Detection of an autoreactive T-cell population within the polyclonal repertoire that undergoes distinct autoimmune regulator (Aire)-mediated selection

Abstract

The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting the display of tissue-specific antigens in the thymus. To study the influence of Aire on thymic selection in a physiological setting, we used tetramer reagents to detect autoreactive T cells specific for the Aire-dependent tissue-specific antigen interphotoreceptor retinoid-binding protein (IRBP), in the polyclonal repertoire. Two class II tetramer reagents were designed to identify T cells specific for two different peptide epitopes of IRBP. Analyses of the polyclonal T-cell repertoire showed a high frequency of activated T cells specific for both IRBP tetramers in Aire(-/-) mice, but not in Aire(+/+) mice. Surprisingly, although one tetramer-binding T-cell population was efficiently deleted in the thymus in an Aire-dependent manner, the second tetramer-binding population was not deleted and could be detected in both the Aire(-/-) and Aire(+/+) T-cell repertoires. We found that Aire-dependent thymic deletion of IRBP-specific T cells relies on intercellular transfer of IRBP between thymic stroma and bone marrow-derived antigen-presenting cells. Furthermore, our data suggest that Aire-mediated deletion relies not only on thymic expression of IRBP, but also on proper antigen processing and presentation of IRBP by thymic antigen-presenting cells.

Fig. 1.

Adult Aire^−/− mice exhibit a high frequency of P2- and P7-specific T cells. (A) Peripheral lymphoid organs were pooled from adult Aire^+/+ or Aire^−/− mice and stained with the P2 and P7 tetramers. Representative FACS plots of tetramer-enriched, peripheral CD4⁺ T cells from an Aire^+/+ mouse, an Aire^−/− mouse with a low frequency of tetramer-positive T cells, and an Aire^−/− mouse with a high frequency of tetramer-positive cells are shown. The absolute number of tetramer-positive cells from the individual mice is noted in each FACS plot. (B) The total number of P2- and P7-specific binding cells from individual mice were quantified for Aire^+/+ and Aire^−/−. Each data point represents one mouse, and the horizontal bar is the average of each group. The dotted line in each graph is the limit of detection (LOD). The LOD is the average number of tetramer-positive CD3⁺CD8⁺ cells (shown on the right side of the graph) plus 3 SDs of this average (16).

Fig. 2.

High frequency of activated P2 and P7 specific T cells correlates with the presence of uveitis in Aire^−/− mice. (A) H&E-stained retina sections (200× magnification) taken from 10- to 20-wk-old Aire^−/− mice with low (Left) and high (Right) frequencies of P2- and P7-specific T cells which correlates with no uveitis (Left) and severe uveitis (Right). (B) The total number of P2 and P7 specific binding cells from 10- to 20-wk-old Aire^−/− mice, as shown in Fig. 1B, were quantified. Each data point represents one mouse, and the horizontal bar is the average of each group. The dotted line in each graph is the LOD, as described previously. (C and D) Individual lymph nodes from 10- to 20-wk-old 5 Aire^+/+ or 5 Aire^−/− mice were pooled and analyzed by flow cytometry after tetramer-binding T-cell enrichment. (C) The absolute number of tetramer-positive cells is noted in each FACS plot. (D) The total number of tetramer-positive cells in the designated peripheral lymphoid organs pooled from five mice was calculated. Data in C and D are representative of three independent experiments.

Fig. 3.

P2-specific CD4⁺ T cells can be expanded in Aire^−/− mice, but not in Aire^+/+ mice, whereas P7-specific cells can be expanded in both Aire^−/− and Aire^+/+ mice. Here, 4- to 7-wk-old Aire^+/+, Aire^−/−, and IRBP^−/− mice were immunized with either P2 peptide or P7 peptide in CFA. At 8 d after immunization, tetramer-positive T cells in the secondary lymphoid organs were quantified. Representative FACS plots (Upper) and a summary of all data collected (Lower) are shown. Each graphed data point represents one mouse. The dotted line in each graph is the LOD.

Fig. 4.

Thymic deletion of P2-specific cells, but not of P7-specific cells, is dependent on Aire expression. Four thymi from Aire^+/+ and Aire^−/− mice were pooled and the tetramer-binding T cells quantified. Representative FACS plots of CD3⁺CD4⁺ SP thymocytes and CD3⁺CD8⁺ SP thymocytes from pooled Aire^+/+ and Aire^−/− thymi are shown. Each data point in the summary graph represents the number of tetramer-positive cells in the four thymi. The dotted line in each graph is the LOD.

Fig. 5.

Thymic APCs preferentially present the P2 peptide epitope over the P7 peptide epitope. P7-specific (A2, E4, and F8) and P2-specific (LB4) hybridoma stimulation was measured by CD69 up-regulation. (A) T-cell hybridomas were incubated with irradiated WT B6 splenocytes and P2 or P7 peptide for 12 h. The empty triangle and circle data points indicate hybridoma stimulated in the presence of anti-class II blocking mAb plus 10 μg/mL of P2 peptide and P7 peptide, respectively. Data are representative of three independent experiments. (B) P7-specific (A2, E4, and F8) and P2-specific (LB4) hybridoma clones were incubated with CD45⁺CD11c⁺ thymic APC and soluble eye antigen from WT or IRBP^−/− mice. Data are representative of two independent experiments. (C) Four- to 5-wk-old WT and Aire^−/− mice were immunized with eye lysate from WT in CFA or CFA alone. At 8 d after immunization, CD44^high tetramer-positive T cells in the secondary lymphoid organs were quantified. Each graphed data point represents one mouse. The dotted line in each graph is the LOD.

Fig. 6.

Thymic deletion of P2-specific cells, but not of P7-specific cells, is dependent on antigen presentation by BM-derived APCs. Irradiated WT mice were reconstituted with either WT or CIITA^−/− BM. Four thymi were pooled and the tetramer-binding T cells quantified. Representative FACS plots of CD3⁺CD4⁺ SP and CD3⁺CD8⁺ SP thymocytes are shown. Each data point in the summary graph represents the number of tetramer-positive cells in the four thymi. The dotted line in each graph is the LOD.