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. 2017 Aug 15;199(4):1250-1260.
doi: 10.4049/jimmunol.1600941. Epub 2017 Jul 12.

Prdm1 Regulates Thymic Epithelial Function To Prevent Autoimmunity

Natalie A Roberts  1   2 , Brian D Adams  3   4 , Nicholas I McCarthy  5 , Reuben M Tooze  6 , Sonia M Parnell  5 , Graham Anderson  5 , Susan M Kaech  7 , Valerie Horsley  8   9
Affiliations

Prdm1 Regulates Thymic Epithelial Function To Prevent Autoimmunity

Natalie A Roberts et al. J Immunol. .

Abstract

Autoimmunity is largely prevented by medullary thymic epithelial cells (TECs) through their expression and presentation of tissue-specific Ags to developing thymocytes, resulting in deletion of self-reactive T cells and supporting regulatory T cell development. The transcription factor Prdm1 has been implicated in autoimmune diseases in humans through genome-wide association studies and in mice using cell type-specific deletion of Prdm1 in T and dendritic cells. In this article, we demonstrate that Prdm1 functions in TECs to prevent autoimmunity in mice. Prdm1 is expressed by a subset of mouse TECs, and conditional deletion of Prdm1 in either Keratin 14- or Foxn1-expressing cells in mice resulted in multisymptom autoimmune pathology. Notably, the development of Foxp3+ regulatory T cells occurs normally in the absence of Blimp1. Importantly, nude mice developed anti-nuclear Abs when transplanted with Prdm1 null TECs, but not wild-type TECs, indicating that Prdm1 functions in TECs to regulate autoantibody production. We show that Prdm1 acts independently of Aire, a crucial transcription factor implicated in medullary TEC function. Collectively, our data highlight a previously unrecognized role for Prdm1 in regulating thymic epithelial function.

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Figures

FIGURE 1.
FIGURE 1.
Conditional deletion of Prdm1 in K14+ epithelium results in spontaneous development of autoimmunity. (A) Quantification of cervical lymphadenopathy presentation in controlK14 (n = 35) and Prdm1 cKOK14 (n = 51). (B) Images of mandibular cervical lymphadenopathy in Prdm1 cKOK14 mice compared with the normal nodes of control mice. Scale bar, 10 mm. (C) Total lymphocyte numbers are elevated in Prdm1 cKOK14 and Prdm1 cKOFoxN1 mandibular and accessory mandibular cervical lymph nodes. (D) Flow cytometric analysis of CD4 T cells isolated from cervical (mandibular and accessory mandibular) lymph nodes shows an increase in proportion of CD44+CD62L− activated CD4 T cells. (E) An increase in CD44+CD62L− activated CD4 and CD8 T cells from Prdm1 cKOK14 cervical (mandibular and accessory mandibular) lymph nodes compared with control. (F) Serum isolated from Prdm1 cKOK14 mice contains ANAs indicated by positive staining of HEp2 cells. Positive control is MRL/Faslpr mouse serum; negative control is secondary Ab alone. Scale bar, 50 μm. (G) The presence of ANAs is significantly more frequent in Prdm1 cKOK14 than in control. (H) Quantification of nuclear patterns identified in sera derived from Prdm1 cKOK14 mice. (I) ELISA of serum Ab isotypes. Note IgG2b and IgG1 are significantly increased in Prdm1 cKOK14 serum. (J) Representative images of Prdm1 cKOK14 kidney sections exhibit more intense IgG staining than those of control mice. Scale bar, 25 μm. (K) The presence of IgG is significantly more frequent in Prdm1 cKOK14 mice than in control. Data are mean ± SEM. n ≥ 8 (C), n ≥ 5 (D and E), n ≥ 13 (F–H), n ≥ 15 (I), and n ≥ 3 (J) for each genotype. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 2.
FIGURE 2.
Expression of Prdm1 in the mouse thymus. (A) Representative flow cytometry plots indicating the gating strategy for the CD45− EPCAM1+ epithelium, (B) cTEC (Ly51+EPCAM1+), mTEC (Ly51−EPCAM1+), (C) mTEClo (Ly51−EPCAM1+CD80−MHCIIlo), and mTEChi (Ly51−EPCAM1+CD80+MHCIIhi) for Prdm1-YFP mice. (D) Representative flow cytometry plots indicating the YFP expression within the populations shown in (A). (E) Quantification of the percentage of YFP+ cells for each of the indicated epithelial cell populations. Data are mean ± SEM. n = 5 mice. (F and G) Sections of the thymus from Prdm1-mGFP mice reveal expression of mGFP in K5+ and involucrin+ cells in the medulla. (H) Human thymus sections illustrating Prdm1 expression within mTECs and TECs within Hassles corpuscles. (I) Sections from human skin epithelium illustrating positive stain for Prdm1 in the granular layer of the epidermis and negative staining below. Scale bars, 5 μm (G); 10 μm (F); 20 μm (H and I). ****p < 0.0001.
FIGURE 3.
FIGURE 3.
Normal TEC and T cell development in the absence of Prdm1. (A) Representative flow cytometry plots indicating the gating strategy for CD45− cTECs (Ly51+EPCAM1+), immature mTECs (Ly51−EPCAM1+CD80−), and mature mTECs (Ly51−EPCAM1+CD80+) for both control and Prdm1 cKOK14 thymus. (B–D) Summary of the absolute cell numbers for each of the indicated epithelial cell populations. n = 3 mice for each genotype. (B) p > 0.9999, (C) p = 0.7, (D) p = 0.7. (E) Representative fluorescence images of control and Prdm1 cKOK14 thymus sections stained for (left) K5 (green), K8 (red), and (right) MHCII (gray). Scale bars, 100 μm. (F) mRNA expression of MHCII in FACS-isolated control and Prdm1 cKOK14 mTECs. Data are mean (± SD) of at least three independent experiments. (G) Representative fluorescence images of control and Prdm1 cKOK14 thymus sections stained for K5 (red) and Aire (green). Scale bars, 25 μm. (H) Quantification of the number of Aire-positive mTECs (K5+). n = 3 mice for each genotype. (I) Quantitative real-time PCR analysis of mRNA transcript levels of Aire in control and Prdm1 cKOK14 mTECs. (J) Representative CD4 and CD8 profiles from control and Prdm1 cKOK14 thymocytes. (K) Quantification of the total cell numbers for each thymocyte population of CD4+ and CD8+ single-positive cells and CD4+CD8+ double-positive cells. (L) Histograms illustrating the expected upregulation of the TCRβ with thymocyte maturation. Data are mean (B–D, H) or mean ± SD (F, I–K). n = 3 mice for each genotype. ns, not significant.
FIGURE 4.
FIGURE 4.
Spontaneous development of autoimmunity is not the result of an absence of Tregs. (A–C) Representative flow cytometric plots showing CD44+CD4+CD25+Foxp3+ Tregs in thymus and cervical (mandibular and accessory mandibular) lymph nodes. Note the levels of Foxp3 and CD25 are similar in control, Prdm1 cKOK14, and Prdm1 cKOFoxN1. (B) Absolute cell numbers of Tregs found within the thymus are not significantly different between control and cKO mice. (D) Absolute cell numbers of Tregs found within the cervical lymph node suggest an increase in cKO mice compared with control. Data are mean. n ≥ 3 mice for each genotype; each point represents an individual mouse. *p = 0.0159. ns, not significant.
FIGURE 5.
FIGURE 5.
Transplantation of Prdm1 cKOK14 thymic stroma leads to autoimmunity. Representative flow cytometry analysis of blood CD4 and CD8 T cells in WT and Nude mice prior to transplantation (A) or 6 wk after grafting of control or Prdm1 cKOK14 embryonic (E15) thymus lobes under the kidney capsule (B). (C) Analysis of CD4 and CD8 T cell development in the grafted thymic epithelial tissue after 5 mo revealed T cell profiles similar to those in WT thymus. (D) Quantification of percentage of T cell subsets between control and Prdm1 cKOK14 grafts. Each bar represents three or more independently analyzed thymic grafts harvested from a single host. (E) Compared with serum isolated from control host mice, Prdm1 cKOK14 host serum contains ANAs as indicated by positive staining of HEp2 cells. Representative images from sera derived from one host, engrafted with thymi from control, or two hosts engrafted with thymi from Prdm1 cKOK14 mice. Scale bars, 50 μm. (F) The presence of ANAs is more frequent in Prdm1 cKOK14 host mice compared with control. (G) Homogenous and speckled nuclear staining patterns were observed from sera isolated from mice engrafted with thymi from Prdm1 cKOK14 mice. (H) ELISA of serum Ab isotypes after 5 wk of engraftment. Note the increase in IgG2b and IgG1 in Prdm1 cKOK14 serum compared with control serum. Data are mean. n = 4 grafted mice for each genotype of transplanted thymi. *p < 0.05.
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