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

Ester Badamia,b,c,1, Olivier N. F. Cexusa,d, and Sonia Quaratinoa,e

aCancer Research UK Oncology Unit, Sciences Division, University of Southampton, SO16 6YD Southampton, United Kingdom; bFondazione Ricerca nel Mediterraneo (RiMED), 90133 Palermo, Italy; cDepartment of Laboratory Medicine and Advanced Biotechnologies, Istituto di Ricovero e Cura a Carattere Scientifico–Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, 90127 Palermo, Italy; dAgency for Science Research and Technology, Institute of Molecular and Cell Biology, 138673 Singapore; and eKymab, Babraham Research Campus, CB22 3AT Cambridge, United Kingdom

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved November 11, 2019 (received for review June 18, 2019)

Activation of self-reactive T cells is a major driver to autoimmunity CD25 (Fig. 1A), a surface marker up-regulated by activated Teff and is suppressed by mechanisms of regulation. In a humanized but also constitutively expressed by Treg (10). We expected that the + + model of autoimmune thyroiditis, we investigated the mechanism detected CD4 CD25 T cells were spontaneously activated self- −/− underlying break of tolerance. Here, we found that a human TCR reactive Teff since the transgenic T cells were on the Rag back- + specific for the self- thyroid peroxidase (TPO) is positively ground (11). Surprisingly, about 15% of CD4 T cells expressed selected in the of RAG KO mice on both T effector (T ) eff high levels of Foxp3, the specific marker for Treg in mice (Fig. 1B) + + + + and T regulatory (Treg) CD4 Foxp3 cells. In vivo Teff are present in (12). The majority of CD4 Foxp3 T cells expressed the all immune organs, whereas the TPO-specific Treg are present in all high + + − of Treg being CD25 CD62L , whereas CD4 Foxp3 cells had the lymphoid organs with the exception of the thyroid-draining lymph − classic phenotype of activated effector memory CD25-/lowCD62L nodes. We suggest that the presence of TPO in the thyroid drain- T cells (Fig. 1C and SI Appendix,Fig.S1). Morphologically, these ing lymph nodes induces the activation of T and the depletion of + + + – eff CD4 CD25 T cells were also smaller compared to the CD4 CD25 Treg via activation-induced cell death (AICD). Our findings provide insights on the failure of the mechanisms of , with population, in keeping with a previously published report potential implications in designing immunotherapeutic strategies. (SI Appendix,Fig.S2) (13). We then questioned whether TAZ10 Treg were naturally oc- regulatory T cells | autoimmunity | tolerance | AICD curring Treg (nTreg) selected in the thymus (12, 14) or induced Treg (iTreg), which develop extrathymically in the periphery (15, 16). + + + egulatory CD4 CD25 Foxp3 T cells (T ) play a key role TAZ10 T cells, which express the human TCR VB1/VA15, can be reg stained by the human TCRBV1 monoclonal antibody (mAb). A in the maintenance of immune tolerance by exerting cell-to-cell + R small proportion of TCRBV1 TAZ10 expressed and -mediated suppression (1, 2), while impaired regulation + Foxp3 mainly within the CD4 single-positive population. TCRβ by Treg triggers multiorgan autoimmunity in both mice and human (3, 4). The mechanisms leading to the loss of immune homeostasis thymocytes from wild type (WT) syngenic mice were used as and breakdown of peripheral tolerance, and the role that T cell control (Fig. 1D). We also confirmed that splenocytes and thy- recognition of immunodominant self-, might play in these mocytes from TAZ10 mice express Foxp3 at the RNA level (Fig. + mechanisms remain still elusive due to the limitations of autoim- 1E), and this expression was confined to CD4 Tcells(Fig.1F). mune animal models (5). Our data strongly indicated that TAZ10 mice presented a The TAZ10 mouse model is transgenic for a human T cell re- population with the phenotype of thymus-derived regulatory + + + ceptor (TCR) isolated from the thyroid infiltrate of a patient with CD4 CD25 Foxp3 T cells. autoimmune thyroiditis and specific for the immunodominant cryptic peptide of thyroid peroxidase (TPO536–547)(6,7).Tcell Significance recognition of cryptic epitopes has been shown to play a pivotal role in triggering break of tolerance, as cryptic epitopes are poorly In the present study, we provide evidence of a cellular mechanism presented in the thymus but aberrantly displayed in the periphery of breach of tolerance in an in vivo model of spontaneous auto- under inflammatory conditions (8). We reported that the + − − immune thyroiditis (AIT). We observe that self-antigen (Ag) spe- TAZ10 mice, both on Rag and Rag / backgrounds, spontane- cific T circulate in all lymphatic tissues except for the lymph ously develop autoimmune hypothyroidism, which resembles reg nodes (LNs) draining the thyroid, which represents the site of in- Hashimoto’s disease with its clinical, hormonal, and histological − − flammation. By contrast, cognate effector T cells accumulate in the signs (7, 9). In TAZ10 Rag / mice, clinical manifestations of the draining LNs and thyroid. The absence of T unleashes cognate disease occur as early as 12 wk of age and are more severe than reg + T , which promote tissue destruction. We demonstrate that the in Rag littermates (7, 9). eff organ-restricted disappearance of T is driven by the self-Ag, Here, we describe a unique and unexpected population of reg + + + − − aberrantly expressed in a highly inflammatory environment, by thymus-derived CD4 CD25 Foxp3 T in the TAZ10 Rag / mice. reg a mechanism of activation-induced cell death. Interestingly, despite displaying strong suppressive functions on effector T cells in vitro, these TPO-specific T fail to protect reg Author contributions: E.B. and S.Q. designed research; E.B. performed research; O.N.F.C. TAZ10 mice from autoimmunity in vivo. In this study, we un- contributed new reagents/analytic tools; E.B. and S.Q. analyzed data; E.B., O.N.F.C., and derpin the mechanism responsible for the initiation and exac- S.Q. wrote the paper; O.N.F.C. produced the mDCs from mouse bone marrow and per- erbation of autoimmunity. formed peptide extraction from mouse thyroids; and S.Q. planned and conceived the study and provided experimental advice, overall guidance, and provided funds. Results The authors declare no competing interest. Presence of CD4+CD25+Foxp3+ T Cells in Lymphoid Tissues of TAZ10 This article is a PNAS Direct Submission. Mice. To underpin the mechanisms leading to the spontaneous Published under the PNAS license. −/− activation of TAZ10 Rag (from now on referred to as TAZ10) 1To whom correspondence may be addressed. Email: [email protected]. TPO-specific T cells, we initially characterized the phenotype of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ peripheral T cells. As expected, in the spleen we observed the doi:10.1073/pnas.1910281116/-/DCSupplemental. + presence of a discrete proportion of CD4 T cells expressing First published December 9, 2019.

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+ + 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 -/low − Treg and CD25 CD62L 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).

TAZ10 CD4+CD25+ T Cells Suppress Effector T Cell Responses In Vitro. α-CD3, except when they have been previously T -depleted (Fig. + + + reg To prove that TAZ10 CD4 CD25 Foxp3 Treg were bona fide 2D and SI Appendix,Fig.S3C) (21). All these data confirm the + + regulatory cells, we first addressed whether their depletion would hypothesis that the isolated CD4 CD25highFoxp3 TAZ10 T cells have any impact on immune responses. TAZ10 splenocytes were are indeed regulatory T cells, not only as per phenotype but also α therefore in vitro-depleted of Treg using the -CD25 mAb anti- function. body clone 7D4 (Fig. 2A). These cells showed a significantly + increased proliferation to α-CD3, thus indicating that the TAZ10 TAZ10 CD25 Treg Suppress Antigen-Specific T Cell Responses In Vitro. + Foxp3 have a suppressive function (Fig. 2B and SI Appendix, We then assessed whether TAZ10 Treg were able to impair Fig. S3A). As expected, Treg-mediated suppression was abro- TAZ10 Teff response to its peptide antigen, the human cryptic gated upon addition of an agonistic α-CD28 (17) (Fig. 2B and SI TPO536–547 peptide. Indeed, in vitro depletion of Treg (7D4) led to Appendix, Fig. S3A). The depletion of Treg also resulted in a a strong increased T cell proliferation compared to unmanipulated change in the cytokine profile in response to α-CD3, with levels splenocytes (C), in an antigen-dependant manner (Fig. 2E and SI of secreted IL-10 drastically reduced and much higher amounts Appendix,Fig.S3D). Similarly, we were able to block proliferation + + of IL-2 (Fig. 2C and SI Appendix, Fig. S3B), further demon- by the reintroduction of sorted TAZ10 CD4 CD25 Treg cells strating that Treg promoted an antiinflammatory microenviron- (Fig. 2F and SI Appendix,Fig.S3E). ment (18). Interestingly, the levels of IFNγ did not significantly change after depletion (Fig. 2C and SI Appendix, Fig. S3B) with Treg Suppress Autoimmune Thyroiditis In Vivo. The data so far −/− Treg unable to abrogate IFNγ release by Teff, possibly due to the highlighted that the transgenic TAZ10 TCR (on the Rag back- large amount of IFNγ produced by the Teff. ground) can select a single TCR on both Treg and classic Teff able to Blockade of the glucocorticoid-induced TNF (GITR), trigger autoimmunity (22). The spontaneous development of severe another functional marker of Treg (19, 20), also abolishes the autoimmune thyroiditis in TAZ10 transgenic mice suggested, + + suppressive activity of CD4 CD25 Treg leading to exacerbation however, that the TAZ10 Treg could not provide complete pro- of autoimmunity in several animal models (20, 21). The selective tection in vivo (7). To understand their function in vivo, 3-wk-old + blockade of GITR, expressed by TAZ10 Treg (SI Appendix,Fig. TAZ10miceweredepletedofCD25 Treg twiceaweekfrom3to S4), has the ability to block the proliferation of TAZ10 T cells to 20 wk of age using the α-CD25 mAb clone PC61 injected

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+ + + + 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)or4(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).

intraperitoneally (IP) (SI Appendix,Fig.S5). Weight gain was used worthy, at 12 wk of age the TAZ10 mice injected with α-CD25 mAb as readout of thyroiditis progression since it correlates with disease significantly gained more weight than the untreated TAZ10 mice severity (7). At 12 wk of age, the TAZ10 transgenic mice were al- (37.4 g ± 3.7 and 29.73 g ± 1.3, respectively) (Fig. 3A). At 20 wk of − − ready overweight compared to the WT Rag / littermates. Note- age, however, the α-CD25–treated and untreated TAZ10 littermates

26790 | www.pnas.org/cgi/doi/10.1073/pnas.1910281116 Badami et al. Downloaded by guest on September 23, 2021 **** **** 50 5 *** A ns -/- B -/- Rag * Rag *** **** -/- -/- TAZ10 Rag TAZ10 Rag ) **** -/- 4 -/- **** TAZ10 Rag TAZ10 Rag

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+ + + 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 IMMUNOLOGY AND INFLAMMATION 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 in- filtrating 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).

presented similar average weights (42.5 g ± 2.1 and 40.1 g ± 2.2, draining lymph nodes (D-LNs) presented a discrete population of respectively) (Fig. 3A). The levels of thyroid-stimulating hormone Treg (Fig. 4A). This absence of Treg in the D-LNs was confirmed at (TSH) in the sera at 20 wk of age were also in line with the the RNA level also in the thyroid and correlated with the inability to changes in weight with both groups of mice presenting a significant detect IL10 and Foxp3. This was in sharp contrast to their detection increase in thyroid stimulating hormone (TSH) compared to WT in the spleen and ND-LNs (Fig. 4B). As positive control, the human − − Rag / littermates (Fig. 3B). TCRBV1 was detected in all immune tissues tested, thus con- These observed clinical and serological signs of hypothyroid- firming the presence of infiltrating pathogenic Teff at the sites of ism were confirmed by histological analysis of thyroids from inflammation (7). untreated and α-CD25–treated TAZ10 mice at 20 wk of age. Indeed, the architecture of thyroid lobes in Treg-depleted mice WT Treg Harbor in D-LNs of TAZ10 Mice and Protect from Autoimmunity. was even more severely compromised with fibrotic follicles and The absence of Treg in D-LNs prompted us to assess whether the extensive cellular infiltrates (Fig. 3C and SI Appendix, Fig. S6). D-LNs of TAZ10 mice represented a hostile microenvironment for their homing, expansion, and survival. We adoptively trans- To demonstrate that cellular infiltrate was represented by + + pathogenic TAZ10 T cells, thyroid sections were stained with the ferred 3-wk-old TAZ10 mice with purified CD4 CD25 Treg from α-human TCRBV1 antibody (Fig. 3D). We found a significantly syngeneic WT mice. Mice were killed 17 wk later and the distri- + bution of Treg in D-LNs and ND-LNs assessed. Interestingly, while higher degree of TCRBV1 cell staining in the thyroid of PC61- + + CD4 Foxp3 WT Treg were present in both ND-LNs and D-LNs, treated mice (Fig. 3E). This in vivo data clearly demonstrated + that TPO-specific TAZ10 Treg were bona fide functional regu- the transgenic TCRBV1 TAZ10 Treg were still not detectable in latory cells capable of in vivo delaying, albeit not entirely pre- D-LNs (Fig. 4C and SI Appendix, Figs. S7 and S8). The impact of venting, the progression of the autoimmune process. the adoptive transfer of WT Treg in TAZ10 mice could be further seen as mice did not gain weight over time compared to age- −/− The Sites of Autoimmune Response Are Devoid of Treg Cells. Con- matched untreated Rag littermates both at early (12 wk of sidering the observed lack of protection from autoimmunity in vivo, age, 27.1 g ± 1.2) and late stages of disease (20 wk, 30.4 g ± 0.6) we questioned if Treg were present at the sites of inflammation (Fig. 4D). Changes in the serum TSH also confirmed the pro- where they would be most needed. Strikingly, contrary to a distinct tection conferred by WT Treg to TAZ10 mice (Fig. 4E). Histo- + + + population of CD4 CD25 Foxp3 Treg present in the spleen (Fig. logical analysis of the thyroid of Treg-treated TAZ10 mice also 1), all nondraining lymph nodes (ND-LNs) but not the thyroid revealed that the architecture was preserved, with follicles filled

Badami et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26791 Downloaded by guest on September 23, 2021 A Gated on Gated on B ND-LNs CD4+Foxp3- CD4+Foxp3+

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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-PCRforFoxp3, IL10, TCRBV1,andβ-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 dis- − + + + + – tribution 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).

26792 | www.pnas.org/cgi/doi/10.1073/pnas.1910281116 Badami et al. Downloaded by guest on September 23, 2021 A B WT TEC C 20000 30000 WT TEC WT TEC 20000

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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 IMMUNOLOGY AND INFLAMMATION T cells were used as negative control. (C) 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 concen- trations of recombinant human TPO [1 and 20 μM]. RT-PCR for the expression or Foxp3, IL10, human TCRBV1,andβ-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).

with colloid and no cellular infiltrate. On the contrary, the follic- tracts from any other lymphoid tissues such as ND-LNs, spleen, ular structure of untreated TAZ10 thyroids appeared compro- and lung or gut (Fig. 5C). mised with abundant cellular infiltrates (Fig. 4F). Altogether, These results show that the immunogenic peptide recognized these data indicated that polyclonal WT Treg could traffic to the by TAZ10 T cells was present in the thyroid and thyroid D-LNs. D-LNs and confer protection to TAZ10 mice against the auto- We assessed whether this peptide had any impact on the survival of TAZ10 Treg and Teff. mRNA expression of Foxp3, IL10, and immune process. This also importantly highlighted that TAZ10 + human TCRBV1 was determined after TAZ10 CD4 T cells Teff were sensible to immune regulation by Treg and, therefore, raised questions on the failure of TPO-specific TAZ10 T to were stimulated for 48 h with either the TPO cognate peptide or reg α naturally prevent the disease. -CD3. While these were expressed ex vivo (Fig. 5D), exposure to the cryptic TPO536–547 peptide, but not to α-CD3, Altered Expression of Foxp3 by CD4+ T Cells in the Presence of TPO prompted a simultaneous loss of Foxp3 and IL10. The continu- ous presence of TCRBV1 independently of the stimulation sug- Self-Ag. The selective absence of TAZ10 Treg in the D-LNs drove us to investigate whether the cognate antigen had any role in gested that Treg cells but not Teff were sensitive to TPO536–547 stimulation (Fig. 5E). Similarly, a strong reduction of expression driving their disappearance at the sites of inflammation (23, 24). + We first investigated if TAZ10 T cells could recognize the of Foxp3 was observed when TAZ10 CD4 T cells were stimu- immunogenic TPO antigen presented in the thyroid and LNs. lated with purified human TPO (Fig. 5F). We could exclude that Interestingly, follicular thyroid epithelial cells (TEC) of TAZ10 any impurity generated during the purification of the TPO pro- mice constitutively express high levels of MHC class II (Fig. 5A) tein from human thyroid was responsible for immunologic re- sponse as the use of recombinant TPO protein (Rec TPO) led to and can act as antigen-presenting cells (APC) inducing the a similar reduction of Foxp3 expression. This observation cor- proliferation of TAZ10 T cells (Fig. 5B). By contrast, WT TEC related to the concentration of Rec TPO (Fig. 5G). needs the addition of IFNγ to up-regulate MHC class II mole- cules and stimulate T (Fig. 5 A and B) (25). These TAZ10 Treg Are Prone to Antigen-Induced Cell Death When Exposed to results confirmed that the TAZ10 T cells can recognize au- eff the Cognate Antigen. The data shown so far suggested that Treg tologous thyroid epithelial cells, as shown in human autoimmune are highly sensitive to the cognate antigen. This was a chal- thyroiditis (26). lenging hypothesis as TAZ10 Teff and Treg express the same It remained to be assessed whether the TPO antigen was pre- TCR, thus leading us to investigate their ultimate fate following sent in the thyroid D-LNs as its expression would not naturally TPO antigen stimulation. After in vitro exposure to TPO536–547 occur in any other LNs. We observed that TAZ10 T lymphocytes or Rec TPO, up to 80% of TAZ10 Treg up-regulated phospha- proliferated when challenged with autologous DC loaded with tidylserine and FAS, initiating a process of activation-induced peptide extracts derived from thyroid and thyroid D-LNs. How- cell death (AICD) (Fig. 6A and SI Appendix, Fig. S9). This was ever, proliferation was not observed in response to peptide ex- in stark contrast to TAZ10 Teff, with only a minority (less than

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m m iu CD3 TPO iu CD3 TPO m ed med eric LN s Spleen A xillary LNInguinal s LNs Mesent C

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+ + + − 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).

15%) of these cells expressing these apoptotic markers (Fig. 6A Discussion and SI Appendix, Fig. S10). Polyclonal Treg from WT mice were Mouse models have been used extensively to provide insight into used as control and showed no major fluctuations in expression the mechanisms underlying many diseases, to explore the efficacy of early death markers when stimulated with TPO antigen of candidate drugs, and to predict patient responses. The (Fig. 6A). TAZ10 transgenic mice, with a human TCR isolated from a thy- To confirm that the Treg are sensitive to AICD, we immunized roiditis patient and specific for the self-antigen TPO, is a mouse the mice at the base of the tail with TPO purified from human model that spontaneously develops autoimmunity (6, 7). Due to thyroid. Five days after immunization with TPO, a significant re- the high degree of homology between the human DQB1*0602 and duction of TAZ10 Treg was observed in all LNs and, in particular, mouse A2-K, the human TAZ10 TCR can recognize the human the mesenteric LNs and spleen of TPO-immunized mice compared TPO peptide and the highly homologous mouse TPO in the to PBS-treated controls (Fig. 6B). To have an insight of the im- context of mouse MHC (7). munological microenvironment at the sites of chronic inflammation, Here we show that the TAZ10 TCR can be naturally selected in the thymus on both T and T subsets. It has been previously we compared the cytokine milieu of D-LNs and ND-LNs from reg eff shown that thymocytes could be directed to differentiate into T TAZ10 mice, looking for common proinflammatory and antiin- reg in response to thymic self-antigen expression (27). flammatory markers. Interestingly, we observed that IFNγ was Studies on Treg with known antigen specificity have however produced in both D-LNs and ND-LNs, but fundamental been hampered by their low frequencies. Furthermore, the de- for the survival, function, recruitment, and expansion of T such as reg velopment of Treg in TCR transgenic mice on a RAG-deficient IL-2, IL-4, IL-5, and IL-10 were not detected ex vivo in the D-LNs, background does not usually occur as endogenous α-chain re- although present in ND-LNs (Fig. 6C). Altogether, these data combination is required to shape the Treg repertoire (11). With the suggest that the selective reduction in TAZ10 Treg observed in the TAZ10 model, we had the unique opportunity to unravel the D-LNs, and their absence in the thyroid of TAZ10 mice, was driven initiation of autoimmunity, in a model in which Teff coexist with by the presence/recognition of the cognate TPO antigen. Treg. These are also naturally occurring Treg (nTreg) as we detected

26794 | www.pnas.org/cgi/doi/10.1073/pnas.1910281116 Badami et al. Downloaded by guest on September 23, 2021 −/− Foxp3 in the thymus of TAZ10 Rag (28). We therefore spec- generation of Treg with high TCR diversity (38), but the use of ulated that the shaping of this Treg repertoire in the thymus is antigen-specific Treg as therapy might raise some issues for the likely orchestrated by the homologous murine TPO, which it is treatment of autoimmune diseases. Current therapies do not known to be also expressed in the thymus (29). Whether TAZ10 only try to dampen proinflammatory cytokines but also restore T cells sharing the same transgenic human TCR are selected in the homeostasis by promoting Treg stability and function (39). In the thymus into Treg and Teff on the same or different peptides re- era of personalised medicine, some strategies are also under mains still unknown, but this is likely to ultimately influence their investigations, such as adoptive transfer of ex vivo expanded self- respective activation status and function in the periphery (22, 30). reactive Treg or in vivo expansion of self-reactive Treg, specific – Self-antigen specific regulatory T cells are difficult to study antigen-driven conversion of Teff into Treg in vivo or ex vivo (40) since suitable specific tools to isolate and characterize these cells or chimeric antigen receptors (CAR)-engineered Treg (41). The are lacking; they comprise only a small fraction of the total T cell identification of the self-antigen landscape in each patient af- population and are difficult to culture and maintain in vitro (31, + fected by autoimmunity could help to select those Treg expanded 32). The compelling demonstration that the CD25highFoxp3 Tcell in vitro, which undergo depletion by AICD or conversion to detected in TAZ10 mice were in fact Treg was that they elicited Th17. This could prove difficult as this landscape is complex, and robust suppression in standard coculture assays. the nature of the antigens generated by epitope spreading is The in vivo disappearance of the Treg only from the thyroid difficult to predict. The strong beneficial effect of the adoptive draining lymph nodes begs the question if the cognate antigen transfer of Treg from WT mice into TAZ10 mice shows that has any role in this process and what are the cells that present the choosing a polyclonal TCR population of Treg from healthy in- antigen to T cells. In this regard, most attention to date has fo- dividual with the same haplotype could be an ideal strategy, cused on DCs as main APCs in the LNs. We have shown, however, especially in conjunction with current immunotherapeutic that in TAZ10 mice, the thyroid epithelial cells aberrantly up- approaches. regulate MHC class II molecules, as also previously demon- strated in patients with autoimmune thyroiditis (33). The aberrant Materials and Methods − − + − − expression of MHC class II molecules makes TEC able to present Mice. WT (CBA), Rag / , and TAZ10 TCR Rag / mice were housed at the the endogenously processed TPO to T cells (6). It is possible that Tenovus animal facility at the University of Southampton (United Kingdom). cells with scavenger function may engulf dead thyrocytes and All mice were used between 3 and 20 wk of age and immunocompromised present TEC-TPO in the draining LNs (34). Upon recognition of transgenic mice maintained under specific pathogen-free conditions. All experiments were carried out according to local ethical committee guide- the cognate TPO peptide in the thyroid-draining LNs, Treg acti- vate a process of AICD. That recognition of the cognate peptide is lines under United Kingdom Home Office license number PIL70/13192. Ad- IMMUNOLOGY AND INFLAMMATION the triggering factor for AICD of T is also confirmed by the fact ditional details are described in SI Appendix, Supplemental Materials and reg Methods. Additional data, associated protocols, code, and materials in the that immunization with whole TPO leads to Treg cell death in paper can be available upon request through the corresponding author or peripheral lymphoid tissues. Immunization with whole TPO has following the link DOI: https://doi.org/10.5281/zenodo.3552497. no effect, however, on the Teff cells. These data indicate that Treg homeostasis is positively regu- Flow Cytometry. Cell suspensions were obtained as previously described (7). lated by encounter with self-antigen expressed on peripheral Briefly, cells obtained from thymuses, spleens, lymph nodes, and bone- μ tissues, a process likely to be relevant for reshaping the Treg marrows of 3- to 20-wk-old mice were passed through a 70- m cell-filter repertoire across the different anatomical locations. This is before red blood cells were lysed in ammonium–chloride–potassium (ACK) buffer. Cells were collected in ice-cold 1% fetal calf serum, 0.05% sodium confirmed by the fact that while TAZ10 Treg have little effect on azide in phosphate-buffered saline (PBS) and stained at 4 °C. Flow cytometry the progression of autoimmunity, adoptive transfer of WT Treg strongly inhibits the onset and progression of disease, as shown analysis was performed using a FACSCalibur flow cytometer, and data were analyzed using Cell Quest or fluorescence-activated cell sorting (FACS) Diva by the lack of weight gain and only weak increase in serum TSH. software (Becton Dickinson). Cells were stained using antibodies to CD4, Furthermore, these data demonstrate that TAZ10 Teff are sensi- CD8α, CD25 (clone 7D4), CD62L, TCRβ, H-2k, CD11c, Annexin V (BD Phar- tive to suppression by WT Treg, strongly suggesting that breach of mingen). Mouse CD25 (clone PC61) and GITR (clone DTA-1) were made in-house. tolerance is more likely due to a dysfunction of the endogenous Anti-human TCRBV1 was obtained from lmmunotech. CD95/Fas, CD86, and Treg pool rather than to a refractoriness of Teff to suppression (35). Foxp3werefromeBioscience. These data confirm but also add complexity to the “buddy hypothesis” that postulates that autoreactive conventional T cells Cell Isolations and Purifications. Thymus, spleen, and lymph nodes of TAZ10 or would have a T cell “buddy,” with the same antigen specificity, WT mice were passed through a 70-μm cell strainer and red blood cells lysed reg + + + + to prevent unwanted T cell activation and autoimmunity (27, 36). in ACK buffer. CD4 , CD8 , and CD4 CD25 T cell populations were purified with by magnetic cell separation (Miltenyi Biotec). In vitro depletion of In this model, we demonstrated that the in vivo depletion of Treg + in very young TAZ10 mice led to a faster onset of autoimmune CD25 T cells was achieved with anti-mouse CD25 clone 7D4 and rabbit complement (Harlan Seralab). Efficiency of depletion was assessed by flow thyroiditis, confirming the active role of Treg in preventing au- cytometry using the anti-mouse CD25 clone PC61. toimmunity. In this transgenic model, and possibly in bona fide autoimmunity, however, the massive imbalance between self- Generation of Bone Marrow-Derived Dendritic Cells. Bone marrow-derived reactive T cell and Treg lead to disease. When this occurs, the dendritic cells (DC) from 4- to 12-wk-old CBA mice were generated as pre- tissue destruction produces even more debris that are engulfed viously described (42). Briefly, bone marrow cells depleted of red blood cells by scavenger cells, and the self-antigen(s) are going to be pro- were cultured with 200–400 ng/mL GM-CSF for 10 d. Then immature DC cessed and presented in the regional/draining LNs. This triggers were harvested and seeded at 0.5 × 106 cells per well and pulsed with 100 ng/mL (0.1 μM) of purified human TPO (kind gift from B. Rapoport, a program of cell death in the Treg, until they finally disappear from these draining LNs. It has been reported that, compared to Department of Medicine, University of California, Los Angeles [UCLA], CA) and then matured overnight with 10 ng/mL LPS (Sigma) before being cocultured conventional T cells, Treg cells are also highly sensitive to apo- ptosis, correlating with lower expression levels of c-FLIP (37). with TAZ10 T cells. The phenotype of the DC generated was assessed by FACS before and after LPS maturation (SI Appendix,Fig.S11). Unleashed by the controls mediated by TAZ10 Treg, TAZ10 Teff would then overcome regulatory and protective mechanisms in T Cell Activation and Proliferation Assays. Proliferation assays were done as the thyroid and leading to an amplification of the autoimmune previously described (9). Untreated or CD25-depleted TAZ10 lymphocytes were

process. pulsed with increasing doses of the human TPO536–547 N-DPLIRGLLARPA-C Our results support the notion that the establishment and peptide (0.1–10 μM), anti-mouse CD3 alone (10 μg/mL) or in with anti-mouse maintenance of tolerance to self-antigen would rely on the CD28 (2 μg/mL). Where specified, titrating amounts of the blocking anti-mouse

Badami et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26795 Downloaded by guest on September 23, 2021 5 GITR antibody (0.1–100 mg/mL) were used. Cells seeded at 10 cells per well in with 10 ng/mL somatostatin (Sigma) and 2 ng/mL glycyl-L-histidyl-L-lysine ace- triplicate were pulsed with [3H] thymidine for the last 16 h of culture. [3H] tate (Sigma). Medium was changed 1 d after the start of the culture and thymidine incorporation was measured with a microplate scintillation replaced after 2 d. IFNγ (Peprotech) was added at 200 U/mL. counter (Packard). Peptide Extraction. Peptides were isolated from thyroids, LNs draining and not ELISA. Primary coating antibodies used were purified anti-mouse IFNγ (rat draining the thyroid, spleens, and other tissues such as the trachea or the gut lgG1, clone R4-6A2); IL-2 (rat lgG2a, clone JES6-1A12); and IL-10 (rat lgG2b, as previously described (45, 46). Briefly, tissues from 80 WT (CBA) mice were clone JES5-16E3). Secondary detecting antibodies used were biotin anti- passed through a cell strainer, cells were lysed by 3 cycles of gentle freeze- mouse IFNγ (rat lgG1, clone XMG1.2); IL-2 (rat lgG2b, clone JES6-5H4), or thaw cycle. Lysed mixes were centrifuged to pellet the membranes, and IL-10 (rat lgG1, clone JES5-2A5) (eBioscience). Each well was incubated with supernatant was passed through a 30-kDa cutoff filter by centrifugation – streptavidin horseradish peroxidase (HRP) and then with 30% H202/TMB (Millipore). peroxidase substrate. The reaction was stopped with 2 M sulfuric acid. Reading at 450 nm was then measured. RNA Extraction, cDNA Synthesis, RT-PCR, and Real-Time PCR. Total RNA isolation, cDNA synthesis, and PCR were performed from up to 5 × 106 total lymphocytes Mouse Serum TSH Assay. Serum TSH was quantified as previously described from cervical, axillary, inguinal, mesenteric lymph nodes, splenocytes, thymus, (7). Briefly, TAZ10 serum TSH was quantified using a mouse TSH kit (Meso- or thyroid from either WT or TAZ10 transgenic mice. scale Discovery-MSD) on 96-well plates coated with anti-mouse TSH Ab. Ten Real-time PCR was done as previously described (12). All reported mRNA microliters of MSD Sulfo-tag Mouse TSH Ab were plated into each well, and levels are normalized to the GAPDH mRNA level (GAPDH = 1). Primer pairs 25 μL of either TSH standard or mouse serum were plated in duplicate. Signal are described in SI Appendix, Table S1. In other experiments, the relative was revealed with high sensitivity Reading Buffer T, and plate was read at expression of Foxp3 was normalized by semiquantitative RT-PCR for mouse the MSD reading machine MS 6000. β-actin. PCRs were performed on a PTC-200TM cycler (MJ Research Inc.). The + primers used are reported in SI Appendix, Table S2. All primers were In Vivo Depletion and Adoptive Transfer of CD25 Treg. Three-week-old designed to span an . PCR products were visualized by ethidium bro- TAZ10 mice (n = 6 per group) were injected twice per week IP with 400 μg of the anti-mouse mAb PC61 (43) or PBS. Readouts of weight was taken at mide 2% agarose gel. 12 and 20 wk of age. Serum TSH levels were determined at 20 wk of age. The efficiency of depletion was assessed by flow cytometry and RT-PCR. Hystological Analysis. on mouse thyroids was done as previously + + Macs purified CD4 CD25 cells (5 × 106 cells, Miltenyi Biotec) from WT described (7, 9). Briefly, thyroids were embedded in OCT. Four- to five- CBA mice were injected i.v. into the lateral tail vein of 3-wk-old TAZ10 mice, micrometer frozen sections were adhered to glass slides. For hematoxylin/ followed a month later by a second injection of 5 × 106 CD4+CD25+ T cells. eosin (H/E) staining, sections were fixed for 10 min 4% PFA, stained in Harris’ Organs and blood were collected 17 wk later for further FACS and RT-PCR Haematoxylin for 10 min and in eosin (BDH) for 10 min. For immunofluo- analysis. The thyroids of treated mice were cryopreserved in OCT for further rescence, air-dry frozen sections were fixed in acetone (BDH) for 10 min, histological or RT-PCR analysis. Readouts of weight was taken at 12 and blocked for Streptavidin/Biotin and for another 30 min with Normal Goat 20 wk of age. Serum TSH levels were determined at 20 wk of age. Serum (Vector Lab). Sections were incubated O/N at 4C with anti-human TCRBV1-FITC mAb (rat IgG1, BL37.2) Pictures of sections were taken with Immunization of TAZ10 Mice with TPO. Three-week-old TAZ10 mice were an Axioskop 2 Plus microscope (Zeiss). The mean infiltration score was cal- immunized at the base of the tail with thyroid-purified TPO protein or PBS in culated using LCmicro Imaging software (Olympus) and expressed as number CFA (n = 3 per group). Mice were culled a week later, and their spleen, of TCRBV1+ cells/300 μm2 area. axillary, inguinal, and mesenteric lymph nodes isolated separately for fur- ther flow cytometry and RT-PCR analysis. Statistical Analysis. Statistical analysis considered the sample size for each data point as well as technical and experimental replicates, if any. Specific Thyroid Digestion and Culture. Thyroid primary cultures were obtained as tests are described in this section as well as in the legends and were per- previously described (44, 45). Briefly, thyroid lobes separated with a scalpel formed using the GraphPad Prism (GraphPad v6 and v8 software). Statistical from the trachea were digested in 112 U/mL of collagenase-A and 1.2 U/mL analysis was performed using 2-tailed unpaired Student’s t test. The in vivo dispase-I (Roche). Digestion was carried out at 37 °C, the single-cell suspension data were analyzed using ordinary 1-way ANOVA with Tukey’s multiple μ was gently passed through a 70- m cell strainer. Thyroid lobes were aseptically comparisons test. Not significant (n.s.), P > 0.05; *P < 0.05; **P < 0.01; ***P < −/− removed from the trachea of WT (CBA), Rag , or TAZ-10 transgenic mice, 0.001; ****P < 0.0001. placed in Eagle’s Minimum Essential Medium (EMEM) (AutogenBioclea) and cut into pieces. Disrupted thyroid lobes were digested in 1 mL of EMEM ACKNOWLEDGMENTS. We thank Dr. Claudia Coronnello (Fondazione containing type I collagenase (5 mg/mL; Sigma) and Dispase I (2.5 mg/mL; RiMED, Palermo, Italy) and Dr. Paolo Vicini (Kymab, Cambridge, UK) for Roche) for 45 min at 37 °C. The digested mix was spun down before the pellet the statistical analysis of the data, Prof. Basil Rapoport (Cedars-Sinai Medical containing partially digested single thyroid follicles was resuspended in com- Center, UCLA) for providing the human TPO and Richard Reid, and staff at plete thyroid medium. The culture medium was Nu-Serum IV (BD Biosciences) the Tenovus Animal Research Facility for technical support. This work was diluted 2.5 times in Hams F-12 medium (AutogenBioclear) and supplemented supported by Cancer Research UK Grants C7056/A3110 and C8624/A4912.

1. J. M. Kim, J. P. Rasmussen, A. Y. Rudensky, Regulatory T cells prevent catastrophic 10. S. Sakaguchi, N. Sakaguchi, M. Asano, M. Itoh, M. Toda, Immunologic self-tolerance autoimmunity throughout the lifespan of mice. Nat. Immunol. 8, 191–197 (2007). maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Break- + + 2. S. Sakaguchi, Naturally arising Foxp3-expressing CD25 CD4 regulatory T cells in down of a single mechanism of self-tolerance causes various autoimmune diseases. – immunological tolerance to self and non-self. Nat. Immunol. 6, 345 352 (2005). J. Immunol. 155, 1151–1164 (1995). 3. R. Khattri, T. Cox, S. A. Yasayko, F. Ramsdell, An essential role for Scurfin in 11. M. Itoh et al., Thymus and autoimmunity: Production of CD25+CD4+ naturally CD4+CD25+ T regulatory cells. Nat. Immunol. 4, 337–342 (2003). anergic and suppressive T cells as a key function of the thymus in maintaining 4. M. Dominguez-Villar, D. A. Hafler, Regulatory T cells in . Nat. immunologic self-tolerance. J. Immunol. 162, 5317–5326 (1999). Immunol. 19, 665–673 (2018). 12. S. Hori, T. Nomura, S. Sakaguchi, Control of development by the 5. J. H. Buckner, Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+)regula- factor Foxp3. Science 299 , 1057–1061 (2003). tory T cells in human autoimmune diseases. Nat. Rev. Immunol. 10,849–859 (2010). 13. Y. Peng, Y. Laouar, M. O. Li, E. A. Green, R. A. Flavell, TGF-beta regulates in vivo 6. S. Quaratino, M. Feldmann, C. M. Dayan, O. Acuto, M. Londei, Human self-reactive + + T cell clones expressing identical T cell receptor beta chains differ in their ability to expansion of Foxp3-expressing CD4 CD25 regulatory T cells responsible for protection – recognize a cryptic self-epitope. J. Exp. Med. 183, 349–358 (1996). against . Proc. Natl. Acad. Sci. U.S.A. 101,4572 4577 (2004). 7. S. Quaratino et al., Degenerate self-reactive human T-cell receptor causes spontaneous 14. J. D. Fontenot, M. A. Gavin, A. Y. Rudensky, Foxp3 programs the development and autoimmune disease in mice. Nat. Med. 10,920–926 (2004). function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003). 8. J. A. Collado, C. Guitart, M. T. Ciudad, I. Alvarez, D. Jaraquemada, The repertoires of 15. M. A. Curotto de Lafaille, J. J. Lafaille, Natural and adaptive + regulatory T cells: peptides presented by MHC-II in the thymus and in peripheral tissue: A clue for More of the same or a division of labor? 30, 626–635 (2009). autoimmunity? Front. Immunol. 4, 442 (2013). 16. E. Badami et al., Defective differentiation of regulatory FoxP3+ T cells by small-intestinal 9. E. Badami, L. Maiuri, S. Quaratino, High incidence of spontaneous autoimmune dendritic cells in patients with . Diabetes 60, 2120–2124 (2011). thyroiditis in immunocompetent self-reactive human T cell receptor transgenic mice. 17. A. Schmidt, N. Oberle, P. H. Krammer, Molecular mechanisms of treg-mediated T cell J. Autoimmun. 24,85–91 (2005). suppression. Front. Immunol. 3, 51 (2012).

26796 | www.pnas.org/cgi/doi/10.1073/pnas.1910281116 Badami et al. Downloaded by guest on September 23, 2021 18. A. Chaudhry et al., Interleukin-10 signaling in regulatory T cells is required for suppression 32. K. S. Smigiel, S. Srivastava, J. M. Stolley, D. J. Campbell, Regulatory T-cell homeostasis: of Th17 cell-mediated inflammation. Immunity 34,566–578 (2011). Steady-state maintenance and modulation during inflammation. Immunol. Rev. 259, 19. R. S. McHugh et al., CD4(+)CD25(+) immunoregulatory T cells: expression 40–59 (2014). analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Im- 33. M. Londei, J. R. Lamb, G. F. Bottazzo, M. Feldmann, Epithelial cells expressing aber- munity 16, 311–323 (2002). rant MHC class II determinants can present antigen to cloned human T cells. Nature – 20. J. Shimizu, S. Yamazaki, T. Takahashi, Y. Ishida, S. Sakaguchi, Stimulation of CD25(+)CD4(+) 312, 639 641 (1984). 34. M. R. Elliott, K. S. Ravichandran, The dynamics of apoptotic cell clearance. Dev. Cell regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3,135– 38, 147–160 (2016). 142 (2002). 35. S. A. Basdeo et al., Polyfunctional, pathogenic CD161+ Th17 lineage cells are resistant 21. M. Gavin, A. Rudensky, Control of immune homeostasis by naturally arising regula- to regulatory T cell-mediated suppression in the context of autoimmunity. J. Immunol. tory CD4+ T cells. Curr. Opin. Immunol. 15, 690–696 (2003). + 195,528–540 (2015). 22. L. Wojciech et al., The same self-peptide selects conventional and regulatory CD4 Tcells 36. N. Kurd, E. A. Robey, T-cell selection in the thymus: A spatial and temporal per- with identical antigen receptors. Nat. Commun. 5, 5061 (2014). spective. Immunol. Rev. 271, 114–126 (2016). 23. S. L. Bailey-Bucktrout et al., Self-antigen-driven activation induces instability of reg- 37. C. Plaza-Sirvent et al., c-FLIP expression in Foxp3-expressing cells is essential for sur- – ulatory T cells during an inflammatory autoimmune response. Immunity 39, 949 962 vival of regulatory T cells and prevention of autoimmunity. Cell Rep. 18,12–22 (2017). (2013). 38. D. Adeegbe, T. Matsutani, J. Yang, N. H. Altman, T. R. Malek, CD4(+) CD25(+) 24. L. F. Su, D. Del Alcazar, E. Stelekati, E. J. Wherry, M. M. Davis, Antigen exposure Foxp3(+) T regulatory cells with limited TCR diversity in control of autoimmunity. shapes the ratio between antigen-specific Tregs and conventional T cells in human J. Immunol. 184,56–66 (2010). peripheral blood. Proc. Natl. Acad. Sci. U.S.A. 113, E6192–E6198 (2016). 39. P. Fasching, M. Stradner, W. Graninger, C. Dejaco, J. Fessler, Therapeutic potential of 25. M. Londei, G. F. Bottazzo, M. Feldmann, Human T-cell clones from autoimmune targeting the Th17/Treg Axis in autoimmune disorders. Molecules 22, E134 (2017). thyroid glands: Specific recognition of autologous thyroid cells. Science 228,85–89 40. M. Miyara, Y. Ito, S. Sakaguchi, TREG-cell therapies for autoimmune rheumatic dis- – (1985). eases. Nat. Rev. Rheumatol. 10, 543 551 (2014). 26. C. M. Dayan et al., Autoantigen recognition by thyroid-infiltrating T cells in Graves 41. D. A. Boardman et al., Expression of a chimeric antigen receptor specific for donor disease. Proc. Natl. Acad. Sci. U.S.A. 88, 7415–7419 (1991). HLA class I enhances the potency of human regulatory T cells in preventing human – 27. C. S. Hsieh, H. M. Lee, C. W. Lio, Selection of regulatory T cells in the thymus. Nat. Rev. skin transplant rejection. Am. J. Transplant. 17, 931 943 (2017). 42. M. B. Lutz et al., An advanced culture method for generating large quantities of Immunol. 12, 157–167 (2012). + + highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 7223,7 –92 28. P. Kraj, L. Ignatowicz, The mechanisms shaping the repertoire of CD4 Foxp3 reg- (1999). ulatory T cells. Immunology 153, 290–296 (2018). 43. M. J. McGeachy, L. A. Stephens, S. M. Anderton, Natural recovery and protection from 29. A. V. Misharin, Y. Nagayama, H. A. Aliesky, B. Rapoport, S. M. McLachlan, Studies in autoimmune encephalomyelitis: Contribution of CD4+CD25+ regulatory cells within mice deficient for the (Aire) and transgenic for the thyro- the central nervous system. J. Immunol. 175, 3025–3032 (2005). tropin receptor reveal a role for Aire in tolerance for thyroid autoantigens. Endo- 44. L. T. Jeker, M. Hejazi, C. L. Burek, N. R. Rose, P. Caturegli, Mouse thyroid primary crinology 150, 2948–2956 (2009). culture. Biochem. Biophys. Res. Commun. 257, 511–515 (1999). 30. J. Pohar, Q. Simon, S. Fillatreau, Antigen-specificity in the thymic development and 45. O. Cexus, “Immunological mechanisms controlling chronic inflammatory diseases,” + + peripheral activity of CD4 FOXP3 T regulatory cells. Front. Immunol. 9, 1701 (2018). PhD thesis, University of Southampton, Southampton, UK (2009).

31. E. M. Shevach, Mechanisms of foxp3+ T regulatory cell-mediated suppression. Im- 46. S. R. Schwab, K. C. Li, C. Kang, N. Shastri, Constitutive display of cryptic translation IMMUNOLOGY AND INFLAMMATION munity 30, 636–645 (2009). products by MHC class I molecules. Science 301, 1367–1371 (2003).

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