Aire Enforces Immune Tolerance by Directing Autoreactive T Cells into the Regulatory T Cell Lineage

Abstract

The promiscuous expression of tissue-restricted antigens in the thymus, driven in part by autoimmune regulator (Aire), is critical for the protection of peripheral tissues from autoimmune attack. Aire-dependent processes are thought to promote both clonal deletion and the development of Foxp3(+) regulatory T (Treg) cells, suggesting that autoimmunity associated with Aire deficiency results from two failed tolerance mechanisms. Here, examination of autoimmune lesions in Aire(-/-) mice revealed an unexpected third possibility. We found that the predominant conventional T cell clonotypes infiltrating target lesions express antigen receptors that were preferentially expressed by Foxp3(+) Treg cells in Aire(+/+) mice. Thus, Aire enforces immune tolerance by ensuring that distinct autoreactive T cell specificities differentiate into the Treg cell lineage; dysregulation of this process results in the diversion of Treg cell-biased clonotypes into pathogenic conventional T cells.

Figure 1. Treg Cells Are Present at Elevated Frequency in the Prostate of Aire^−/− Mice

(A) Representative flow cytometric analyses of 9-week-old Aire^+/+ and Aire^−/− males on the B6 background. Top, CD8 vs. CD4 expression by T cells isolated from the spleen, lymph nodes, and prostate. Bottom, for the CD4⁺CD8α^neg cells in the top panels, plots of Foxp3 vs. CD4 expression. The percentage of cells falling within the denoted gates is indicated. pLN, periaortic lymph nodes; cLN, cervical lymph nodes; mLN, mesenteric lymph nodes. (B and C) Summary plots of (B) the percentage of Foxp3⁺ cells among CD4⁺ T cells and (C) the total number of CD4⁺Foxp3⁺ T cells isolated from the indicated sites of Aire^+/+ (open circles) or Aire^−/− (closed circles) males depicted in panel A. N = 5 for Aire^+/+ samples, N = 5–8 for Aire^−/− samples. Data are pooled from N = 2 experiments. The mean +/− SEM is indicated. Asterisks denote P < 0.05. For (B), ANOVA and Tukey’s multiple comparison test were used to compare the different organ sites within Aire^+/+ mice and the different organ sites within Aire^−/− mice. For (C), t-tests were used to compare Aire^+/+ vs. Aire^−/− mice at each organ site. (D) Representative flow cytometric analyses of CD69 and CD44 expression by CD4⁺ Tconv cells from >9- week-old Aire^+/+ and Aire^−/− males (indicated). Bold histograms denote CD4⁺ Tconv cells from the prostate, filled gray histograms denote CD4⁺ Tconv cells from the spleen. The percentage of cells expressing the indicated markers is shown.

Figure 2. Aire Impacts the CD4⁺ T Cell Repertoire in the Secondary Lymphoid Organs

CD4⁺Foxp3⁺ Treg cells and CD4⁺Foxp3^neg Tconv cells were purified by FACS from 9-week-old TCRβTg Foxp3^GFP males on an Aire^+/+ and Aire^−/− background, and subjected to complete TCRα sequencing. (A and B) The impact of Aire on the Treg cell repertoire (A) and Tconv cell repertoire (B) was analyzed via complete TCRα sequencing of T cell populations isolated from the pooled spleen and lymph nodes using the iRepertoire platform (see Experimental Procedures). For the TCRα chain sequences, volcano plots of false discovery rate (FDR) versus differential TCR representation (log₂ fold-change) in Aire^+/+ mice vs. Aire^−/− mice are plotted for Treg cells (A) and Tconv cells (B). 7,955 TCRs are plotted in A, and 6,422 TCRs are plotted in B. Comparisons were made using EdgeR and were adjusted for multiple comparisons (see Experimental Procedures). Red dots denote TCRs with fold-change > 4 and FDR < 0.05. The arms of the volcano plots denoting TCRs that are overrepresented or underrepresented in Aire^−/− mice are indicated in gray above each plot. Data points with FDR < 10⁻⁶ are plotted at 10⁻⁶. Horizontal dashed lines indicate FDR cutoff. N = 4 for Aire^+/+ Treg cell samples, N = 5 for Aire^−/− Treg cell samples, N = 4 for Aire^+/+ Tconv cell samples, N = 5 for Aire^−/− Tconv cell samples. See also Figure S1 and Table S1.

Figure 3. The Autoimmune Defect in Aire^−/− Mice Maps to the Tconv Cell Compartment

(A and B) A mixture of 7×10⁶ CD4⁺Foxp3^neg Tconv cells and 1×10⁶ CD4⁺Foxp3⁺ Treg cells, isolated from the pooled spleen and lymph nodes of 4–5-week-old CD45.2⁺ Aire^+/+ or Aire^−/− males, as indicated, was transferred into 6–8-week-old male CD45.1⁺ Rag1^−/− recipients. 3 weeks post-transfer, the fate of CD45.2⁺ donor cells was assessed at different sites. (A) Representative flow cytometric plots of Foxp3 vs. CD4 expression are shown for CD4⁺CD45.2⁺ donor T cells isolated from the indicated organs. The percentages of cells falling within the denoted gates are shown. pLN, periaortic lymph nodes; cLN, cervical lymph nodes; bLN, brachial lymph nodes. (B) Summary plot of the number of CD45.2⁺ donor Tconv cells recovered from the prostate. The source (Aire^+/+ or Aire^−/−) of donor Treg cells and Tconv cells is indicated. Data are pooled from N = 4 experiments. The mean +/− SEM is denoted. Asterisks indicate P < 0.05; ns, not significant. Statistical comparisons of all groups were made using ANOVA and Tukey’s multiple comparison test. (C and D) A fixed number of CD4⁺Foxp3^neg Tconv cells (1.5×10⁶) from the pooled spleen and lymph nodes of 4–5-week-old CD45.2⁺ Aire^−/− males, were transferred alone, or together with the indicated percentages of Treg cells from CD45.2⁺ Aire^+/+ males, into 6–8-week-old male CD45.1⁺ Rag1^−/− recipients. 3 weeks post-transfer, the fate of CD45.2⁺ donor cells was assessed at different sites. (C) Representative flow cytometric plots of Foxp3 vs. CD4 expression are shown for CD4⁺CD45.2⁺ donor T cells isolated from the indicated organs. (D) Summary plot of the number of CD45.2⁺ donor Tconv cells recovered from the prostate. Data are pooled from N > 3 experiments. The mean +/− SEM is denoted. Asterisks indicate P < 0.05; ns, not significant. Statistical comparisons of all groups were made using ANOVA and Tukey’s multiple comparison test. See also Figures S2 and S3.

Figure 4. A Large Proportion of Tconv Cell Clonotypes Overrepresented in Aire^−/− Mice Are Preferentially Expressed by Treg Cells in Aire^+/+ Mice

For the TCR clonotypes that are recurrently overrepresented in the Tconv cell subset of Aire^−/− mice relative to the Tconv cell subset of Aire^+/+ mice (denoted in red in left arm of volcano plot in Fig. 2B), the mean frequencies of these clonotypes in the Tconv cell and Treg cell catalogs from Aire^+/+ mice (left) and Aire^−/− mice (right) are plotted. The shift of many TCRs from below the diagonal in the left plot to above the diagonal in the right plot reflects a shift from preferential representation in the Treg cell subset in Aire^+/+ mice to preferential representation in the Tconv cell subset in Aire^−/− mice. N = 246 TCRs. The red arrow denotes 15 superimposed data points, representing TCRs that are not detected (ND) in both the Tconv cell and Treg cell catalogs from Aire^+/+ mice, suggestive of clonotypes that are deleted in Aire^+/+ mice. Of the 246 TCRs shown, 183 TCRs (74%) are biased to the Treg cell lineage (i.e. lie below the diagonal) in the Aire^+/+ catalogs. See also Table S1.

Figure 5. The Predominant TCR Clonotypes Expressed by Tconv Cells Infiltrating the Prostates of Aire^−/− Mice are Preferentially Expressed by Treg Cells in Aire^+/+ mice

Tconv cells were FACS-purified from the prostates of nine 9-week-old Aire^−/− TCRβTg Foxp3^GFP males, and subjected to complete TCRα sequence analysis (see Experimental Procedures). These data were then compared to TCR frequency data from “catalogs” generated via complete TCRα sequencing of Tconv cell and Treg cell populations isolated from the pooled spleen and lymph nodes of Aire^+/+ and Aire^−/− mice, described in the text and depicted in Fig. 2. (A) For the 10 most prevalent Tconv cell TCRs in each Aire^−/− prostate sample, the average frequency (log₁₀) of each TCR in the Tconv cell catalog is plotted vs. the average frequency (log₁₀) in the Treg cell catalog for catalogs derived from Aire^+/+ mice (top row) and Aire^−/− mice (bottom row). The size (area) of each data point is proportional to the frequency of this TCR in the Tconv cell subset of the Aire^−/− prostate sample. For each prostate sample, the frequency of the most prevalent TCR clonotype is indicated (as a percentage of sequence reads). For example, in Aire^−/− prostate 1, 47% of Tconv cell TCR sequence reads encode the ALSMSVNYQLI TCR (magenta), indicating predominance of this clonotype. This TCR is preferentially expressed by Treg cells in the Aire^+/+ catalog (top row), but is preferentially expressed by Tconv cells in the Aire^−/− catalog (bottom row). TCRs that are found among the 10 most prevalent clonotypes in more than one Aire^−/− prostate sample are color-coded as indicated in the legend. TCRs that are found only once among the 10 most prevalent Tconv cell TCRs in these nine samples are shown in gray. ND, not detected in catalog. Data points centered within the dashed boxes were not detected in both the Tconv cell and Treg cell catalogs for the indicated Aire genotype. See also Table S2. (B) Summary of the data plotted in A. For each Aire^−/− prostate sample, the cumulative percentage of Tconv cell clonotypes exhibiting representation patterns suggestive of “diverted Treg cell clonotypes” or “clonotypes evading deletion” are plotted. TCRs that fall below the diagonal in the Aire^+/+ catalog and above the diagonal in the Aire^−/− catalog are classified as “diverted Treg cell clonotypes”. TCRs that are not detected in the Aire^+/+ catalog, but are observed in the Aire^−/− catalog are classified as “clonotypes evading deletion”. Lines denote paired samples from a given mouse. The mean +/− SEM is indicated. Statistical analysis was performed using a paired t-test. Asterisks indicate P < 0.05.

Figure 6. In the Absence of Aire, MJ23 T Cells Are Diverted Into the Tconv Cell Lineage And Dominate the Prostatic TRAV14⁺ Tconv Cell Infiltrate

CD4⁺Foxp3^neg Tconv cells and CD4⁺Foxp3⁺ Treg cells were FACS-purified from the prostate and prostatedraining lymph nodes (pLN) of 9-week-old TCRβTg Foxp3^GFP males on the Aire^+/+ or Aire^−/− background. cDNA was then subjected to TRAV14 TCRα sequencing. (A) In many mice, the prostate-specific MJ23 TCRα LYYNQGKLI is the predominant TCRα expressed by TRAV14⁺ Tconv cells infiltrating the Aire^−/− prostate. For Tconv cells at this site, the percentages of TRAV14⁺ TCR encoding the indicated TCRα chains are plotted for each sample. The five most abundant TRAV14⁺ TCRs found in two or more samples are shown. The mean +/− SEM is indicated. N = 6 for Aire^+/+ and Aire^−/− groups. (B) In the pLN, the MJ23 TCRα LYYNQGKLI is skewed to the Treg cell lineage in Aire^+/+ mice, but found in both Tconv cell and Treg cell subsets in Aire^−/− mice. For Tconv cells (left plot) or Treg cells (right plot) isolated from the pLN of Aire^+/+ or Aire^−/− mice, the percentage of TRAV14⁺ TCRs encoding the MJ23 TCRα is plotted. The mean +/− SEM is indicated. Asterisks indicate P < 0.05; ns, not significant. Statistical comparisons between Tconv cell and Treg cell subsets were made using the Mann-Whitney nonparametric test. N > 8 for Aire^+/+ and Aire^−/− groups.