Autoimmune regulator (AIRE)-deficient CD8+CD28low regulatory T lymphocytes fail to control experimental colitis

Abstract

Mutations in the gene encoding the transcription factor autoimmune regulator (AIRE) are responsible for autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome. AIRE directs expression of tissue-restricted antigens in the thymic medulla and in lymph node stromal cells and thereby substantially contributes to induction of immunological tolerance to self-antigens. Data from experimental mouse models showed that AIRE deficiency leads to impaired deletion of autospecific T-cell precursors. However, a potential role for AIRE in the function of regulatory T-cell populations, which are known to play a central role in prevention of immunopathology, has remained elusive. Regulatory T cells of CD8(+)CD28(low) phenotype efficiently control immune responses in experimental autoimmune and colitis models in mice. Here we show that CD8(+)CD28(low) regulatory T lymphocytes from AIRE-deficient mice are transcriptionally and phenotypically normal and exert efficient suppression of in vitro immune responses, but completely fail to prevent experimental colitis in vivo. Our data therefore demonstrate that AIRE plays an important role in the in vivo function of a naturally occurring regulatory T-cell population.

Fig. 1.

AIRE° and WT mice have normal numbers of transcriptionally and phenotypically unaltered CD8⁺CD28^low Treg with similar but apparently distinct TCR repertoires. (A) Definition of WT (AIRE^+/+) and AIRE° (AIRE°^/°) C57BL/6 CD8⁺CD28^low cells from spleen. Flow cytometry was performed using the indicated antibodies. (B) WT and AIRE° littermates had similar percentages of splenic CD8⁺CD28^low Treg (gated as in A). Indicated are mean values ± SD (n = 3). (C) Phenotype of CD8⁺CD28^low cells, electronically gated as shown in A. Expression profiles of markers indicated in the figure. Control (ctrl) stainings were performed using isotype-matched antibodies. Shown are typical results of three independently performed experiments. (D) Heatmap of the differentially expressed genes (according to fold change) in resting compared with activated, WT versus AIRE° CD8⁺CD28^low Treg. Genes with statistical differential average expression (adjusted P < 0.05) with a fold change >2 when comparing activated and native cells are represented in this heatmap. Red indicates increased expression; blue indicates decreased expression. The dendrogram shows that WT and AIRE° (KO) samples are not clustering within the activated and native states. (E) Vα or Vβ segment use of WT versus AIRE° CD8⁺CD28^low splenic Treg, as measured by semiquantitative RT-PCR. Indicated are mean values ± SD (n = 3 samples consisting of pooled cells from three mice). (F) CDR3 length distribution of indicated variable domains. Shown are typical results for indicated Vα and Vβ CDR3s. “10” indicates a CDR3 length of 10 amino acids; other peaks are separated by 3 nucleotides = 1 amino acid. Immunoscope results for all Vα and Vβ CDR3s for all analyzed mice are shown in Fig. S2 D and E. The arrow indicates the relatively increased signal for the 9-amino acid-long Vα12 CDR3 in AIRE° Treg.

Fig. 2.

AIRE° and WT CD8⁺CD28^low cells have identical activity in in vitro suppression assays and produce similar levels of IL-10. (A) CD8⁺CD28^low Treg isolated from WT (AIRE^+/+) and AIRE° (AIRE°^/°) C57BL/6 (B6) mice were cultured with CFSE-labeled responder B6 CD4⁺ T cells and APC in the presence of anti-CD3ε antibody. Proliferation of CD4⁺ cells was assessed by FACS analysis of CFSE dilution. (B) As in A, but responder CD4⁺ T cells were analyzed by flow cytometry for IFN-γ production. (C) B6 CD4⁺ T cells were cultured with DBA/2 APC in the presence of B6 AIRE° or WT CD8⁺CD28^low Treg (as indicated) at indicated Treg:CD4 ratios. Proliferation in these mixed lymphocyte reactions was assessed by measuring incorporation of [³H]thymidine. (D) CD8⁺CD28^low Treg were activated in vitro with anti-CD3ε antibody and APC over 5 d and then intracellularly stained with antibody specific for IL-10 or with isotype-matched control antibody (dotted line). (E) As in D, but supernatants of cultures were analyzed by ELISA for IL-10. The broken line indicates background value, determined in the absence of anti-CD3ε antibody in the in vitro culture. Results are representative of those obtained in at least three independent experiments.

Fig. 3.

AIRE° CD8⁺CD28^low T cells fail to prevent colitis. RAG-2° hosts were i.v. injected with syngeneic CD4⁺CD45RB^high colitogenic T cells with or without the indicated CD8⁺CD28^low Treg (“wt”, AIRE^+/+; “AIRE°”, AIRE°^/°). (A) Evolution of weight of animals. Shown is the mean weight ± SD as a percentage of weight at the start of the experiment (*P < 0.05, **P < 0.01, Mann–Whitney test; n = 9 per group; three independent experiments). (B) Mice were euthanized 6 wk after injection of T cells. Microscopic sections of distal colon were stained with hematoxylin and eosin and examined for histological signs of colitis. Shown results are representative of those obtained in three independent experiments. (C) Colons of mice were examined as in B and clinical scores of colitis were attributed (n = 9 from three independent experiments).

Fig. 4.

AIRE° CD4⁺CD25^high Treg do not show any defect in prevention of colitis. Colitis was induced as in Fig. 3. (A) Evolution of weight of animals. Shown is the mean weight ± SD as a percentage of weight at the start of the experiment (*P < 0.05, **P < 0.01, Mann–Whitney test; n = 6 without Treg, n = 8 with WT Treg, n = 8 with AIRE° Treg; two independent experiments). (B) Mice were euthanized 7 wk after injection of T cells. Microscopic sections of distal colon were stained with hematoxylin and eosin and examined for histological signs of colitis. Shown results are representative of those obtained in two independent experiments. (C) Colons of mice were examined as in B and clinical scores of colitis were attributed (n values as indicated, from two independent experiments).