Activation-induced cell death of self-reactive regulatory T cells drives autoimmunity

Abstract

Activation of self-reactive T cells is a major driver to autoimmunity and is suppressed by mechanisms of regulation. In a humanized model of autoimmune thyroiditis, we investigated the mechanism underlying break of tolerance. Here, we found that a human TCR specific for the self-antigen thyroid peroxidase (TPO) is positively selected in the thymus of RAG KO mice on both T effector (Teff) and T regulatory (Treg) CD4+Foxp3+ cells. In vivo Teff are present in all immune organs, whereas the TPO-specific Treg are present in all lymphoid organs with the exception of the thyroid-draining lymph nodes. We suggest that the presence of TPO in the thyroid draining lymph nodes induces the activation of Teff and the depletion of Treg via activation-induced cell death (AICD). Our findings provide insights on the failure of the mechanisms of immune tolerance, with potential implications in designing immunotherapeutic strategies.

Keywords: AICD; autoimmunity; regulatory T cells; tolerance.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Phenotypic and genotypic characterization of CD4+CD25+ T cells from TAZ10 mice. (A) Flow cytometry of TAZ10 spleen stained for CD4 and CD25. Density plot is gated on FSC/SSC. (B) Staining for surface CD4 and intracellular Foxp3 of splenocytes gated on the CD4+ population. (C) Staining of TAZ10 splenocytes for CD4, CD25, CD62L, and Foxp3. Cells were gated on the CD4+Foxp3+ and CD4+Foxp3− populations, further gated on CD25highCD62L+ Treg and CD25-/lowCD62L− T effector (Teff) cells. (D) Staining of TAZ10 and WT thymus for human TCRBV1 or mouse TCRβ, respectively, CD4, CD8, and Foxp3. TAZ10 and WT thymocytes are gated on human TCRBV1+ and mouse TCRβ+, respectively. (E) Real-time PCR for Foxp3 in the spleen and thymus of TAZ10 (blue, n = 3) and WT mice (red, n = 3). CD4+CD25+ splenocytes from WT mice were used as positive controls (n = 3). Foxp3 mRNA expression was normalized against GAPDH and CD8+CD25− sorted cells were used as calibrator. Dots represent individual mice. (F) RT-PCR for the expression of mouse Foxp3 and β-Actin on cellular extract of whole thymus, spleen, or purified CD4+ and CD8+ T cells from TAZ10 mice. Data are from 1 experiment representative of more than 10 mice analyzed (A–C) or 4 independent experiments (D and F). (E) Statistical analysis comparing the trends of expression of Foxp3 between WT and TAZ10 was performed using 2-tailed unpaired Student’s t test (nonsignificant [n.s.], P > 0.05; **P < 0.01). Cell percentages are indicated in each quadrant (A–D).

Fig. 2.

TAZ10 CD4+CD25+Foxp3+ Treg suppress Teff proliferation in vitro. (A) Depletion of CD25+ T cells was done by incubating TAZ10 LNs and splenocytes (from pooled mice, n = 6) with the anti-CD25 monoclonal antibody clone 7D4 in conjunction with rabbit complement. Complement only was used as control. Depletion’s efficiency was verified by costaining with CD4 and CD25 (clone PC61). Numbers in quadrants indicate cell percentages. Untreated (C, red) or CD25 depleted (7D4, blue) TAZ10 splenocytes were challenged with: plate-bound CD3 antibody alone or in conjunction with CD28 for 18 h (B), and the levels of IFNγ, IL-2, and IL-10 were assessed by ELISA (C); CD3 in the presence of increasing concentrations of blocking anti-mouse DTA-1 mAb [0–100 mg/mL] (D); increasing concentrations of TPO peptide (TPO536–547) [0.1–10 μM] (E); TPO536–547 [10 μM] in the presence or absence of sorted TAZ10 CD4+CD25+ Treg (green) (F). (B–F) Data show 1 representative of 3 (B and D–F) or 4 (C) independent experiments, each done in triplicate with 3 independent wells (mean + SEM). Proliferation was assessed by 72-h thymidine incorporation assay adding Tritiated Thymidine in the last 18 h of the assay. (B–F) Statistical analysis was performed by using 2-tailed unpaired Student’s t test (nonsignificant [n.s.], P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001).

Fig. 3.

CD4+CD25+Foxp3+ Treg cells suppress autoimmune thyroiditis in vivo. (A) Three-week-old TAZ10 mice (n = 6; purple) were injected periodically with anti-mouse CD25 mAb (clone PC61) and weighted at 12 and 20 wk of age. PC61-treated TAZ10 mice showed a significantly higher weight at 12 wk of age (mean 37.4 ± 3.7 g), than age-matched nontransgenic (n = 6; orange; mean 22.2 ± 1.2 g) and untreated TAZ10 littermates (n = 6, green, mean 29.7 ± 1.3 g). No significant difference was observed between untreated and PC61-treated TAZ10 mice at 20 wk, scoring close average weights (mean 40.1 ± 2.2 g and 42.5 ± 2.1 g, respectively), and both significantly higher than WT Rag−/− littermates. Age-matched WT Rag−/− mice showed no significant weight increase at 12 or 20 wk (mean 22.2 ± 1.2 g and 22.8 ± 0.9 g, respectively). (B) Serum TSH levels of 20-wk-old Rag−/− (orange), untreated (green), and PC61-treated TAZ10 mice (purple) (n = 6). PC61-treated and PC61-untreated transgenic mice showed significantly higher TSH levels than Rag−/− littermates. (C and D) At 20 wk, mice were killed and thyroids observed by H/E staining for sign of thyroiditis (D) and immunofluorescence with anti-human TCRBV1-FITC antibody to mark infiltrating pathogenic T cells. (Magnification: C and D, Left, 20×; D, Right, 40×.) The thyroid of 1 representative mouse of 6 is shown. (E) Mean infiltration score of TCRBV1+ cells of thyroid sections of TAZ10 mice untreated or injected with PC61 mAb (n = 6). (A and B) Data are done on the same mice at 12 and 20 wk of age (mean + SEM). Statistical analysis was calculated using ordinary 1-way ANOVA with Tukey’s multiple comparisons test (A and B) or 2-tailed unpaired Student’s t test (E) (nonsignificant [n.s.], P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001).

Fig. 4.

WT Treg home to the sites of inflammation and protect TAZ10 mice from autoimmunity. (A) CD4, CD25, and Foxp3 expression in ND-LNs and D-LNs of TAZ10 mice. Numbers indicate cell percentages. (B) RT-PCR for Foxp3, IL10, TCRBV1, and β-Actin in the spleen, ND-LNs, D-LNs, and thyroid of TAZ10 mice. (C) Adoptive transfer of CD4+CD25+ cells from WT syngeneic mice into 3-wk-old TAZ10 Rag−/− mice (n = 5). Density plots of 1 representative mouse show the distribution of WT TCRBV1− or TAZ10 TCRBV1+ CD4+Foxp3+ Treg and CD4+Foxp3– Teff in ND-LNs and D-LNs of Treg-injected TAZ10 mice. (D) Weight record at 12 and 20 wk of age of Rag−/− (orange), TAZ10 (green), and Treg-injected TAZ10 mice (purple). At 12 wk and 20 wk, Treg-injected TAZ10 mice showed no significant weight gain compared to age-matched nontransgenic Rag−/− (mean 23.88 ± 4.1 g; P = 0.3339, ns; and 26.5 ± 5.9 g; P = 0.1901, ns, respectively). Weight of untreated transgenic littermates increased as to 29.96 ± 1.4 g at 12 wk and 40.66 ± 5.9 g at 20 wk being significantly higher of Rag−/− and Treg-injected littermates. (E) Serum TSH levels of Rag−/− (orange), untreated (green), and Treg-injected TAZ10 mice (purple) at 20 wk of age. Untreated TAZ10 mice had the highest TSH levels, but injection of Treg was accompanied by a drastic reduction of TSH levels compared to the untreated mouse control. TSH levels in Treg-injected TAZ10 compared to control Rag−/− littermates was also increased. (F) H/E staining of thyroids from untreated and Treg-injected TAZ10 mice. (Magnification 20×.) (A and B) One representative mouse of 10 is shown. (D) Data are done on the same mice at 12 and 20 wk of age (mean + SEM). (D and E) Statistical analysis was performed with ordinary 1-way ANOVA with Tukey’s multiple comparisons test (nonsignificant [n.s.], P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 5.

Stimulation with the TPO cognate antigen induces a reduction of Foxp3 expression. (A) Medullary thyroid epithelial cells (mTEC) of WT mice express H2-Ak molecules under stimulation with IFNγ (dotted line), while TAZ10 mice express H2-Ak constitutively (shaded line). (B) Seventy-two-hour proliferation assay of TAZ10 T cells stimulated with TAZ10 mTEC (purple) and WT mTEC activated with IFNγ (green). No proliferation is observed with resting WT mTEC (orange). WT T cells were used as negative control. (C) Protein extract obtained from tissue homogenate of thyroid, cervical D-LNs, ND-LNs, spleen, lung, and gut from 80 mice was loaded on professional mDCs and used as APC on a 72-h thymidine incorporation assay to stimulate TAZ10 T cells. Coated aCD3 antibody was used as positive control. The error bars represent variation of 3 technical replicates. (D–G) Purified CD4+ T cells isolated from mesenteric LN and spleen of pooled TAZ10 mice (n = 6) were added to autologous mature DC challenged with αCD3, TPO536–547 peptide [0.1 μM], thyroid purified human TPO [0.1 μM], or different concentrations of recombinant human TPO [1 and 20 μM]. RT-PCR for the expression or Foxp3, IL10, human TCRBV1, and β-actin ex vivo (D) and after stimulation with α-CD3 or human TPO (E–G). (B) Statistical analysis using 2-tailed unpaired Student’s t test (nonsignificant [n.s.], P > 0.05; ****P < 0.0001).

Fig. 6.

Treg display higher sensitivity to the cognate TPO antigen than effector T cells. (A) Proportion (%) of CD4+Foxp3+ Treg (red) and CD4+Foxp3− Teff cells (blue) positive for Annexin V and FAS after 48-h stimulation with aCD3 or mDCs loaded with TPO536–547 peptide [0.1 μM]. The experiment was repeated 3 times in similar conditions. Each dot represents the experimental replicate. (B) Proportion (%) of CD25+CD4+Foxp3+ Treg in different lymphoid districts of TAZ10 mice immunized with thyroid purified human TPO protein (n = 3, red) or PBS (n = 3, blue). (C) RT-PCR for IL2, IL4, IL5, IL10, IFNγ, and Foxp3 in D-LNs and ND-LNs of 1 TAZ10 mouse representative of 3. (A and B) Statistical analysis was performed using 2-tailed unpaired Student’s t test (nonsignificant [n.s.], P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).