Immune tolerance. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance

Abstract

Aire is an important regulator of immunological tolerance, operating in a minute subset of thymic stromal cells to induce transcripts encoding peptides that guide T cell selection. Expression of Aire during a perinatal age window is necessary and sufficient to prevent the multiorgan autoimmunity characteristic of Aire-deficient mice. We report that Aire promotes the perinatal generation of a distinct compartment of Foxp3(+)CD4(+) regulatory T (Treg) cells, which stably persists in adult mice. This population has a role in maintaining self-tolerance, a transcriptome and an activation profile distinguishable from those of Tregs produced in adults. Underlying the distinct Treg populations are age-dependent, Aire-independent differences in the processing and presentation of thymic stromal-cell peptides, resulting in different T cell receptor repertoires. Our findings expand the notion of a developmentally layered immune system.

Fig. 1. A perinatal Treg population that is Aire-dependent and guards against the autoimmune manifestations typical of Aire-KO mice

(A) Summary data for fractional representation (left) and numbers (right) of Foxp3⁺CD4⁺CD8⁻ thymocytes from Aire-WT or -KO mice of increasing age. P-values from the Student’s t test: *, P≤ 0.05; **≤ 0.01; ns = not significant. n=5. Examples of corresponding dot plots can be found in Fig. S2A. (B) Treg depletion in perinates. Perinatal (0.5 days after birth) NOD.Foxp3-DTR⁺ mice or DTR⁻ littermates were treated every other day until day 10 with DT, and then followed for manifestations of autoimmune disease. Perinates had to be examined <24 days after birth due to wasting in the DTR⁺ littermates. Upper left: weight curves. Upper right: survival curves; mice were sacrificed if their weight fell to <20% of that of their DTR⁻ littermates. Lower left: presence (shaded) or absence of organ infiltrates; “i” indicates that insulitis replaced infiltration of the exocrine pancreas. Lower right: severity of organ infiltration (scored as per the Methods section). n=9. (C and D) NOD.Foxp3.DTR⁺ mice perinatally depleted of Tregs as per panel B were supplemented on days 12 and 19 with Tregs isolated form 20-day-old Aire-WT (C) or -KO (D) littermates. Cohorts were followed until 70 days of age. n = 9. Otherwise set up as per panel B.

Fig. 2. Stability and function of perinate- versus adult-tagged Tregs

(A) Tamoxifen was administered from 0–10 or 35–45 days of age; at various times later, splenocytes were analyzed for GFP and YFP expression by flow cytometry. Left: representative flow-cytometric dot-plots. Numbers represent percentages of CD4⁺3⁺ cells in the designated gates. Center: summary data on numbers of GFP⁺YFP⁻ bulk Tregs. Right: corresponding data on GFP⁺YFP⁺ perinate-tagged or adult-tagged Tregs from the same mice. n=5. (B–E) 1.5x10⁵ Tregs were transferred into Aire-KO mice on days 0.5, 3 and 7 after birth, and the recipients were followed until 16 weeks of age. A four-way comparison as schematized in Fig. S8A: GFP⁺YFP⁺ Tregs tagged from 35–45 days of age and isolated from a 60-day-old mouse (C), GFP⁺YFP⁻ bulk Tregs from the same mouse (B), GFP⁺YFP⁺ Tregs tagged from 0–10 days of age and isolated from a 60-day-old mouse (E), and GFP⁺YFP⁻ bulk Tregs from the same mouse (D). Data organized as per Fig. 1B. The key comparison is boxed.

Fig. 3. A distinct transcriptome in perinate-tagged Tregs

The same type of four-way comparison employed in Fig. 2 was conducted except that the sorted cells were analyzed for diverse phenotypic features. (A) FC/FC plots comparing perinate-tagged GFP⁺YFP⁺ cells vs bulk GFP⁺YFP⁻ cells from the same mice (x-axis) and adult-tagged GFP⁺YFP⁺ cells vs bulk GFP⁺YFP⁻ cells from the same mice (y-axis). Pink dots denote transcripts over-represented in perinate-tagged GFP⁺YFP⁺ cells; green dots indicate under-represented transcripts. (B) P-value vs FC volcano plot comparing gene expression of perinate-tagged GFP⁺YFP⁺ and adult-tagged GFP⁺YFP⁺ cells. Red and blue dots indicate up- and down-regulated Treg signature genes, respectively (31). P-values from the chi-squared test (C) Classical in vitro suppression assay on the four sorted Treg populations. P-values from the Student’s t test. **, p≤ 0.01; ***, p≤0.001. (D) Same volcano plot as in panel B, except up-(red) and down-(blue) regulated activation signature genes (31) are superimposed. (E) Summary data on late activation marker (CD44^hiCD62L^lo) expression in the four Treg populations. n=5. P-value from the Students’ t test ***, P≤0.001. (F) EdU uptake (left) and Ki67 expression (right) by the four Treg populations. ***, P≤ 0.001. (G) GSEA of transcripts increased in the perinate-tagged GFP⁺YFP⁺ vis-à-vis the adult-tagged control Treg populations. NES, normalized enrichment score. FDR q-val, false discovery rate. Representative transcripts showing increased expression are shown on the right. (H) Flow cytometric confirmation of gene over-expression in perinate-tagged Tregs. For Fgl2 and PD1: Left = representative flow-cytometric histograms; red, perinate-tagged; blue, adult-tagged; black, control bulk populations; gray shading, isotype-control antibody; bar indicates marker positivity. Center = summary data for % of the four Treg populations expressing the marker; Right = summary data for marker MFI in the marker-positive population.

Fig. 4. Age-dependent, Aire-independent differences in the processing and presentation of MEC-generated peptides

(A) Microarray-based quantification of transcripts encoding a set of proteins involved in processing/presentation of MHCII-bound peptides. (B) Microarray-based quantification of DOa and DOb in MEC^hi from Aire-WT or -KO adults or perinates. (C) Intracellular expression of DOb protein. Left = representative flow-cytometric histograms. Red, perinate; blue, adult; gray shading, negative control staining. Right = summary MFI data. (D and E) Same as panels B and C except DMa and DMb were examined. (F) Coordinate intracellular staining of DOb and DMab. (G) Surface expression of Ab:CLIP complexes on MEC^hi. Left = representive flow-cytometric histograms. Red, perinate; blue, adult; gray shading, negative control staining. Center = summary data for % MEC^hi expressing little or no CLIP. Right = summary data for MFI. (H) Flow cytometric quantification of MHC^hiCD8α⁺ DCs in perinatal vs adult thymus (left) and spleen (right). Summary data for representation in the CD11c⁺ (left) and CD45⁺ (right) compartments. (I) High-frequency Vα2⁺ TCRs from 5wk-old (upper) and 4d-old (lower) BDC2.5/NOD females. These sequences correspond to those in Table S2. Bars represent frequency of each sequence. Except for panel I, P-values are from the Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. n=3–6.