Diversity of TCRs on natural Foxp3+ T cells in mice lacking Aire expression

Abstract

Medullary thymic epithelial cells expressing the Aire gene play a critical role in the induction of tolerance to tissue-specific Ags (TSAs). It was postulated that recognition of Aire-controlled TSAs by immature thymocytes results in the selection of natural CD4+Foxp3+ regulatory T cells (Tregs) and enriches this repertoire in self-reactive receptors, contributing to its vast diversity. In this study, we compared the TCRs on individual Tregs in Aire+ and Aire- mice expressing a miniature TCR repertoire (TCRmini) along with GFP driven by the Foxp3 promoter (Foxp3GFP). The Treg TCR repertoires in Aire+ and Aire- TCRminiFoxp3GFP mice were similar and more diverse than their repertoires on CD4+ Foxp3- thymocytes. Further, TCRs found on potentially self-reactive T cells, with an activated phenotype (CD4+Foxp3-CD62Llow) in Aire- TCRminiFoxp3GFP mice, appear distinct from TCRs found on Tregs in Aire+ TCRminiFoxp3GFP mice. Lastly, we found no evidence that TSAs presented by medullary thymic epithelial cells in Aire+TCRmini mice are often recognized as agonists by Treg-derived TCR hybridomas or CD4+CD25+ thymocytes, containing both natural Tregs and precursors. Thus, positive selection and self-reactivity of the global Treg repertoire are not controlled by Aire-dependent TSAs.

FIGURE 1

CD4⁺Foxp3⁺ thymocytes from Aire⁺ and Aire⁻ TCR^mini Foxp3^GFP mice express similarly diverse repertoires of TCRs. A, FACS analysis of Aire⁺ and Aire⁻ TCR^miniFoxp3^GFP thymocytes. The numbers in the quadrants represent the percentage of cells in the indicated quadrant. Results are representative of at least three independent experiments, each using two or more mice per group. B and C, 2D-F-SSCP analysis comparing the TCRα-CDR3 regions of CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ thymocytes from Aire⁺ and Aire⁻ TCR^mini Foxp3^GFPmice (total naive spots: 131 versus 145, respectively) (total regulatory spots: 235 versus 232, respectively). The intensity of individual spots corresponds to the real frequency of a particular VαJα rearrangement in the sample. Each spot represents one CDR3α region, except on rare occasions in which more than one sequence may be represented, if their conformation is the same. Nevertheless, the pattern of migration of that sequence would be the same in each population compared. Spots migrating with different speeds on the gel represent different rearrangements. Overlapping spots between two populations are shown in the middle panel colored blue, representing identical VαJα rearrangements, and unique spots are red. Results are representative of three independent experiments, each using two mice per group. D and E, TCRα-CDR3 region amino acid sequences obtained from single cell-sorted RT-PCR products comparing the frequencies of dominant (found five or more times) CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ TCRs from Aire⁺ and Aire⁻ TCR^miniFoxp3^GFP mice (total regulatory sequences: 492 versus 201, respectively) (total naive sequences: 417 versus 425, respectively). F, Computed Morisita-Horn similarity index based on sequences from D and E. The Morisita-Horn index assesses the probability that any two randomly chosen sequences from two populations will be the same. This index ranges from 0 (no similarity) to 1 (total similarity). Results are representative of two independent experiments using a total of nine mice to retrieve ~2225 thymic regulatory and naive sequences.

FIGURE 2

Dominant TCRs expressed on CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ thymocytes in Aire⁺ and Aire⁻ TCR^miniFoxp3^GFP mice are asymmetrically distributed. A and B, The frequencies of the most dominant CD4⁺Foxp3⁻ and Treg clones found in Aire⁺ and Aire⁻ TCR^miniFoxp3^GFP mice. Shown amino acid sequences represent fragments of TCRα-CDR3 regions, beginning with the third amino acid after the invariant C residue in all TCRAV genes (Y-L/F-C-A-X-1) and spanning the amino acid immediately preceding the TCRAJ motif (2-F/W-G-X-F-G-T). Clones shared between populations are highlighted by gray boxes. C, TCRα-CDR3 region amino acid length distribution of naive and regulatory sequences obtained from single-cell RT-PCR from Aire⁺ and Aire⁻ TCR^miniFoxp3^GFP mice. The most common CDR3 length is 8 aa.

FIGURE 3

Aire⁻ TCR^miniFoxp3^GFP mice develop autoimmune manifestations in the salivary gland. Representative histopathology of the salivary glands and lungs in Aire⁺ and Aire⁻ TCR^miniFoxp3^GFP mice (12–15 wk). A and B, Healthy lung (×20) and salivary gland (×10) from Aire⁺ TCR^miniFoxp3^GFP mice. C and D, Lung (×20) with little infiltration and salivary gland (×20) showing mild infiltration without tissue destruction. Organs were fixed with 10% neutral buffered formalin, sections were cut at 8 μm and stained with H&E.

FIGURE 4

Many TCRs expressed on CD4⁺Foxp3⁺ thymocytes in Aire⁺ TCR^miniFoxp3^GFP mice are also found on peripheral CD4⁺Foxp3⁺ T cells in Aire⁻ TCR^miniFoxp3^GFP mice. A, FACS analysis of CD4⁺Foxp3⁺ cells in salivary gland and lung of Aire⁻ TCR^miniFoxp3^GFP mice. B, Cross-comparison of the frequencies of total regulatory TCRα-CDR3 region amino acid sequences in Aire⁺ TCR^miniFoxp3^GFP thymii to Aire⁻ TCR^miniFoxp3^GFP thymus, lymph node, salivary gland, and lung. The table below the graph shows the total and unique/different number of sequences analyzed and shared. The percentage shared of total was calculated by dividing the total shared by the total analyzed; the percentage of unique shared was calculated by dividing the unique shared by the unique analyzed; and percentage of unique of total was calculated by dividing the unique by the total analyzed. Results are representative of three independent experiments using a total of 11 mice to retrieve 1075 regulatory sequences.

FIGURE 5

Self-Ags presented by mTECs in Aire⁺ mice are infrequently recognized as agonists by Treg cells. A, FACS analysis of dGuo-treated neonatal thymic cultures from Aire⁺ thymii used to isolate fresh EpCAM⁺Ab⁺ mTECs. B, IL-2 secretion/T cell activation assay using hybridomas derived from Tregs isolated from wild-type, TCR^mini, and A^bEp single-peptide mice or from autoreactive CD4⁺ T cells from Scurfy mice. Hybridomas were cocultured for 24 h in the presence of anti-CD3 mAb or with freshly isolated mTECs. C, FACS analysis of CD69 upregulation by CD4⁺CD25⁺ thymocytes from A^bEp, Aire⁺, and Aire⁻ mice cocultured with fresh mTECs isolated from Aire⁺ mice or with immobilized anti-CD3 mAb. Results are representative of two independent experiments with hybridomas using the indicated number of hybridomas each time and three independent experiments for CD69 up-regulation using at least five mice per experiment for mTEC isolation and at least two mice per experiment per group for thymocytes.

FIGURE 6

In nonlymphoid organs of Aire⁻ TCR^miniFoxp3^GFP mice, dominant activated and Treg clones express nonoverlapping TCRs and do not appear to be diverted from self-reactive thymocytes in Aire⁺ TCR^miniFoxp3^GFP mice. A, FACS analysis of activated CD4⁺ CD62L^low T cells found in the salivary gland and lung from Aire⁻ TCR^miniFoxp3^GFP mice. B, Extrapolation of activated TCRα-CDR3 region amino acid sequences from Aire⁻ TCR^miniFoxp3^GFP mice lung and salivary gland back to Treg TCRα-CDR3 region amino acid sequences in Aire⁺ TCR^mini thymii (also marked by asterisks in C). C, Comparison of the frequencies of dominant TCRs (found three or more times) on activated and Tregs from Aire⁻ TCR^miniFoxp3^GFP mice. Clones shared between populations are highlighted by gray boxes.