Active suppression of intestinal CD4(+)TCRαβ(+) T-lymphocyte maturation during the postnatal period

Abstract

Priming of the mucosal immune system during the postnatal period substantially influences host-microbial interaction and susceptibility to immune-mediated diseases in adult life. The underlying mechanisms are ill defined. Here we show that shortly after birth, CD4 T cells populate preformed lymphoid structures in the small intestine and quickly acquire a distinct transcriptional profile. T-cell recruitment is independent of microbial colonization and innate or adaptive immune stimulation but requires β7 integrin expression. Surprisingly, neonatal CD4 T cells remain immature throughout the postnatal period under homeostatic conditions but undergo maturation and gain effector function on barrier disruption. Maternal SIgA and regulatory T cells act in concert to prevent immune stimulation and maintain the immature phenotype of CD4 T cells in the postnatal intestine during homeostasis. Active suppression of CD4 T-cell maturation during the postnatal period might contribute to prevent auto-reactivity, sustain a broad TCR repertoire and establish life-long immune homeostasis.

Figure 1. Kinetic of immune cell composition in the neonatal small intestine.

(a) Immune cells were isolated from whole small intestinal tissue (SI) at the indicated age and absolute numbers of CD45⁺ cells were determined by FACS analysis normalizing to the simultaneously counted Trucount beads. Total recovered CD45⁺ immune cell numbers were divided by the average weight of the total organ at the indicated age and are presented as CD45⁺ cells per g tissue (mean±s.d.). (b) Major immune cell subsets were determined over time and are presented as percentage of viable CD45⁺ cells (solid line, left y axis) and absolute cell numbers per g tissue (broken line, right y axis). Cell subsets were defined by the following markers: TCRβ⁺, αβ T lymphocytes; TCRδ⁺, γδ T lymphocytes; CD19⁺, B lymphocytes; SiglecF⁺SSC^hi, eosinophils; MHCII⁺CD11b⁺F4/80⁺, Macrophages; MHCII⁺CD11c⁺, DC. (c) αβ T lymphocyte subset analysis represented as FACS plots (CD4 and CD8α) from total small intestine of 11- and 56-day-old mice (n=6, mean±s.d., two experiments) and (d) as an age kinetic presented as percentage of viable CD45⁺ cells. Total TCRβ⁺ (dark blue), TCRβ⁺CD8αβ⁺ (turquoise), TCRβ⁺CD4⁺ (blue), TCRβ⁺CD8αα⁺ (light blue) and TCRβ⁺CD4⁺CD8αα⁺ (grey blue) subsets are represented. For all time points in a,b,d, n=4–11 pooled from three to five independent experiments. (e) Immunofluorescence staining of TCRβ (green) on SI tissue sections at the indicated ages. Images in the upper row are representative for the LP; images in the lower row illustrate PPs dissected using the binocular. Magnification, × 100; counterstaining with EpCAM (red) and DAPI (blue) (scale bar 100 μm). Bar graph: PPs were depleted by injection of the anti-IL7Rα anti body in pregnant dams at E14.5 gestation. Small intestinal CD4⁺ T lymphocytes were analysed in 11-day-old neonates from dams that received an anti-IL7Rα antibody (aIL7Ra) or an isotype control antibody (iso). (n=1 litter, mean±s.d.). d, days.

Figure 2. Transcriptional profiling and functional characterization of neonatal mucosal CD4 T lymphocytes.

Transcriptome analysis of flow cytometry-sorted CD4⁺CD8⁻TCRβ⁺ cells isolated from the indicated organs/age of the animals (days, d). The array was performed in quadruplicates with each group comprising sorted cell populations pooled from one litter of neonate animals (d6 and day 11) and two to three adult mice per sample. Thymus (Th) d6, SI d6, SI d11, mLN d6, mLN d56, LP d28, LP d56 and PP d56. (a) Principal coordinate analysis of the groups based on 10,000 differentially regulated individual genes using multigroup comparison (ANOVA analysis) and a false discovery rate (FDR) of <10⁴. (b) Functional analysis of up- (red) and downregulated (green) genes of d6 SI compared with d6 mLN using two group analysis, FDR=0.05 and a fold-change cutoff of 2. Analysis was performed in PANTHER using the gene ontology term biological process. (c) Transcriptome analysis of signature T-helper genes d6 SI and d56 PP. (d) Fold-change values (normalized to the mean intensity of all samples) of CD44 and CD69 mRNA measured by microarray analysis in CD4 T lymphocytes from thymus (Th) and small intestine (SI) of 6-day-old mice, SI of 11-day-old mice and PPs and LP of 56-day-old mice (left panels) and comparative FACS analysis of the CD44 or CD69 versus CD62L surface expression on CD4 T lymphocytes in PP of 11-day-old mice and PP and LP of 56-day-old mice (right panels; n=4 for left and right panels, mean±s.d.). (e) Intracellular FACS staining for IFNγ and IL-17A in CD4 T cells isolated from PPs from 11- and 56-day-old mice restimulated with PMA/Ionomycin (n=4, representative of three experiments, mean±s.d.). (f) Cytokine detection by Multiplex Bead Array analysis in the supernatant of FACS-sorted CD4 T cells isolated from PPs of 11- and 56-day-old mice and restimulated with PMA/Ionomycin. (n=4, representative of two experiments, mean±s.d.; two-way ANOVA, Bonferroni's post test, ***P<0.001; *P<0.05).

Figure 3. Postnatal homing of CD4 T cells to the neonatal intestine.

(a) Comparative analysis of the percentages of CD4⁺TCRβ⁺ T lymphocytes among total CD45⁺ immune cells in intestinal tissue of conventional (cv) and germ-free (gf) mice at the indicated age (n=8–11 from two experiments, mean±s.d.; one-way ANOVA, Bonferroni's post test, ***P<0.001; NS, not significant). (b) Comparative analysis of the percentage of CD4⁺TCRβ⁺ T lymphocytes among total CD45⁺ immune cells in wild type (wt), Tlr4^−/−, Trif^Lps2/Lps2, MyD88^−/− and Nod2^−/− mice at 6 days after birth (n=4–11, mean±s.d.; one-way ANOVA, Bonferroni's post test; NS, not significant). (c) Comparative analysis of the frequency of Vα2⁺ and Vα2⁻ cells among TCRβ⁺CD4⁺ lymphocytes in the total small intestine (SI), spleen (Spl) and thymus (Th) of 12-day-old and PPs, LP, spleen (Spl) and thymus (Th) of adult OTII transgene mice (n=7 and 3, respectively; representative of two independent experiments, mean±s.d.). (d) Comparative analysis of the percentage of CD4⁺TCRβ⁺ T lymphocytes among total CD45⁺ immune cells in the small intestine (SI) and Spleen (Spl) of wild type (wt), Ccr9^−/− and Itgb7^−/− mice at 6 days after birth (n=7–11 from two experiments, mean±s.d.; one-way ANOVA, Bonferroni's post test, ***P<0.001; NS, not significant). Note that the same data set from the neonatal wt/cv group is shown in a–c because the figures represent pooled data. (e) Immunofluorescence staining of MadCAM-1 (red) in combination with CD31 (green) on PP tissue sections of gf and cv 11-day-old neonates dissected using the binocular. Magnification, 1 × 200; counterstaining with DAPI (blue; scale bar, 100 μm; n=1, representative of two experiments).

Figure 4. Maturation of neonatal CD4 T lymphocytes following mucosal challenge.

(a) Lymphocyte maturation status in the adult lymphopenic host after the induction of Rag in InduRag mice. FACS analysis (left panel) and gMFI of CD44 (right panel) on CD4⁺TCRβ⁺ lymphocytes obtained from the SI of 11- and 56-day-old wt (wt d11 and wt d56) and adult InduRag mice 11 days after Tamoxifen administration (InduRag d56+TAM11). (n=3–4, representative of two experiments, mean; one-way ANOVA, Bonferroni's post test, ***P<0.001; NS, not significant). (b) Ex vivo activation of CD4⁺TCRβ⁺ lymphocytes from PP of 11- and 56-day-old mice after 3 days of culture in presence of anti-CD3/CD28 beads (n=4, one experiment). (c) H&E staining and (d) percentage of infiltrating CD4⁺TCRβ⁺ lymphocytes of CD45⁺ in the colonic tissue of adult Rag2 recipients 5–6 weeks after transfer of 1–5 × 10⁴ SI CD4⁺ T cells from 11-day-old or adult Foxp3-GFP donor mice after T_Reg depletion (T-cell transfer colitis model). Magnification, × 40 (bar graph 100 μm). (n=3–4, representative of two experiments, mean±s.d.; unpaired Student's t-test, ***P<0.001). (e) Percentage of CD44^hi cells among CD4⁺TCRβ⁺ lymphocytes (using the 3% of CD4 T cells in the non-infected control mice with the highest CD44 expression as a reference gate) in the neonate SI after infection with rotavirus (infected at d4 post parturition, pp and analysed at 8 d.p.i.), S. Typhimurium (infected at d4 pp and analysed at 4 d.p.i.) and Giardia lamblia (infected d4 pp and analysed at 8 d.p.i.). (One litter (n=6–11) from the infected group and four age-matched non-infected controls were analysed per experiment; representative of two experiments, mean±s.d.; unpaired Student's t-test, *P<0.05, ***P<0.001; NS, not significant). (f) Quantification of rotavirus antigen by ELISA in colon homogenate of wt and TCRα^−/− neonates at 8 d.p.i. infected at 4d pp (n=12–13 from two experiments, mean; Mann–Whitney U-test, ***P<0.001). (g) FACS plots (left panel) and percentages (right panel) of CD44^hiCD62L⁻ of CD4⁺TCRβ⁺ lymphocytes in PPs. 11-day-old DO11.10 neonates were gavaged daily with 10 mg OVA or PBS starting at d3 pp (n=5, representative of two experiments, mean±s.d.; unpaired Student's t-test, ***P<0.001).

Figure 5. Mechanisms that maintain immaturity of intestinal CD4 T cells during the homeostatic postnatal period.

(a) Percentage of CD44^hi CD4 T cells in the SI of 11-day-old B-cell-sufficient (μMT^+/− or μMT^+/+) (left panel) and IgA-sufficient (pIgR^+/− or pIgR^+/+)(right panel) neonates fed by B-cell or IgA-sufficient (wt mother) or deficient (μMT^−/− or pIgR^−/− mother) dams. (μMt: n=4 litters from four experiments; pIgR: n=2 litters from two experiments, mean; unpaired Student's t-test, **P<0.01, ***P<0.001). (b) Comparative proliferation assay culturing OVA-loaded BMDCs, eFluor670-labelled OTII cells together with neonatal or adult PP cells for 3 days at the indicated ratio (ratio PP:OTII; n=4 technical replicates, representative of five similar independent experiments, mean±s.d.; one-way ANOVA, Bonferroni's post test, ***P<0.001). (c) Comparative proliferation assay culturing OVA-loaded BMDCs, and eFluor670-labelled OTII T lymphocytes together with FACS-sorted subgroups of neonatal PP cells for 3 days at the ratio of 4:1 (PP:OTII: B and T cells) or 2:1 (PP:OTII: stroma and rest). (n=3–4 technical replicates, representative of four similar independent experiments, mean±s.d.; one-way ANOVA, Bonferroni 's post test, ***P<0.001). (d) Comparative proliferation assay culturing OVA-loaded BMDCs, eFluor670-labelled OTII T lymphocytes together with FACS sorted neonatal regulatory T cells (T_Reg, from Foxp3 reporter mice) for 3 days at the ratio of 4:1 (ratio PP:OTII). (n=2 replicates from two experiments with T_Regs pooled from 20 and 8 neonates per experiment, respectively, mean±s.d.; one-way ANOVA, Bonferroni 's post test, ***P<0.001, NS, not significant). (e) Percentage of CD44^hi cells among CD4 T lymphocytes (using non-transgenic littermate controls as a reference gate) in the SI of 11-day-old DEREG mice and non-transgenic littermate controls all treated with DT on days 1/2/5/6 (n=5 litters from five experiments, mean; unpaired Student's t-test, **P<0.01.