Epithelial Cells Differentiation of Medullary Thymic Thymocytes Regulate the Terminal Lymphotoxin Signals from Positively Select

Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells This information is current as of September 29, 2021. Andrea J. White, Kyoko Nakamura, William E. Jenkinson, Manoj Saini, Charles Sinclair, Benedict Seddon, Parth Narendran, Klaus Pfeffer, Takeshi Nitta, Yousuke Takahama, Jorge H. Caamano, Peter J. L. Lane, Eric J. Jenkinson and Graham Anderson Downloaded from J Immunol 2010; 185:4769-4776; Prepublished online 22 September 2010; doi: 10.4049/jimmunol.1002151 http://www.jimmunol.org/content/185/8/4769 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells

Andrea J. White,* Kyoko Nakamura,* William E. Jenkinson,* Manoj Saini,* Charles Sinclair,† Benedict Seddon,† Parth Narendran,‡ Klaus Pfeffer,x Takeshi Nitta,{ Yousuke Takahama,{ Jorge H. Caamano,* Peter J. L. Lane,* Eric J. Jenkinson,* and Graham Anderson*

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and Downloaded from cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune speciﬁcities. Despite this role, the mechanisms of Aire+ mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall’s corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally sepa- rable events that are controlled by distinct mechanisms. We ﬁnd that whereas mature thymocytes are not essential for Aire+ http://www.jimmunol.org/ mTEC development, use of an inducible ZAP70 transgenic mouse line—in which positive selection can be temporally controlled— demonstrates that the emergence of involucrin+ mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire+ mTECs depends upon RANK signaling, continued mTEC development to the involucrin+ stage maps to activation of the LTa–LTbR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs. The Journal of Immunology, 2010, 185: 4769–4776.

fter their recruitment to the thymus, T cell precursors I, respectively (1, 2). Newly selected thymocytes are then screened proliferate and differentiate to produce a large pool of further for their ability to recognize self-peptide/MHC complexes, by guest on September 29, 2021 A immature CD4+8+ thymocytes that resides within the with negative selection purging the abTCR repertoire of potentially thymic cortex. As a result of the random recombination of gene autoreactive specificities (3). Such combined selection events en- segments at TCR-a and TCR-b loci, CD4+8+ thymocytes express sure that T cell production in the thymus leads to the generation of a diverse repertoire of abTCRs (abTCR), and so are required to a naive T cell pool that recognizes self-MHC molecules but is able undergo selection events based on abTCR specificity. Positive to discriminate between self and nonself Ags. selection rescues thymocytes capable of self-MHC recognition Positive and negative selection events occur within specialized from cell death, with the lineage commitment mechanism ensur- intrathymic environments defined by distinct stromal components ing that changes in coreceptor expression result in the generation (4). Positive selection signals are provided by cortical thymic of CD4+ and CD8+ cells recognizing MHC class II and MHC class epithelial cells (cTECs), a function that is at least in part due to their expression of a unique MHC-bound self-peptide repertoire generated by cTEC-specific expression of Prss16 (the gene *Medical Research Council Centre for Immune Regulation; ‡School of Clinical and encoding thymus-specific serine protease) and the b5t thymo- Experimental Medicine, Institute for Biomedical Research, Medical School, Univer- proteosomal subunit (5–7). In contrast, the medulla provides sity of Birmingham, Edgbaston, Birmingham; †Division of Immune Cell Biology, Medical Research Council National Institute for Medical Research, London, United a microenvironment where self-tolerance is imposed, through both Kingdom; xInstitute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine negative selection and FoxP3+ regulatory T cell (Treg) production. University, Dusseldorf, Germany; and {Division of Experimental Immunology, In- stitute for Genome Research, University of Tokushima, Tokushima, Japan Moreover, autoimmune regulator (Aire) expressing medullary thymic epithelial cells (mTECs) have been shown to play a role in both Received for publication June 29, 2010. Accepted for publication August 18, 2010. processes (8–10), highlighting their importance in the avoidance of This work was supported by a Medical Research Council Programme grant (to G.A. and E.J.J.) and by core facilities of the Medical Research Council Centre for Immune autoimmunity. Several advances have recently been made in un- Regulation. derstanding the regulation of Aire+ mTEC development. For ex- Address correspondence and reprint requests to Prof. Graham Anderson, Floor 4, ample, the TNFRs RANK and CD40 are key to the generation of Institute for Biomedical Research, Medical School, University of Birmingham, Edg- Aire+ mTECs (11–15), a process involving lymphostromal cross- baston, Birmingham B15 2TT, United Kingdom. E-mail address: g.anderson@bham. + + ac.uk talk with RANKL lymphoid tissue inducer cells and RANKL / + + Abbreviations used in this paper: Aire, autoimmune regulator; BM, bone marrow; CD40L CD4 thymocytes. An additional TNFR family member, cTEC, cortical thymic epithelial cell; dox, doxycycline; HC, Hassall’s corpuscle; LTbR, also plays a pivotal yet poorly understood role in mTEC IRES, internal ribosomal entry site; MSCV, murine stem cell virus; mTEC, medullary development (16–20). Interestingly, the medulla is also recognized thymic epithelial cell; Treg, regulatory T cell; WT, wild type. as a dynamic thymic compartment, with mature mTECs displaying Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 a turnover time of 2–3 wk (21, 22). Collectively, such findings www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002151 4770 POSITIVE SELECTION AND mTEC DIFFERENTIATION suggest that Aire+ mTEC production is a continuous process stem- involucrin (Covance, Maidenhead, U.K.), rabbit anti-Aire (M-300; Santa ming from a TEC progenitor pool that is under hemopoietic cell Cruz Biotechnology, Santa Cruz, CA), AlexaFluor 647 EpCAM-1 (G8.8; control. Despite these advances, critical to our understanding of kind gift from A. Farr, University of Washington, Seattle, WA), biotinylated anti-CD80 (clone Ly53; eBioscience), anti-IAb MHC class II alloantigen mTEC development and homeostasis are events that occur in mTEC (clone AF6-120.1; Becton Dickinson, San Jose, CA), and mouse anti-keratin post-Aire expression. Whereas initial studies suggested that Aire 10 (clone SPM261; Covance). Biotinylated Abs were detected using strep- may induce apoptosis as a means to regulate mTEC homeostasis tavidin conjugated to either AlexaFluor 555 or AlexaFluor 646 (Invitrogen, (23), others suggest that it plays a role in further mTEC differenti- Paisley, U.K.). Involucrin and Aire Abs were detected using donkey anti- rabbit AlexaFluor 488 (Invitrogen), whereas keratin 10 Abs were detected ation (24, 25), with recent cell fate mapping approaches directly with anti-mouse AlexaFluor 594 (Invitrogen), and anti-IAb MHC class II demonstrating that mTEC differentiation continues beyond the alloantigen was detected using an FITC amplification method described later. Aire+ stage (26). Moreover, late-stage mTEC differentiation has Simultaneous two-color staining of Aire and involucrin was performed using been shown to involve expression of involucrin (25, 26), a compo- rabbit anti-involucrin and goat anti-Aire (clone D-17; Santa Cruz Bio- nent of the cross-linked cornified envelope and a marker of terminal technology). Human thymic sections were stained with FITC anti-CD4 (RPA- T4; eBioscience), biotinylated anti-CD8 (HIT8a; eBioscience), mouse anti- differentiation in keratinocytes (27). However, whether progression involucrin (SY5; Abcam, Cambridge, U.K.), and goat anti-Aire (D-17; Santa to this stage is a cell-autonomous process post-Aire expression or Cruz Biotechnology). Anti-CD4 was detected using rabbit anti-FITC (Invi- also requires cross-talk and regulation by hemopoietic elements is trogen) then goat anti-rabbit FITC (Southern Biotechnology, Birmingham, unknown. This is a key issue in understanding the regulation of AL), and anti-CD8 was detected using Streptavidin-AlexaFluor 646 (Invi- trogen). Anti-involucrin was detected using goat anti-mouse AlexaFluor 594 mTEC homeostasis and hence the efficiency of self-Ag presentation (Invitrogen), and anti-Aire was detected using donkey anti-goat AlexaFluor for central tolerance. 594 (Invitrogen). Sections were mounted using 1,4-Diazabicyclo[2.2.2]oc-

In this study, we have analyzed cellular and molecular regulation tane solution (Sigma Aldrich, Dorset, U.K.), and all images were obtained Downloaded from of the mTEC lineage, including the requirements for developmental stages post-Aire expression. We show an ordered ontogenetic appearance of distinct Aire+ and involucrin+ mTEC subsets in the thymus medulla, the latter appearing postnatally at a time point correlating with the initial accumulation of positively selected

thymocytes. By analyzing mTEC development in mouse models http://www.jimmunol.org/ where positive selection is either absent or can selectively be restored by an inducible Zap70 transgene, we provide direct evidence that whereas the absence of positive selection does not preclude generation of Aire+ mTECs, development of terminally differentiated involucrin+ mTECs strictly depends upon the presence of mature thymocytes. Finally, we show that the requirement for positive selection in mTEC terminal differentiation cannot be met by provision of RANKL and instead maps to activation of LTbR signaling. Collectively, our data deﬁne cross-talk mechanisms in by guest on September 29, 2021 mTEC terminal differentiation, help explain the poorly understood role of LTbR in thymus medulla development, and highlight the temporal requirement for distinct TNFR signaling during initial and late-stage mTEC differentiation.

Materials and Methods Mice Wild-type (WT) C57BL/6 (B6) mice at 4–6 wk of age were used as indicated. Zap702/2 (28), Ltbr2/2 (29), Lta2/2 (30), Cd40lg2/2 (31) mice have been described previously. Tetracycline-inducible Zap70 transgenic mice (Zap70Tre rtTAhuCD2 Zap702/2; TetZap70 hereon) have been described in detail elsewhere (32). Zap70 expression in adult mice was in- duced by administration of tetracycline derivative doxycycline (dox) in food (3 mg/g). Mice were bred and maintained in the Biomedical Services Unit, University of Birmingham, with the exception of TetZap70 mice, which were bred and maintained at the National Institute for Medical Research (Mill Hill, London, U.K.). All experiments were carried out with institution and U.K. Home Ofﬁce approval. Human thymus samples

Fresh thymic tissue was provided by Birmingham Children’s Hospital FIGURE 1. Involucrin expression defines terminally differentiated (Birmingham, U.K.) from pediatric patients undergoing routine cardiac mTECs that are distinct from the Aire+ subset. A–D, Frozen tissue sections of surgery. Tissues were obtained after informed consent and with the ethical adult mouse and neonatal human thymus were stained with Abs to CD4, approval of National Health Service North Staffordshire Local Research CD8, and the epithelial terminal differentiation marker involucrin using an Committee. LSM 510 Meta microscope, and images were analyzed using Zeiss LSM Immunofluorescent confocal analysis and mTEC quantitation software. C and D show higher-power magnification to demonstrate the characteristic swirled morphology of involucrin+ mTEC in both murine and Freshly isolated mouse and human thymus tissue was embedded in OCT human thymus. Dual analysis of Aire and involucrin expression in mouse (E) compound (Sakura Fintek UK, Thatcham, U.K.) and frozen on dry ice. Sections were cut at 5-mm thickness, fixed in acetone, and stained with Abs and human (F) thymus identifies nonoverlapping mTEC subsets that are as described previously (12). For analysis of mouse tissue, the following often found in close anatomical association. Original magnification 340; reagents were used: AlexaFluor 647 anti-CD4 (L3TA; eBioscience, Hat- scale bars, 20 mm. Data shown are representative of at least three separate field, U.K.), biotinylated anti-CD8 (ebioH35-17.2; eBioscience), rabbit anti- experiments. The Journal of Immunology 4771 using an LSM 510 Meta microscope, with image analysis performed using irradiated recipients (900 rad for WT B6 and 700/800 rad for Zap702/2 Zeiss LSM software (Zeiss, Welwyn Garden City, U.K.). To quantitate the hosts), and mice were analyzed 4–5 wk after injection. frequency of Aire+ and involucrin+ mTECs, medullary areas were initially identified on the basis of CD4, CD8 staining to identify regions containing Flow cytometry + 2 2 + + + CD4 8 and CD4 8 cells but not CD4 8 cortical thymocytes. For each Freshly prepared WT neonatal and adult thymocyte samples were centri- mouse, a minimum of three separate thymus sections at least 10 sections apart fuged and then analyzed by flow cytometry as described (12). Cells were m 2 were analyzed. From each section, medullary areas of up to 200,000 m acquired using CellQuest software on a Becton Dickinson LSR flow were imaged, and the number of involucrin+ and Aire+ cells was determined + + cytometer. For analysis of TetZap70 mice, thymocyte suspensions were to give the number of involucrin and Aire cells per square millimeter. analyzed on a FACSCanto II (Becton Dickinson) using FACS diva soft- Statistical analysis ware v6.1.2. All samples were then analyzed postacquisition with Flo-Jo software (Tree Star, Ashland, OR). Analysis was performed using a two-tailed unpaired Student t test with Prism software (GraphPad, San Diego, CA). Significance is denoted with asterisks (i.e., pp , 0.05; ppp , 0.01; pppp , 0.001). Results Analysis of Aire and involucrin expression identifies temporal Retroviral transduction and bone marrow chimeras regulation in the development of distinct mTEC compartments To generate RANKL-expressing retrovirus, PCR-cloned cDNA fragments of The keratinocyte terminal differentiation marker involucrin, a com- murine RANKL, using the primers 59-CTCGAGGAAGGGAGAGAACG- ATC-39 and 59-CTCGAGTCAGTCTATGTCCTGAACTTTG-39, were ponent of the cornified envelope in skin, has recently been reported cloned into XhoI site of MIGR1 (a gift from Dr. W.S. Pear, University of to identify a subset of mTEC in adult mouse thymus (25, 26). Pennsylvania, Philadelphia, PA). Retroviral supernatants were prepared as Importantly, cell fate mapping studies show that mTEC maturation described (33) using the packaging cell line Plat-E (34). To generate bone + Downloaded from 2/2 extends beyond the Aire stage and further suggest that involucrin marrow (BM) chimeras, adult WT B6 or Zap70 mice were treated with expression may follow Aire expression, a relationship that is sug- 150 mg/kg 5-fluorouracil, and 4 d later, Sca1+ progenitors were isolated from femurs and tibias by magnetic sorting (Miltenyi Biotec, Auburn, CA). gestive of a terminal differentiation program in mTECs (26). To Retrovirus infection of sorted Sca1+ cells was performed as described (13). investigate further these poorly defined stages in thymus medulla Briefly, freshly sorted BM cells were cultured in IMDM supplemented with development, we initially analyzed the phenotype and ontogenetic 20% FCS, L-glutamine, sodium pyruvate, nonessential amino acids, peni- timing of the appearance of Aire+ and involucrin+ mTECs in both cillin, streptomycin, 50 ng/ml stem cell factor, 50 ng/ml IL-6, and 10 ng/ml IL-3. After 48, 72, and 96 h, cells were spin-infected with retrovirus by adult murine and human pediatric thymus. In human thymus (Fig. http://www.jimmunol.org/ centrifuging the culture plates in the presence of 10 mg/ml polybrene for 90 1B,1D,1F), involucrin identifies Hassall’s corpuscles (HCs), rep- min. Following infection, total cells were intravenously injected into resenting swirled epithelial structures formed from terminally by guest on September 29, 2021

FIGURE 2. Phenotypic characterization of involucrin+ mTECs. Frozen tissue sections of adult mouse thymus were stained with Abs to involucrin and either EpCAM1 (A), CD80 (B), MHC class II (C), or keratin 10 (D). The images show typical examples of medullary areas within thymic sections. Original magnification 340; scale bars, 20 mm, and data are representative of at least three separate experiments. 4772 POSITIVE SELECTION AND mTEC DIFFERENTIATION differentiated epithelial cells (25, 35). In agreement with a recent were found to be absent in embryonic thymus (data not shown) study (25), analysis of adult mouse thymus (Fig. 1A,1C,1E)shows and immediately after birth, being first detected at postnatal day 5 a similar staining pattern, with small, medullary-located involucrin+ (Fig. 3A,3C), a time point coinciding with the appearance of cells displaying an organization reminiscent of HC-like structures in positively selected CD4+abTCRhigh and CD8+abTCRhigh thy- human thymus. Interestingly, in both mouse (Fig. 1E) and human mocytes (Fig. 3D). Collectively, these findings identify an ordered (Fig. 1F) thymus, Aire and involucrin expression were consistently appearance of distinct Aire+involucrin2 and Aire2involucrin+ found to be nonoverlapping. However, although we failed to detect mTEC subsets during thymus development, with the emergence of mTECs that coexpressed involucrin and Aire, Aire+involucrin2 the latter correlating with the accumulation of mature thymocytes mTECs and Aire2involucrin+ mTECs were often found in close within thymic medullary areas. proximity to one another (Fig. 1E,1F). Further phenotypic analysis (Fig. 2) with a panel of epithelial markers demonstrated that in Positive selection is essential for mTEC terminal contrast with Aire+ mTECs, which are enriched for CD80+MHCII+ differentiation keratin102 cells, involucrin+ mTECs are uniformly CD802MHCII2 To determine whether the emergence of involucrin+ mTECs is and express keratin10+, an additional marker of epithelial differen- functionally linked to the presence of positively selected thymo- tiation in skin (36). Thus, our data suggest that Aire+ mTECs and cytes, we next analyzed Aire and involucrin expression by mTECs terminally differentiated involucrin+ mTECs represent distinct sub- in Zap702/2 mice, where thymocyte development is blocked at sets of epithelial cells within the thymic medulla. the immature CD4+8+ stage due to an absence of abTCR- We next investigated the timing of appearance of Aire+ and mediated positive selection signals (28). In Zap702/2 mice, involucrin+ mTECs in mouse thymus development. By quantita- medullary thymus areas were still readily detectable, identified as Downloaded from tive confocal microscopy, we found that Aire+ mTECs, which regions surrounded by cortical areas containing CD4+8+ thymo- were more abundant than involucrin+ mTECs at all stages ana- cytes (Fig. 4A). Whereas Aire+ mTECs were clearly detectable in lyzed, were readily detectable at postnatal day 0 (Fig. 3A,3B), both adult WT and Zap702/2 thymic medullary areas, albeit at a finding that is in agreement with the initial emergence of Aire+ reduced frequency in the latter (Fig. 4A,4B), the absence of cells at E16 of gestation (12). In contrast, involucrin+ mTECs mature CD4+ and CD8+ cells correlated with the absence of http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Ordered appearance of involucrin+ and Aire+ mTECs in thymus ontogeny identifies a postnatal phase of terminal differentiation. A, Thymus sections from newly born postnatal day (P) 0 and 5 mice were FIGURE 4. Positively selected thymocytes are required for mTEC ter- analyzed for expression of combinations of either CD4 and CD8 or Aire minal differentiation. A, Adult WT and Zap702/2 thymic sections were and involucrin. Original magnification 340; scale bars, 20 mm. Quanti- analyzed for expression of CD4 (blue) and CD8 (green) together with tative analysis of Aire+ and involucrin+ mTECs at the indicated ages is either Aire (red, upper panels) or involucrin (red, lower panels). Original shown in B and C, respectively. Ontogeny of thymocyte development in P0 magnification 340; scale bars, 20 mm. B and C, Quantitative analysis of and P5 thymus was analyzed by flow cytometry using Abs to CD4, CD8, the frequency of Aire+ (B) and involucrin+ (C) mTEC subsets in WT (red and abTCR. D, Quantitative analysis of CD4+8-abTCRhigh (green circles) circles) and Zap702/2 (green circles) mice, with each point representing and CD428+abTCRhigh (red circles) subsets. In the graphs shown in B–D, a single mouse. The ratio of Aire+/involucrin+ mTECs is 23.2:1 6 9.6 SD each point represents a single mouse. Data are representative of three and 65.1:1 6 24.6 SD in WT and Zap702/2, respectively. Data shown are separate experiments. pp , 0.05; ppp , 0.01; pppp , 0.001. representative of at least three separate experiments. ppp , 0.01. The Journal of Immunology 4773 involucrin+ mTECs in Zap702/2 mice (Fig. 4A,4C), suggesting that signals from CD4+ and/or CD8+ thymocytes are important for late stages of mTEC development. To test this possibility directly, we analyzed mTEC development in TetZap70 mice, in which thymocyte positive selection in Zap70-deficient animals can be restored by controlled expression of a tetracycline-inducible Zap70 transgene within the T cell lineage (32). Fig. 5A shows that, as reported previously, compared with untreated controls (2dox), treatment of adult TetZap70 mice with dox (+dox) for 14 d resulted in the restoration of positive selection and the appearance of mature CD4+82 and CD428+ thymocytes. Subsequent confocal analysis of medullary microenvironments showed that the restoration of positive selection by dox treatment was ac- companied by an increase in Aire+ mTEC frequency (Fig. 5B,5C) and the emergence of involucrin+ mTECs (Fig. 5B,5D) at a frequency similar to that seen in unmanipulated adult WT mice (Fig. 4C). Collectively, these findings suggest that whereas mature thymocytes are not essential for the generation of Aire+ mTECs, they are an essential requirement to provide signals that drive later Downloaded from stages of mTEC differentiation, including generation of Aire- involucrin+ mTECs. Terminal differentiation of mTEC is regulated by LTa–LTbR signaling

Several studies have identiﬁed a role for various TNFRs in thymus http://www.jimmunol.org/ medulla development, including RANK, CD40, and LTbR. Of these, the requirement for RANK signaling during Aire+ mTEC development in adult thymus has shown to map to the presence of positively selected thymocytes (13). However, whether RANK signaling explains the requirement for single positive thymocytes in post-Aire involucrin+ mTEC development has not been addressed. Due to the dramatic reduction in Aire+ mTECs in Rank2/2 thymus (11, 14), such mice cannot be readily used to determine the role of RANK in mTEC development post-Aire by guest on September 29, 2021 expression. Thus, we next determined whether RANKL could replace the requirement for single positive thymocytes in the development of involucrin+ mTECs. To this end, we established chimeras in which BM progenitors from Zap702/2 mice were transduced with retroviral constructs, resulting in constitutive expression of RANKL by thymocytes that are blocked at the CD4+8+ stage. Sca1+ BM progenitors from adult Zap702/2 mice were subjected to multiple rounds of retroviral transduction in vitro with either mouse stem cell virus (MSCV)–internal ribosomal entry site (IRES)–GFP or MSCV–RANKL–IRES–GFP biscistronic retroviral constructs. Retrovirally infected cells were then 2/2 injected i.v. into sublethally irradiated Zap70 hosts, and thy- FIGURE 5. Restoring positive selection rescues mTEC terminal dif- mus tissue was collected and analyzed 4 to 5 wk later. In agree- ferentiation. A, TetZap70 mice were fed dox for 14 d, resulting in the ment with Hikosaka et al. (13), RANKL expression by thymocytes rescue of positive selection and the emergence of mature CD4+ and CD8+ in the absence of single positive cells (data not shown) resulted in thymocytes. B, Confocal analysis of untreated (left panels) or dox-treated an increase in the sizes of thymic medullary areas (data not (right panels) TetZap70 mice for CD4 (blue) and CD8 (green) together shown) and an increase in the numbers of Aire+ mTECs (Fig. 6A), with either Aire (red, upper panels in B) or involucrin (red, lower panels). indicating that retroviral RANKL in Zap702/2 BM is expressed at Original magniﬁcation 340; scale bars, 20 mm. Arrow in B indicates a typical involucrin+ HC-like structure. C and D, Quantitative analysis of a functional level. In marked contrast, however, despite these + + effects on the thymus medulla, RANKL expression in MSCV– Aire and involucrin mTEC subsets, respectively, in untreated TetZap70 2 2 (red circles) and TetZap70 + dox (green circles). Results are representative RANKL–IRES–GFP Zap70 / chimeras failed to induce the ap- of three separate experiments. ppp , 0.01; pppp , 0.001. pearance of involucrin+ mTECs (Fig. 6B). Thus, despite the in- creased availability of RANKL, the absence of single positive thymocytes still resulted in a block in later stages of mTEC dif- when RANKL was overexpressed, there was an increase in the ferentiation. We next made chimeras using WT BM precursors frequency of Aire+ mTECs (Fig. 6C), again showing that retroviral and WT hosts, in which positive selection and generation of CD4+ RANKL expression in WT BM is expressed at a functional level. and CD8+ thymocytes occurred (data not shown). Flow cytometric Importantly, in contrast with chimeras generated using Zap702/2 analysis shows comparable levels of RANKL expression on CD4+ BM (Fig. 6B), involucrin+ mTECs were detected when WT BM was 8+ thymocytes in WT and Zap702/2 chimeras and CD4+ and transduced with either MSCV–IRES–GFP or MSCV–RANKL– CD8+ thymocytes in WT chimeras (data not shown). As expected, IRES–GFP constructs (Fig. 6D). Moreover, no difference was ob- 4774 POSITIVE SELECTION AND mTEC DIFFERENTIATION

begun to uncover the developmental program of mTEC maturation, which begins with bipotent cTEC/mTEC progenitors (37, 38) and leads to the generation of postmitotic Aire+ mature mTECs via a RANK-dependent mTEC progenitor stage (11). In addition, several studies have now addressed the mechanisms regulating the generation of Aire+ mTECs (reviewed in Ref. 39). Several models of mTEC development have been proposed, including those that are postulated to involve either a “terminal differentiation” or a “progressive restriction” model (reviewed in Ref. 40). While the former model involves a linear differentiation program in which Aire+ mTECs expressing the most tissue-restricted Ags represent the most mature cells, the progressive restriction model suggests that expression of Aire and Aire-dependent tissue-restricted Ags occurs within immature mTECs, which under the inﬂuence of Aire be- come progressively restricted to stages that mimic gene expression patterns seen in other epithelia. Whereas evidence for both sce- narios exists, little is known about the program of mTEC differentiation post-Aire expression. Relevant to this, the role of Aire

itself in mTEC differentiation is not clear, and several models have Downloaded from been proposed in which Aire is involved in either the initial (41) or later stages (23, 25, 26) of terminal differentiation. In our study, we provide evidence that the generation of Aire+ mTECs is followed by a program of terminal differentiation that is associated with the loss of Aire expression and the acquisition of the terminal differ-

entiation marker involucrin. Thus, the data presented in this study http://www.jimmunol.org/ support a linear terminal differentiation program of Aire+ mTEC development and provide clues about the regulation of mTEC development post-Aire expression. First, the ontogenetic appearance initially of Aire+ mTECs and then involucrin+ mTECs is strongly FIGURE 6. RANK–RANKL signaling is insufficient to induce mTEC suggestive of a precursor-product relationship between these cells, 2 + terminal differentiation. The frequency of Aire+ and involucrin+ mTECs as was shown previously for CD80 and CD80 mTEC subsets was calculated in chimeras established from Zap702/2 (A, B)orWT(C, D) BM precursors, transduced with either MSCV–IRES–GFP (d)or MSCV-RANKL-IRES-GFP (:) biscistronic retroviral constructs. Each by guest on September 29, 2021 point represents a single mouse, and data are representative of three separate experiments. pp , 0.05; pppp , 0.001. served in the frequency of involucrin+ mTECs in both types of WT BM chimeras (Fig. 6D). Overall, these experiments suggest that the requirement for single positive thymocytes is a rate-limiting step in mTEC terminal differentiation and cannot be simply explained by provision of RANKL alone. Instead, these data indicate that positively selected thymocytes provide additional signals that regulate the appearance of involucrin+ mTECs. To investigate the nature of these signals, we next analyzed mTEC development in mice ge- netically deficient in additional TNFR ligands that are provided by mature thymocytes, namely CD40L (12, 15) and LTa (16). Although comparison of thymus medullary areas in WT and Cd40lg2/2 adult mice showed no differences in the frequency of involucrin+ mTECs (Fig. 7), we found striking differences in mTEC development in mice with disrupted LTa–LTbR signaling. Thus, despite a relatively normal frequency of Aire+ mTECs, Lta2/2 mice showed a marked and specific decrease in the frequency of involucrin+ mTECs, a phenotype that was also evident in Ltbr2/2 adult thymus (Fig. 7). Collectively, these findings suggest that activation of the LTa–LTbR axis by LT-expressing mature thymocytes drives post-Aire mTEC FIGURE 7. Terminal differentiation of mTEC is regulated by LTa– b 2/2 2/2 development and the appearance of involucrin+ mTECs. LT R signaling. Thymus sections from adult WT, Lta , Ltbr , and Cd40lg2/2 mice were stained with Abs to CD4, CD8, and either Aire or involucrin. The frequency of Aire+ (A) and involucrin+ (B) mTECs was Discussion determined as described in Materials and Methods, with each point rep- The importance of intrathymic medullary microenvironments in the resenting a separate mouse. The ratio of Aire+/involucrin+ mTECs is establishment of T cell tolerance is well established. In particular, 23.2:1 6 9.6 SD (WT), 81.6:1 6 19.9 SD (Ltbr2/2), 92:1 6 38.7 SD mTECs expressing Aire have been linked to both negative selection (Lta2/2), and 19.7:1 6 3.9 SD (Cd40lg2/2). Data shown are representative and the induction of Treg development (3). Recent studies have of three separate experiments. ppp , 0.01. The Journal of Immunology 4775

(11). Second, that the development of Aire+ and involucrin+ mTEC LTbR results in only a very modest induction of Aire mRNA ex- subsets appears to be controlled by cross-talk mechanisms in- pression (14). Thus, although the role of LTbR signaling in Aire+ volving distinct TNFR argues for a series of distinct steps in mTEC development remains controversial, our observation that a program of differentiation. Importantly, this scenario is supported Ltbr2/2 thymus lacks Aire2involucrin+ mTECs extends our un- by data from recent cell-fate mapping studies demonstrating con- derstanding of the role of LTbR in thymus medulla development tinued differentiation of Aire-lineage cells (26) and also by studies and fits well with previous studies suggesting that medullary ab- reporting the direct involvement of Aire expression in mTEC dif- normalities in the absence of LTbR signaling cannot be fully ferentiation, as indicated by altered p63 and cytokeratin expression explained by a block in the generation of Aire+ mTECs (16–19). in mTECs from Aire-deficient mice (24, 41). That involucrin- Instead, the requirement for LTbR signaling in late-stage mTEC expressing epithelial cells in murine thymus are reminiscent of development described in this study suggests that alterations in the involucrin+ HCs in the human thymic medulla is further suggestive thymus medulla of Ltbr2/2 mice may occur at least in part as of a terminal differentiation mTEC program that continues post- a result of disrupted mTEC homeostasis following a block in mTEC Aire expression. Interestingly, it is currently not clear whether terminal differentiation post-Aire expression. Finally, we also find involucrin represents a universal marker of mTEC terminal dif- that thymic positive selection is an essential requirement for ter- ferentiation or is specifically indicative of terminal differentiation minal differentiation and the appearance of involucrin+ mTECs. of the Aire+ mTEC lineage. Indeed, the relative paucity of invo- This link between positively selected thymocytes and mTEC ho- lucrin+ cells in the adult mouse thymus is of interest, as it may meostasis may then help to ensure that medullary environments suggest that its expression represents terminal differentiation of expressing the appropriate spectrum of self-Ags are available to a distinct mTEC subset, rather than the total mTEC lineage. screen each newly generated cohort of mature thymocytes for po- Downloaded from Moreover, this low frequency of involucrin+ clusters may indicate tential autoreactivity. Interestingly, however, our data demonstrate that they do not represent long-lived structures but are efficiently that, unlike earlier stages in mTEC development, late-stage mTEC cleared as progressively apoptotic cells by the thymic phagocyte development is driven by LTa-LTbR, but not RANKL–RANK, system. The functional significance of involucrin+ mTEC clusters is signaling. Thus, we propose that following their involvement in the also currently unclear, and study of this is currently hampered by induction of Aire+ mTEC differentiation by provision of RANKL,

the lack of methods for their isolation. While in human thymus the mature thymocytes continue to influence late-stage mTEC terminal http://www.jimmunol.org/ generation of Tregs has been linked to HCs (42), which resemble differentiation by their expression of LTaand stimulation of LTbR. involucrin+ mTEC clusters in the mouse, FoxP3+ Tregs develop Such a model further underscores the importance of lymphostromal normally in Ltbr2/2 mice (17), which we show in this study to lack cross-talk between mature CD4+ and CD8+ thymocytes and involucrin+ mTECs. The finding that mTECs have a turnover time epithelial cells in the thymus medulla and identifies a stage-specific of 2–3 wk may suggest an alternative functional significance of requirement for distinct thymocyte-expressed TNFR ligands at involucrin+ mTEC differentiation that is perhaps linked to epithe- different stages of mTEC development. lial homeostasis in the thymic medulla. For example, as tissue- restricted Ags can be divided into at least three distinct “gene pools,” the expression of which is linked to progressive mTEC Acknowledgments by guest on September 29, 2021 differentiation (43), a normal program of terminal differentiation We thank Dr. Art Weiss for Zap70 knockout mice. We thank Dr. Valerie Horsley for valuable discussion and critical review of the manuscript. We and mTEC homeostasis may help to ensure an adequate represen- also acknowledge the help of Dr. David Barron, pediatric cardiac surgeon tation of “early” and “late” self-Ags within the thymic medulla. at the Birmingham Children’s Hospital. Our data also show that Aire+ mTEC development and the ap- + pearance of involucrin terminally differentiated mTECs are sep- Disclosures arable depending upon the availability of signals provided by The authors have no financial conflicts of interest. positively selected thymocytes. 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