (A) Splenocytes from WT (n = 3) and IκBα∆N (n = 6) mice were cultured for 3 d in the presence (open symbols) or absence (filled symbols) of 1 ng/mL IL-7. Percentages of CD4⁺CD44^lo and CD8⁺CD44^lo live (7AAD-negative) cells were assessed by flow cytometry. (B) WT and IκBα∆N splenocytes were cultured in vitro for 30 min in the presence or absence of 1 ng/mL IL-7, and (Y694) STAT5 phosphorylation in CD4⁺CD44^lo and CD8⁺CD44^lo was assessed by intracellular flow cytometry. Data are representative of n = 6 mice each. (C) Splenocytes from WT (n = 3) and IκΒαΔN (n = 3) mice were cultured in the presence (open symbols) or absence (filled symbols) of IL-7, and 24 h later, expression of Bcl-2 was assessed by intracellular flow cytometry in CD4⁺CD44^lo and CD8⁺CD44^lo cells. Data were analyzed by two-way ANOVA with Bonferroni posttests. (D) Equal numbers of CD45.1/2 WT, CD45.2 WT, and IκBα∆N or IκBα∆NxBcl-2^Tg CD4⁺CD44^lo and CD8⁺CD44^lo cells were coadoptively transferred into CD45.1 recipients. Results were analyzed as in Fig. 2A. The graph represents recipient mice for WT:WT (n = 8), IκBαΔN:WT (n = 9), and IκBαΔNxBcl-2^Tg:WT (n = 9). Data are pooled from three independent experiments and analyzed by Kruskal–Wallis test with Dunn’s posttest. All experiments were performed at least three times. MFI, mean fluorescence intensity; ns, not significant. *P < 0.05. ***P < 0.001.

Fig. 4.

Basal NF-κB is required for IL-7Rα expression. (A) Histograms displaying (Top) IL-7Rα/CD127, (Middle) γ_c/CD132, and (Bottom) CD3ε as analyzed by flow cytometry in WT (solid line) and IκBαΔN (broken line) CD4⁺CD44^lo (Left) and CD8⁺CD44^lo (Right) T cells. IC, isotype control. (B) Expression of CD127 in (Left) CD4⁺CD44^lo and (Right) CD8⁺CD44^lo splenocytes from CD4^CreIKKβ^+/fl or CD4^CreIKKβ^fl/fl mice. Data were analyzed by Student t test. (C) Expression of CD127 in WT (n = 4), IκBαΔN (n = 5), p50^−/− (n = 5), and p52^−/− (n = 8) mice as assessed by flow cytometry and one-way ANOVA with Bonferroni posttests for pairwise comparisons. Results are representative of at least two independent experiments. **P < 0.01. ***P < 0.001.

Fig. 5.

Basal NF-κB controls transcription of Il7r in naïve T cells. (A) Il7r mRNA from WT and IκBαΔN CD4⁺CD44^lo and CD8⁺CD44^lo cells was assessed by RT-qPCR, and triplicates were normalized to Actb. RU, relative units. Results are shown as mean ± SD of triplicates analyzed by Student t test, and they are representative of three independent experiments. ***P < 0.001. (B) WT splenocytes were cultured for 24 h with 1 ng/mL IL-7, washed, and incubated for another 24 h with 1 ng/mL IL-7, no IL-7 (medium), or 5 nM NF-κBi. CD127 expression was analyzed by RT-qPCR in FACS cell-sorted CD4⁺CD44^lo live cells with one-way ANOVA and Bonferroni posttests. **P < 0.01. ***P < 0.001. (C) In silico analysis of murine Il7r locus highlighting ECRs upstream of the 5′ TSS. ECR2 (−3.6 kb) and ECR3 (−5.6 kb) contain sequences with putative NF-κB binding sites. (D) ChIP for ECR2 and ECR3 using anti-RelA antibody or isotype control (IgG) in WT and IκBαΔN naïve T cells as assessed by RT-qPCR. (E) Semiquantitative PCR after ChIP for ECR2 using anti-RelA, anti-p50 antibodies or isotype control (IgG) in WT and IκBαΔN naïve T cells. ^∞Two hundred eighty-two–base pair expected PCR product. ^#Nonspecific band. MW, molecular weight. (F) Supershift EMSA for NF-κB binding site contained in ECR2 using antibodies against RelA, p50, or isotype control. *Supershifted bands. ^ΦRelA/p50. ^θp50/p50 dimers. **P < 0.01. (G) Dual luciferase assay of lysates of 293T cells transfected for 48 h with the luciferase reporter plasmid pGL4.23 alone or containing ECR2 and ECR3 sequences from Il7r gene plus pRL-TK and control plasmid (EV) or plasmids encoding IKKβ-CA and RelA. Results were analyzed by two-way ANOVA with Bonferroni posttests for pairwise comparisons. Results are representative of at least two independent experiments. ***P < 0.001.

Fig. 6.

Constitutive active IKKβ enhances IL-7Rα expression. (A) Expression of GFP was assessed by flow cytometry in CD4⁺CD44^lo and CD8⁺CD44^lo T cells from WT and Lck^Cre IKKβ^CA mice. (B) CD127 expression was assessed by flow cytometry in CD4⁺CD44^lo and CD8⁺CD44^lo T cells from WT (n = 4) and Lck^Cre IKKβ^CA (n = 6; gated on GFP⁻ and GFP⁺ events) mice. Results were analyzed with one-way ANOVA and Bonferroni posttests. ***P < 0.001.

Fig. 7.

Pharmacological inhibition of NF-κB reduces expression of IL-7Rα in human peripheral T cells. PBMCs from healthy individuals were cultured for 24 h with 10 ng/mL recombinant human IL-7, washed, and incubated for 24 h with increasing doses of NF-κBi. Expression of CD127 was assessed in CD3⁺CD4⁺CD45R0⁻ and CD3⁺CD8⁺CD45R0⁻ live-gated cells and represented compared with maximum (NF-κBi = 0 nM). Results are representative of two independent experiments.