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Thymic Medullary Epithelium and Thymocyte Self-Tolerance Require Cooperation between CD28−CD80/86 and CD40−CD40L Costimulatory Pathways This information is current as of September 28, 2021. Joy A. Williams, Jingjing Zhang, Hyein Jeon, Takeshi Nitta, Izumi Ohigashi, David Klug, Michael J. Kruhlak, Baishakhi Choudhury, Susan O. Sharrow, Larry Granger, Anthony Adams, Michael A. Eckhaus, S. Rhiannon Jenkinson, Ellen R. Richie, Ronald E. Gress, Yousuke Takahama and Richard J. Hodes Downloaded from J Immunol 2014; 192:630-640; Prepublished online 13 December 2013; doi: 10.4049/jimmunol.1302550

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Thymic Medullary Epithelium and Thymocyte Self-Tolerance Require Cooperation between CD28–CD80/86 and CD40–CD40L Costimulatory Pathways

Joy A. Williams,*,1 Jingjing Zhang,*,1 Hyein Jeon,* Takeshi Nitta,† Izumi Ohigashi,† David Klug,* Michael J. Kruhlak,* Baishakhi Choudhury,* Susan O. Sharrow,* Larry Granger,* Anthony Adams,* Michael A. Eckhaus,‡ S. Rhiannon Jenkinson,* Ellen R. Richie,x Ronald E. Gress,{ Yousuke Takahama,† and Richard J. Hodes*

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from Downloaded from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28–CD80/86 and CD40– CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTa,LTb, and receptor activator for NF-kB in the thymus and are necessary for medullary development. Combined absence of CD28–CD80/86 and CD40–CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single- positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR–ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice http://www.jimmunol.org/ are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28–CD80/86 and CD40–CD40L pathways is required for normal medullary epithelium and for maintenance of self- tolerance in thymocyte development. The Journal of Immunology, 2014, 192: 630–640.

n important aspect of thymic T cell development is the autoimmune regulator (AIRE) gene (1, 2). The critical role of generation of a functional T cell repertoire that is capable AIRE-expressing mTEC in tolerance induction is evidenced by of responding to a broad universe of foreign Ags but that the severe autoimmune disease that occurs when expression of

A by guest on September 28, 2021 is not reactive to self. Self-tolerance is accomplished by a variety of AIRE protein is disrupted (2, 3). The dialogue between CD4 SP mechanisms, including deletion of thymocytes with high affinity thymocytes and stromal cells, termed thymic crosstalk, is in turn for self and diversion of developing self-reactive thymocytes to important in supporting development of a normal mTEC com- a T regulatory (Treg) cell fate. The thymic medulla plays a central partment (4–7). There has therefore been considerable interest in role in this tolerization process, providing an environment in defining the molecular interactions that mediate this critical cross- which developing thymocytes are exposed to a spectrum of self Ags talk. We undertook studies to determine whether molecular inter- during maturation and selection of CD4 and CD8 single-positive actions known to be important in the thymic tolerization process, by (SP) lineages. Medullary thymic epithelial cells (mTEC) are es- both promoting negative selection and supporting generation of sential to successful induction of self-tolerance, expressing a wide Treg cells, might also have critical roles in maintaining a normal array of peripheral Ags, at least in part due to expression of the thymic medulla. Thymic Treg cell development is critically dependent on CD28–CD80/86 interactions, whereas negative selection of self-reactive SP thymocytes is mediated by both CD28– *Experimental Immunology Branch, National Cancer Institute, National Institutes CD80/86 (8, 9) and CD40–CD40L (10, 11) interactions. We there- of Health, Bethesda, MD 20892; †Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima 3-18-15, Japan; fore addressed the role of CD28–CD80/CD86 and CD40–CD40L ‡Division of Veterinary Resources, Office of Research Services, National Cancer costimulatory pathways in interactions between thymocytes and Institute, National Institutes of Health, Bethesda, MD 20892; xScience Park Research Division, Department of Carcinogenesis, University of Texas MD Anderson Cancer thymic stromal cells during thymic development. Center, Smithville, TX 78957; and {Experimental Transplantation and Immunology We found that thymic epithelial cell (TEC) development was Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD only modestly perturbed by inactivation of the CD40–CD40L or 20892 1 CD28–CD80/86 pathway alone, whereas, in striking contrast, the J.A.W. and J.Z. contributed equally to this work. combined absence of CD28–CD80/86 and CD40–CD40L inter- Received for publication September 23, 2013. Accepted for publication November actions in CD40/CD80/86 knockout (KO) mice resulted in a de- 15, 2013. crease in mTEC numbers as profound as that observed in the Address correspondence and reprint requests to Dr. Richard J. Hodes, National Can- cer Institute/National Institutes of Health, Building 10, Room 4B10, 10 Center Drive, complete absence of SP thymocytes. Examination of thymocyte Bethesda, MD 20892. E-mail address: [email protected] development in the altered thymic environment of CD40/CD80/86 The online version of this article contains supplemental material. KO mice revealed that CD40/CD80/86-deficient SP thymocytes Abbreviations used in this article: AIRE, autoimmune regulator; cTEC, cortical were highly autoreactive, responding strongly in vitro to synge- thymic epithelial cell; DP, double-positive; iNKT, invariant NKT; KO, knockout; neic APCs, in contrast to the minimal responses of either CD80/ mTEC, medullary thymic epithelial cell; RANK, receptor activator for NF-kB; RANKL, RANK ligand; SP, single-positive; TEC, thymic epithelial cell; tg, trans- 86- or CD40-deficient thymocytes, and causing accelerated death genic; Treg, T regulatory; WT, wild-type. when transferred into congenic nude mice. These findings dem- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302550 The Journal of Immunology 631 onstrate that a strong cooperativity between CD28–CD80/86 and Sequential images were acquired using the multidimensional mosaix algo- CD40–CD40L pathways is required for both normal epithelial and rithm in AxioVision software (Carl Zeiss MicroImaging). The resultant thymocyte development; in their absence, the tolerance-inducing matrix of images, covering the entire area of the thymus section, was aligned and stitched using AxioVision software. The stitched image was converted to thymic medullary compartment fails to properly develop and SP a single large TIFF file. In preparing figures for display, a copy of each TIFF thymocytes are autoreactive. file was rescaled and compiled using Adobe Photoshop.

Materials and Methods Quantitative PCR Mice Total cellular RNA was DNase I treated and reverse transcribed with oligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen). Quantitative BALB/c (BALB) mice were obtained from the Frederick Cancer Research real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa) and Facility (Frederick, MD) and maintained at Bioqual (Rockville, MD). Light Cycler DX400 (Roche). Amplified products were confirmed to be BALB CD40-deficient mice were obtained from The Jackson Laboratory single bands by gel electrophoresis. The primer sequences are described (Bar Harbor, ME) and maintained at Bioqual; and BALB CD80/86-deficient previously (6, 18). mice were a gift of A. Sharpe (Boston, MA). BALB CD40/CD80/86 KO mice were generated through crosses of CD40 KO and CD80/86 KO mice Adoptive transfer to athymic nude recipients on a BALB background. BALB CD40L-deficient mice were generated by backcrossing C57BL/6 (B6) CD40L KO mice from The Jackson Labora- Thymocytes were prepared from wild-type (WT), CD40 KO, CD80/86 KO, tory for five generations onto the BALB background. BALB CD28/CD40L and CD40/CD80/86 KO mice on a BALB background and injected i.v. into KO mice were generated by crossing the BALB CD40L KO mice with BALB nu/nu recipient mice. Mice were weighed weekly and monitored BALB CD28 KO mice obtained from The Jackson Laboratory. LTbRKO daily for signs of morbidity. (12) mice have been previously described and were crossed to either B6 Downloaded from CD40 KO or B6 CD28 KO (Jackson Laboratory) mice to generate LTbR/ Histopathology CD40 KO or LTbR/CD28 KO mice, respectively. DO11 TCR transgenic For histological analysis, organs (liver, small intestine, colon, stomach, heart, (tg) mice (13) were crossed to BALB CD40/CD80/86 KO or to B6 CD40/ spleen, lymph nodes, and lung) were fixed in 10% neutral buffered formalin CD80/86 KO mice to generate DO11 tg+ mice lacking expression of 3 (Sigma-Aldrich), embedded in paraffin, sectioned, and stained with H&E. CD40, CD80/86, or CD40/CD80/86 on H2d or H2d b backgrounds, respectively. TCRa KO mice have been previously described (14). Athymic Statistical analysis BALB nu/nu mice were obtained from Frederick National Laboratory for

Cancer Research. Student t test (unpaired, two tailed) was used to determine p values. http://www.jimmunol.org/ Cell proliferation assays CD8-depleted thymocytes (.80% CD4+ SP thymocytes) and T-depleted Results splenocytes were prepared using CD8-specific, or CD4- and CD8-specific Normal thymic medullary development requires CD40–CD40L magnetic beads (Miltenyi Biotec), respectively. T-depleted splenocytes and CD28–CD80/86 interactions were irradiated at 500 rad, and then 2 3 105 CD8-depleted thymocytes were added to titrated numbers of irradiated splenocyte APCs in 96-well Previous work has documented the importance of thymocyte/ round-bottom plates. Cultures were incubated for 3–4 d prior to addition of stromal cell crosstalk in establishing normal thymic medullary 3 [ H]thymidine for 16 h. structure (4, 5). Both CD28–CD80/86 and CD40–CD40L path- by guest on September 28, 2021 Preparation of thymic stromal cells for flow cytometric ways are known to be important interactions for deletion of au- analysis and sorting toreactive thymocytes and mediation of self-tolerance in the thymus (8–11), which prompted us to examine the roles of these Thymic stromal cell preparations were made using methods modified from costimulatory pathways in the thymic crosstalk that results in those reported by Gray et al. (15). Following release of thymocytes by gen- tle teasing of the thymus, thymic fragments were digested with collagenase/ development of the thymic medulla. To accomplish this, we an- dispase at 0.25% w/v plus DNase I at 0.125% w/v (Roche) in four se- alyzed thymic sections from BALB WT, CD80/86 KO, CD40 KO, quential 15-min incubations at 37˚C. Reactions were stopped by addition of and CD40/CD80/86 KO mice. As can be seen in Fig. 1, deletion of FCS to 20%. For TEC analysis, single-cell suspensions were stained with CD40 alone or of CD80/86 alone had little effect on cortico- anti-CD45.2 FITC (104; BD Pharmingen), anti-Ly51 PE (BP-1; BD Phar- mingen), anti-MHC class II allophycocyanin (M5-114; eBioscience), and medullary architecture as defined using either anti-keratin 14 or UEA-1 biotin (Vector). Dead cells were excluded with propidium iodide the lectin UEA-1 to stain thymic medulla, and Ly-51 (cortex staining. Intracellular staining for AIRE was performed using Foxp3 fix/ specific) or anti-keratin 8 (stains cortex and some medullary epi- permeabilization (eBioscience) and rat anti-AIRE (clone 5H12), a gift of thelium) to reveal the cortex. In contrast, the medullary epithelium H. Scott (Walter and Eliza Hall Institute of Medical Research, Melbourne, was much reduced in the CD40/CD80/86 KO thymus relative to Australia) (16), and anti-rat IgG2c-PE (clone 2C-8F1; Southern Biotech- nology Associates). For mTEC sorting, enriched TEC preparations were other genotypes and was comparable to the profound reduction made by discontinuous density gradient fractionation (17). Enriched TEC seen in TCRa KO thymus lacking SP thymocytes. This analysis were stained with anti-CD45.2, anti-MHC class II, and UEA-1. CD45.2- was extended by flow cytometric characterization. TEC were + + negative, MHC II UEA cells (mTEC) were collected using a FACSAria defined as CD45negMHCII+ and assigned further as mTEC and flow cytometer (BD Biosciences) and analyzed using FlowJo (Tree Star, cortical thymic epithelial cell (cTEC) based on staining with UEA San Carlos, CA) FACS analysis software. Gating strategies are included in + neg + figure legends. and Ly51 (mTEC = UEA ; cTEC = UEA Ly51 ) (Fig. 2A). The CD40 KO had reduced numbers of mTEC relative to both WT and Immunohistology CD80/86 KO mice (Fig. 2B). Strikingly, however, the CD40/80/86 Sections (6 mm) of OCT-embedded frozen tissue were air dried for 15 min KO had a profound reduction in mTEC numbers relative to all andthenincubated2hwithoptimaldilutionsoftheprimaryrabbit other groups, including the CD40 KO. Notably, the number of polyclonal anti-keratin 14 (Covance Research Products) and rat anti- CD4 or CD8 SP thymocytes in CD40/80/86 KO mice was not keratin 8 (Troma-I; Developmental Studies Hybridoma Bank, Iowa Uni- versity) or anti-Ly51 PE (BD Pharmingen) and UEA-1 FITC (Vector). decreased, indicating that the defect in mTEC development does Tissues were washed and, after an amplification step (anti-rabbit Alexa 546 not result from a decrease in SP cells known to play a role in for K14, anti-rat Alexa 488 for K8, or biotinylated anti-PE, followed by medullary development (Fig. 2C). CD4 SP cells were in fact in- streptavidin Alexa 546 for Ly51), were mounted on microscope slides and creased in CD80/86 and CD40 KO thymuses, consistent with imaged using a Zeiss axiovert 200M inverted epifluorescence microscope equipped with appropriate fluorescence filters (Chroma Technologies), previous observations (19–21). A significant reduction in mTEC a motorized scanning stage, a 103 plan-apochromat (N.A. 0.45) objective numbers was also observed in BALB CD40L/CD28 KO mice lens, and a Photometrics CoolsnapES CCD camera (Roper Scientific). relative to WT, CD28 or CD40L single KOs, confirming that it is 632 THYMIC mTEC AND SELF-TOLERANCE REQUIRE CD80/86 AND CD40

several days after birth, a time at which the number of SP thymocytes begins to dramatically increase. Costimulation inﬂuences expression of genes critical for mTEC development and SP thymocyte recruitment to the medulla A number of the factors implicated in mTEC development are members of the TNF/TNFR family known to signal through the nonclassical NF-kB pathway central to lympho-organogenesis and mTEC formation [reviewed in (23)]. Therefore, to identify the mechanisms underlying collaborative effects of CD28 and CD40 pathways in mTEC development, we determined whether disruption of CD28–CD80/86 and CD40–CD40L interactions affected expression of a panel of TNF/TNFR family members in both CD4 SP cells and mTEC. Relative to WT CD4 thymocytes, levels of LTa and LTb mRNA were signiﬁcantly decreased in the CD80/86 KO and CD40/CD80/86 KO but not in CD40 KO CD4 SPs (Fig. 3A). In addition, expression of the chemokine CCL19, known to be upregulated in mTEC in response to LTa/b (24), was decreased in

CD40L/CD28 KO relative to WT mTEC. Expression of mTEC Downloaded from CCL21 was not signiﬁcantly decreased (p = 0.1). CD4 SP thymocyte expression of receptor activator for NF-kB ligand (RANKL) was not reduced in the absence of CD40–CD40L and/or CD28– CD80/86 interactions, whereas comparison of WT and CD40L/ CD28 KO mTEC revealed that expression of receptor activator

for NF-kB (RANK) was significantly decreased in CD28/CD40L http://www.jimmunol.org/ KOs (Fig. 3B). Expression of LTbR and AIRE in WT and CD28/ CD40L KO mTEC was comparable. Taken together, the gene expression data from CD4 SP thymocytes and from mTEC indicate that mice lacking both CD28–CD80/86 and CD40–CD40L interactions have deficiencies in at least three TNFR family pathways that are known to promote mTEC development: CD40L–CD40, LT– LTbR, and RANKL–RANK, as well as reduction in the CCL19 chemokine important for recruiting SP thymocytes to the medulla, FIGURE 1. Altered thymic microenvironment in CD40/CD80/86 KO where they are subject to negative selection. by guest on September 28, 2021 mice. Thymic cryosections from WT, CD80/86 KO, CD40 KO, CD40/ a b CD80/86 KO, and TCRa KO were stained with a combination of (left CD28 acts via regulation of LT / expression in cooperating panel) anti-keratin 14 (medullary epithelium, red) and anti-keratin 8 (cor- with CD40 signals to promote mTEC development tical and some medullary epithelium, green) or (right panel)withUEA1 The observations that CD28–CD80/86 interactions contribute sig- (medullary epithelium, green) and anti-Ly51 (cortical epithelium, red). Scale nificantly to production of LTab in CD4 SP thymocytes and that bars, 2 mm. Thymic cross sections from three mice per strain were stained, CD40/CD80/86 KO mice have a profound reduction in mTEC and representative images are shown. Mice were all 4 wk of age. prompted us to test the hypothesis that CD28 signals cooperate with CD40 signals via regulation of the LTbR pathway to promote simultaneous disruption of the CD40–CD40L and CD28–CD80/ mTEC development. To do this, we examined the medullary phe- 86 pathways that results in a deficient mTEC compartment (Sup- notype of LTbR/CD40 KO mice. As previously described (25, 26), plemental Fig. 1). we observed a significant reduction in the number of UEA+ mTEC mTEC are a heterogeneous population consisting of MHCIIlow in LTbR KOs relative to WT mice (Fig. 4). Interestingly, mTEC CD80low and MHCIIhighCD80high cells. A subpopulation of the were significantly further reduced when the LTbRKOiscombined MHCIIhighCD80high cells is AIRE+ and has a critical role in the with CD40 KO in LTbR/CD40 KO mice but not when combined induction of thymic tolerance [reviewed in (22)]. Interestingly, with CD28 KO in LTbR/CD28 KO mice (Fig. 4). The failure to whereas the total number of UEA+ mTEC was significantly de- observe a further deficit in mTEC numbers in LTbR/CD28 KOs creased in CD40/CD80/86 KO mice, the frequencies of MHCIIhigh relative to LTbR KO mice suggests that the major contribution of and MHCIIlow mTEC and the frequency of AIRE+ MHCIIhigh cells CD28–CD80/86 interactions to mTEC development is via increased within the mTEC population were similar to that of WT mice SP thymocyte expression of LTab. Conversely, the finding that the (Supplemental Fig. 2A, 2B). Overall, however, there was a dra- LTbR/CD40 KO does have a more profound deficit in mTEC rela- matic reduction in the absolute number of AIRE+ mTEC in CD40/ tive to the LTbR KO is consistent with CD28–CD80/86 cooperating CD80/86 KO mice (Supplemental Fig. 2C). with CD40–CD40L interactions to facilitate mTEC development by To further characterize the decrease in mTEC observed in CD40/ promoting SP thymocyte production of LTab. CD80/86 KO mice, we compared the number of mTEC present in WT and CD40/CD80/86 KO mice at various time points after birth. Expression of a TCR with high affinity for self does not Numbers of mTEC were comparable in WT and CD40/CD80/86 overcome the requirement for costimulation to promote mTEC KO mice shortly after birth (Fig. 2D). With increasing age, the development number of mTEC increased progressively in WT mice, but not in Requirements for costimulation in the activation of peripheral KO mice. Thus, the absence of CD40–CD40L and CD28–CD80/ T cells are influenced by the strength of TCR–ligand interactions, 86 interactions does not affect the numbers of mTEC present until such that strong TCR signaling can reduce or obviate the re- The Journal of Immunology 633 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Number of mTEC is decreased in CD40/CD80/86 KO mice, whereas number of CD4 SP thymocytes is increased. (A) Thymic stromal cells from BALB WT, CD80/86 KO, CD40 KO, and CD40/CD80/86 KO mice were prepared, as described in Materials and Methods, and stained with anti- CD45, UEA-1, anti-Ly51, and anti–I-A/I-E. After first gating on CD45neg, I-A/I-E+ cells, mTEC were identified as UEA+ and cTEC as UEAnegLy51+. Flow cytometry dot plots are representative of .9 mice per strain. (B) Thymic stromal cells were prepared as in (A). After gating on CD45neg, I-A/I-E+ cells, numbers of mTEC (UEA+) and cTEC (UEAneg, Ly51+) were calculated for each group. Data shown are mean 6 SE for $9 mice per strain. All mice were between 3 and 5 wk of age. **p # 0.01, ***p # 0.001. (C) Thymocytes from 4-wk-old WT, CD80/86 KO, CD40 KO, and CD40/CD80/86 KO mice were stained with anti-CD4 and anti-CD8, and numbers of CD4 and CD8 SP were calculated after gating on the respective single-positive populations. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001. (D) mTEC population in CD40/CD80/86 KO mice fails to expand after birth. Thymic stromal cells from BALB WT and CD40/80/86 KO mice were prepared from mice 1–32 d of age and stained as in (B). After gating on CD45neg, I-A/I-E+ cells, numbers of mTEC (UEA-1+) were determined. Data shown are the mean 6 SE for $3 mice per strain. quirement for costimulation (27, 28). It has also been reported that in promoting mTEC development. To this end we generated DO11 high-affinity TCR–ligand interactions are important in driving TCRtg+ mice on either a H2d or H2b3d background and compared mTEC development (7). We therefore asked whether expression development of mTEC in the presence or absence of CD40– of a TCR with known high affinity for self Ag would overcome the CD40L and CD28–CD80/86 interactions. The DO11 TCR trans- requirement for CD40–CD40L and CD28–CD80/86 interactions gene (tg) has been shown to have a high affinity for self I-Ab li- 634 THYMIC mTEC AND SELF-TOLERANCE REQUIRE CD80/86 AND CD40 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Quantitative RT-PCR analysis of sorted BALB WT, CD80/86 KO, CD40 KO, and CD40/CD80/86 KO CD4 SP thymocytes, and WT and CD28/CD40L mTEC. (A) mRNA expression of LTa,LTb, RANKL, and CCR7 in CD4 SP thymocytes was normalized to GAPDH mRNA, and values for the WT DP samples were arbitrarily set to 1. Data shown are mean 6 SE for five independent measurements for each strain (*p # 0.05, **p # 0.01, ****p # 0.0001). All mice were 4 wk of age. (B) mTEC were sorted, as described in Materials and Methods, by gating on CD45neg, MHC II+, UEA+ cells. mRNA expression of RANK, CCL19, CCL21, AIRE, and LTbR was normalized to GAPDH. Data shown are mean 6 SE for n = 5 (WT) or n = 4 (CD28/ CD40L K0) independent measurements. For each independent measurement, RNA was prepared from mTEC sorted from a minimum of four pooled thymuses per strain. All mice were 2 wk of age (p = 0.1 for CCL21, **p , 0.01, ****p , 0.0001). gand, as evidenced by increased CD5 levels on DO11 tg+ in to those found in DO11tgneg H2b3d mice, whereas numbers of comparison with DO11tgneg CD4+ SP thymocytes in I-Ab– mTEC are profoundly reduced in DO11tg+ H2d mice compared expressing H2b3d mice (29, 30), consistent with results shown with DO11tgneg H2d mice. These results are consistent with in Fig. 5A (left panel). The lower affinity of the DO11 tg for H-2d an enhanced ability of high-affinity TCR to mediate the robust ligand is reflected in the reduced CD5 levels of DO11 tg+ T cells interactions required to support mTEC development (Fig. 5B). in H2d mice (Fig. 5A, right panel). When we examined the thymic Interestingly, we found that generation of the mTEC compartment epithelial compartment of DO11tg+ H2b3d mice, we observed no even in the presence of high-affinity TCR is strongly dependent on significant difference in the numbers of mTEC recovered relative CD40–CD40L and CD28–CD80/86 costimulatory interactions, as The Journal of Immunology 635

that defects in mTEC development were not the result of decreased SP thymocytes (Fig. 5B). Thus, the requirement for CD40–CD40L and CD28–CD80/86 costimulatory interactions is stringently maintained even in the presence of a high-affinity TCR–ligand interaction. Altered thymocyte subpopulations in CD40/CD80/86 KO mice: increased CD4 SP cells and severe reductions of Treg and invariant NKT cell subpopulations As shown above in Fig. 2C, the proportion and number of CD4 SP thymocytes were increased in CD40 KO and CD80/86 KO mice, FIGURE 4. Number of mTEC in CD40/LTbR KO mice is reduced and were further increased in CD40/CD80/86 KO mice relative to relative to LTbR KO mice. Thymic stromal cells from 3- to 5-wk-old mice WT, CD40 KO, and CD80/86 KOs, whereas numbers of CD8 SP were prepared, as described in Materials and Methods, and stained with thymocytes were not significantly different. The significant changes neg anti-CD45, UEA-1, anti-Ly51, and anti–I-A/I-E. After gating on CD45 , in the thymic medullary compartment of CD40/CD80/86 KO mice + + neg + I-A/I-E cells, numbers of mTEC (UEA ), and cTEC (UEA , Ly51 ) prompted us to further characterize the thymocyte subpopulations 6 were calculated for each group. Data shown are mean SE for $3 mice present in these animals. per strain (*p , 0.05, **p , 0.01, ****p , 0.0001; ns, not significant). Using a CD1 tetramer to identify thymic invariant NKT (iNKT)

cells, we observed a significant reduction in these cells in CD80/ Downloaded from mTEC numbers in DO11tg+ CD40/CD80/86 KO H2b3d mice are 86 KO mice, as previously reported (31, 32), and, interestingly, dramatically reduced relative to DO11tg+ WT H2b3d mice. No- also found that the absence of CD40 was accompanied by a sig- tably, the number of CD4 SP thymocytes in DO11tg+ CD40/ nificant decrease in thymic iNKTs, a finding, to our knowledge, CD80/86 KO H2b3d mice was not reduced, and in fact was sig- not previously reported (Fig. 6). A significantly more profound nificantly increased, in costimulation-deficient mice, indicating decrease of iNKT cells occurred in CD40/CD80/86 KO mice. http://www.jimmunol.org/

FIGURE 5. High-affinity TCR– MHC interactions are not sufficient to overcome requirement for costimulation. (A)ThymocytesfromCB6F1 DO11tgneg (H2dxb), CB6F1 DO11 TCRtg+ (H2dxb), BALB DO11tgneg by guest on September 28, 2021 (H2d), and BALB DO11 TCR tg+ (H2d) mice were stained with anti- CD4, anti-CD8, KJ126 (DO11 TCRtg+ samples only), and anti-CD5 and analyzed by flow cytometry. Histo- grams show CD5 expression on gated CD4 SP thymocytes from CB6F1 DO11tgneg (H2d3b) and BALB DO11tgneg (H2d) and KJ126+ CD4 SP thymocytes from CB6F1 DO11 TCRtg+ (H2d3b) and BALB DO11 TCR tg+(H2d)mice.(B) Preparations of thymic stromal cells from costimulation-intact as well as CD40/CD80/86- deficient CB6F1 DO11tgneg (H2d3b), CB6F1 DO11 TCRtg+ (H2d3b), BALB DO11 TCR tgneg (H2d), and BALB DO11 TCR tg+ (H2d)mice were stained with anti-CD45, UEA-1, anti-Ly51, and anti–I-A/I-E to identify mTEC (CD45neg, I-A/I-E+,UEA+) and cTEC (CD45neg, I-A/I-E+, UEAneg, Ly51+). Thymocytes were stained with anti-CD4 and anti-CD8 to identify CD4 SP cells. Numbers of mTEC, cTEC, and CD4 SP thymocytes for each strain are shown. Bar graphs show the mean 6 SE for $4 mice per strain (*p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001). 636 THYMIC mTEC AND SELF-TOLERANCE REQUIRE CD80/86 AND CD40

FIGURE 6. Changes in number (left panels) and frequency (right panels) of thymocyte subpopulations in the absence of costimulatory interactions. Thymocytes from 4-wk-old BALB WT, CD80/86 KO, CD40 KO, and CD40/CD80/86 KO mice were stained with anti-CD3 and CD1- PBS-57 tetramers to identify iNKT cells (top panels), or anti-CD4, anti- CD8, and anti-Foxp3 to identify Treg cells (bottom panels), and analyzed by ﬂow cytometry. Bar graphs show the mean 6 SE for four to nine mice per strain (*p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001). Downloaded from

CD4+Foxp3+ (Treg) thymocytes were clearly decreased in the upon expression of CD40 and CD80/86 costimulatory ligands on absence of CD80/86, as previously reported (33, 34) (Fig. 6). Also APC, it was possible that the absence of in vivo manifestations of http://www.jimmunol.org/ consistent with previous reports, we found a decrease in the fre- autoreactivity in CD40/CD80/86 KO mice was due to the absence quency of CD4+ Treg thymocytes in CD40 KO mice (35, 36), but of CD40–CD40L and CD28–CD80/86 costimulatory interactions we did not see a decrease in absolute Treg numbers. Notably, required to drive an autoimmune response. To overcome the ab- however, both Treg cell frequency and number were decreased in sence of costimulatory interactions and to test whether the auto- CD40/CD80/86 KO relative to WT, CD40 KO, and even CD80/86 reactive response displayed by CD40/CD80/86 KO thymocytes KOs (Fig. 6). Thus, iNKT and Treg thymic populations are de- in vitro would be manifest in vivo, thymocytes from BALB WT, pendent on both CD28–CD80/86 and CD40 signaling. CD40 KO, CD80/86 KO, and CD40/CD80/86 KO mice were adoptively transferred to congenic athymic BALB nude CD40+/

CD40/CD80/86 KO thymocytes are autoreactive by guest on September 28, 2021 CD80/86+ recipients. Transfer of CD40/CD80/86 KO thymocytes The increase in CD4 SP thymocytes observed in CD40/CD80/86 resulted in death of the majority of recipient mice by 2 mo KO mice suggested that tolerance induction via clonal deletion posttransfer, a time point at which no deaths had occurred in may not be occurring normally in these mice. The deficiency in recipients of BALB WT, CD40 KO, or CD80/86 KO thymocytes regulatory iNKT cells and Treg further suggested that defects in (Fig. 8A). Detailed histological examination revealed no consistent tolerance might occur in this setting. To determine whether these cause of death, but striking lymphadenopathy and splenomegaly defects in CD40/CD80/86 KO mice result in the appearance of were significantly more pronounced in recipients of CD40/CD80/ an autoreactive T cell repertoire, we first examined the TCR Vb 86 KO thymocytes, and splenic CD8 T cells from these mice were repertoire of the BALB CD40/CD80/86 KO to assess endogenous significantly increased in numbers relative to BALB nudes re- superantigen-mediated negative selection. Superantigen-mediated ceiving WT, CD80/86 KO, or CD40 KO thymocytes (Fig. 8B). In deletion of CD4 SP cells expressing Vb5, Vb11, and Vb12 was addition, the proportion of CD69+ CD4 and CD8 T cells was severely compromised in BALB CD40 KO, consistent with pre- significantly higher in spleens of recipients of CD40/CD80/86 KO vious reports (Supplemental Fig. 3) (10, 11). BALB CD40/CD80/ thymocytes relative to recipients of individual KO or WT thy- 86 KO mice exhibited a similarly severe impairment. We next mocytes. Taken together, the autoproliferative phenotype observed assessed self-reactivity in MLCs with responder CD4-enriched in the BALB CD40/CD80/86 KO thymocytes in vitro and the high thymocytes derived from BALB WT, CD40 KO, CD80/86 KO, mortality rates accompanied by significant T cell activation ob- or CD40/CD80/86 KO animals and syngeneic BALB WT APCs or served in in vivo transfer indicate that there has been a breakdown allogeneic B6 WT APCs. CD4-enriched thymocytes from BALB of functional self-tolerance in the CD40/CD80/86 KO thymus. WT mice did not proliferate in response to BALB APC (Fig. 7A). Strikingly, in contrast to the limited magnitude of syngeneic re- Discussion sponses of CD40KO or CD80/86 KO thymocytes, CD40/CD80/86 In the studies reported in this work, we found that simultaneous KO thymocytes generated a much stronger syngeneic response. All disruption of CD28–CD80/86 and CD40–CD40L costimulatory four BALB background strains responded vigorously to allogeneic pathways results in a profound defect in mTEC development that B6 APCs. CD40/CD80/86 KO CD4 thymocytes failed to generate is as severe as that which results from absence of all SP thymo- a proliferative response to BALB CD40 KO or CD40/CD80/86 KO cytes. Moreover, CD4 SP thymocytes that matured in the altered splenic APCs and mounted a much reduced response to CD80/86 thymic epithelial environment of CD40/CD80/86 KO mice were KO APCs, indicating that both CD40–CD40L and CD80/86-CD28 highly autoreactive in in vitro proliferative responses and were interactions are required for this syngeneic response (Fig. 7B). lethal when adoptively transferred to congenic athymic recipients CD40/CD80/86 KO mice appeared healthy, with no apparent in vivo. Collectively, these findings demonstrate a critical and signs of overt autoimmunity, despite the in vitro autoreactivity of cooperative role for these costimulatory pathways in the bidirec- these cells. However, because in vitro autoreactivity was dependent tional interactions between medullary epithelium and developing The Journal of Immunology 637

FIGURE 7. CD40/CD80/86 KO thymocytes proliferate in syngeneic MLRs in a costimulation-dependent manner. (A) CD8-depleted thymocytes from BALB WT, CD80/86 KO, CD40 KO, and CD40/CD80/86 KO mice were cul- Downloaded from tured with T cell–depleted BALB or B6 WT splenocytes for 72 h prior to addition of [3H]thymidine for 16 h. Results shown are means of triplicate wells 6 SE and are representative of eight independent experiments. (B) CD8- depleted BALB CD40/CD80/86 KO thymo- http://www.jimmunol.org/ cytes were cultured with T-depleted BALB WT, CD80/86 KO, CD40 KO, and CD40/CD80/86 KO splenocytes for 72 h prior to addition of [3H]thymidine for 16 h. Results shown represent means of triplicate wells 6 SE and are representative of two independent experiments. by guest on September 28, 2021

thymocytes that are critical for self-tolerance as well as thymic detectable until the appearance of CD4 SP thymocytes (40) and is epithelial development. not detectable on LTi cells (41). Although CD28 is expressed on Consistent with previous reports, we observed that disruption of late double-negative and double-positive (DP) thymocytes, it is the CD40–CD40L pathway alone resulted in a significant defect in unlikely that these immature thymocytes would have access to or mTEC development, but one that was far less severe than that in mice interact with CD80/86-expressing mTEC or thymic dendritic cells deficient in both CD40–CD40L and CD28–CD80/86. We found no that are primarily localized to the medulla (42). The migration effect of disruption of CD28–CD80/86, although a recent report of developing thymocytes to the medulla occurs only once de- employing an elegant technique of three-dimensional reconstruction veloping DP thymocytes receive a positive selection signal and the did identify an effect of CD80/86 deletion on mTEC (37). chemokine receptor, CCR7, is upregulated, allowing positively Interestingly, the deficit that we observed in CD40/CD80/86 KO selected thymocytes to migrate toward CCL19/21-producing mTEC mTEC numbers was not evident in newborn mice and did not (43). Thus, CD80/86–CD28 and CD40–CD40L interactions are become apparent until ∼day 6 after birth, following the appearance expected to become critical components of the circuitry that pro- of SP thymocytes. In the embryonic thymus, mTEC are critically motes mTEC development only after CD4 SP thymocytes begin to dependent on RANKL-expressing LTi cells (38) and Vg5 gd T appear, precisely as we observed when tracking mTEC numbers at cells (39). Our data suggest that this early LTi-dependent mTEC birth and shortly thereafter. development is occurring normally in the CD40/CD80/86 KO mTEC express a number of TNFR family members, including mice, with defects in the mTEC compartment only being manifest CD40, LTbR,andRANK(6).AfeaturesharedbytheTNFR in 5- to 6-d-old thymus. Expression of CD40L in the thymus is not family members expressed on mTEC is the capacity to activate 638 THYMIC mTEC AND SELF-TOLERANCE REQUIRE CD80/86 AND CD40

FIGURE 8. Transfer of CD40/80/86 KO thymocytes into syngeneic BALB nu/nu mice results in rapid death. Total thymocytes were puriﬁed from BALB WT, CD80/86 KO, CD40 KO, and CD40/ 80/86 KO mice, and 1 3 107 (13)or 0.5 3 107 (0.53) cells were transferred i.v. to BALB nu/nu recipient mice. (A) Mice were monitored daily for 13.5 wk,

and the proportion of surviving mice is Downloaded from shown plotted against days posttransfer. Fifteen to 20 mice were included in each group. (B) Splenomegaly and increased numbers of activated CD8 T cells in BALB nu/nu mice receiving CD40/CD80/86 KO thymocytes. Total thymocytes were puri-

ﬁed from BALB WT, CD80/86 KO, http://www.jimmunol.org/ CD40 KO, and CD40/80/86 KO mice and transferred i.v. to BALB nu/nu recipient mice as in (A). Single-cell suspensions were prepared from spleens harvested from BALB nu/nu recipient mice 4–6.5 wk after receiving thymocytes and stained with anti-CD4, anti-CD8, and anti-CD69. Data shown are means 6 SE for WT (n = 9), CD80/86 KO (n = 6), CD40 KO (n = 5), and CD40/CD80/86 KO (n = 17). A by guest on September 28, 2021 total of 1 3 107 thymocytes was transferred for WT, CD80/86 KO, and CD40 KO, whereas the data for the CD40/CD80/ 86 KO transfers represent both 13 (1 3 107)and0.53 (0.5 3 107) cells transferred (*p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001).

RelB (44–46), a critical component of the nonclassical NF-kB partment defects of varying severities (6, 25, 38, 40, 41). Con- pathway. The importance of this pathway in mediating develop- sistent with recent reports (37, 52), we found that expression of ment of a normal mTEC compartment is evidenced by the absence LTab on CD4 SP thymocytes is decreased in the absence of of UEA+ medullary epithelial cells in mice deficient for crucial CD28–CD80/86 interactions. Our finding that LTbR/CD40 com- components involved in the alternative NF-kB pathway, including bined KO mice have a more profound mTEC defect than either the RelB, IKKa, and NF-kB–inducing kinase (47–51). The mecha- LTbR or CD40 single KOs suggests that CD28–CD80/86 coop- nism(s) by which SP thymocytes promote mTEC development has erates with CD40–CD40L interactions to promote mTEC develop- been shown to include important roles for members of the TNFR ment, in part via increased LTab production by CD4 SP thymocytes. family ligands. In particular, CD40L, RANKL, and LTab have all These findings are consistent with the recent report that combined been shown to be upregulated on SP thymocytes relative to double- stimulation of 2-dGUO–treated thymic lobes with agonist anti- negative and DP thymocytes (6); and mice lacking RANK–RANKL, LTbR Ab and CD40L synergized in the in vitro induction of mTEC LTab–LTbR, or CD40–CD40L interactions have mTEC com- (52), importantly extending these findings by demonstrating that The Journal of Immunology 639

LTbR and CD40L pathways in fact interact cooperatively during References in vivo thymic development. Furthermore, we found that RANK 1. Heino, M., P. Peterson, J. Kudoh, K. Nagamine, A. Lagerstedt, V. Ovod, expression is decreased on mTEC in mice deficient for both A. Ranki, I. Rantala, M. Nieminen, J. Tuukkanen, et al. 1999. Autoimmune regulator is expressed in the cells regulating immune tolerance in thymus me- CD40–CD40L and CD28–CD80/86 interactions, possibly a re- dulla. Biochem. Biophys. Res. Commun. 257: 821–825. flection of the decrease in LTab observed in the absence of CD28 2. Derbinski, J., A. Schulte, B. Kyewski, and L. Klein. 2001. Promiscuous gene signals (37, 52). Thus, in the CD40/CD80/86 KO thymus, RANK– expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2: 1032–1039. RANKL, LTab–LTbR, and CD40–CD40L interactions, all shown 3. Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. P. Berzins, S. J. Turley, to cooperate in promoting development of mTEC, are either sig- H. von Boehmer, R. Bronson, A. Dierich, C. Benoist, and D. Mathis. 2002. Projection of an immunological self shadow within the thymus by the aire nificantly reduced (RANK–RANKL and LTab–LTbR) or elimi- protein. Science 298: 1395–1401. nated (CD40–CD40L), providing a basis for explaining the dramatic 4. Shores, E. W., W. Van Ewijk, and A. Singer. 1991. Disorganization and resto- reduction in mTEC numbers we observe in these mice. ration of thymic medullary epithelial cells in T cell receptor-negative scid mice: evidence that receptor-bearing lymphocytes influence maturation of the thymic Strong in vitro and lethal in vivo autoreactivity was observed in microenvironment. Eur. J. Immunol. 21: 1657–1661. CD4 SP thymocytes that develop in CD40/CD80/86 KO thymus. A 5. Surh, C. D., B. Ernst, and J. Sprent. 1992. Growth of epithelial cells in the number of factors may contribute to the observed failure to induce thymic medulla is under the control of mature T cells. J. Exp. Med. 176: 611– 616. self-tolerance in the absence of these costimulatory pathways. 6. Hikosaka, Y., T. Nitta, I. Ohigashi, K. Yano, N. Ishimaru, Y. Hayashi, Autoreactivity may result, at least in part, from the defective thymic M. Matsumoto, K. Matsuo, J. M. Penninger, H. Takayanagi, et al. 2008. The cytokine RANKL produced by positively selected thymocytes fosters medullary medullary epithelial compartment found in these costimulation- thymic epithelial cells that express autoimmune regulator. Immunity 29: 438– deficient animals, as defective tolerance induction has been shown 450. to accompany disruption of the mTEC compartment. RelB-, Traf6-, 7. Irla, M., S. Hugues, J. Gill, T. Nitta, Y. Hikosaka, I. R. Williams, F. X. Hubert, Downloaded from H. S. Scott, Y. Takahama, G. A. Holla¨nder, and W. Reith. 2008. Autoantigen- Bcl3/NFkB2-, and CD40/RANK-deficient mice all provide exam- specific interactions with CD4+ thymocytes control mature medullary thymic ples of this; in each of these cases, severe defects in development of epithelial cell cellularity. Immunity 29: 451–463. the mTEC compartment are coupled with an autoreactive T cell 8. Punt, J. A., B. A. Osborne, Y. Takahama, S. O. Sharrow, and A. Singer. 1994. Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apo- repertoire (40, 47, 48, 53, 54). In the RelB-, Traf6-, and Bcl3/NF- ptosis requires a costimulatory signal that can be provided by CD28. J. Exp. kB2–deficient animals, clear signs of autoreactivity were present in Med. 179: 709–713. 9. Pobezinsky, L. A., G. S. Angelov, X. Tai, S. Jeurling, F. Van Laethem, the knockouts themselves, whereas, in the CD40/RANK-deficient L. Feigenbaum, J. H. Park, and A. Singer. 2012. Clonal deletion and the fate of http://www.jimmunol.org/ mice, similar to our CD40/CD80/86 KOs, autoreactivity was re- autoreactive thymocytes that survive negative selection. Nat. Immunol. 13: 569– vealed only when the T cells were placed in an environment in 578. 10. Foy, T. M., D. M. Page, T. J. Waldschmidt, A. Schoneveld, J. D. Laman, which the ligands for T cell activation (i.e., CD40L and CD80/86) S. R. Masters, L. Tygrett, J. A. Ledbetter, A. Aruffo, E. Claassen, et al. 1995. An were supplied (40). However, although the severe disruption of essential role for gp39, the ligand for CD40, in thymic selection. J. Exp. Med. the thymic medullary compartment may have a role in allow- 182: 1377–1388. 11. Williams, J. A., S. O. Sharrow, A. J. Adams, and R. J. Hodes. 2002. CD40 ligand ing self-reactive thymocytes to develop in the CD40/CD80/86 functions non-cell autonomously to promote deletion of self-reactive thymo- KOs, interactions between the costimulatory receptor–ligand pairs cytes. J. Immunol. 168: 2759–2765. 12. Fu¨tterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, and K. Pfeffer. 1998. The CD28–CD80/CD86 and CD40–CD40L have additional roles in lymphotoxin beta receptor controls organogenesis and affinity maturation in shaping the repertoire of developing thymocytes that could, in peripheral lymphoid tissues. Immunity 9: 59–70. by guest on September 28, 2021 their absence, lead to the emergence of autoreactive thymocytes. 13. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250: CD40–CD40L interactions play an essential role in negative 1720–1723. selection of autoreactive thymocytes (10, 11), whereas CD28– 14. Philpott, K. L., J. L. Viney, G. Kay, S. Rastan, E. M. Gardiner, S. Chae, CD80/CD86 interactions are required for thymic development of A. C. Hayday, and M. J. Owen. 1992. Lymphoid development in mice con- + genitally lacking T cell receptor alpha beta-expressing cells. Science 256: 1448– Foxp3 Treg cells (33, 34) as well as a reported role in negative 1452. selection (8, 19). It is therefore possible that the failure to ap- 15. Gray, D. H., A. L. Fletcher, M. Hammett, N. Seach, T. Ueno, L. F. Young, J. Barbuto, R. L. Boyd, and A. P. Chidgey. 2008. Unbiased analysis, enrichment propriately tolerize developing thymocytes in CD40/CD80/86 KO and purification of thymic stromal cells. J. Immunol. Methods 329: 56–66. mice is multifaceted and reflects a failure of negative selection 16. Hubert, F. X., S. A. Kinkel, K. E. Webster, P. Cannon, P. E. Crewther, compounded by a deficit in mTEC and the absence of a Treg cell A. I. Proeitto, L. Wu, W. R. Heath, and H. S. Scott. 2008. A specific anti-Aire antibody reveals aire expression is restricted to medullary thymic epithelial cells population. and not expressed in periphery. J. Immunol. 180: 3824–3832. The results reported in this work demonstrate that the effects of 17. Aschenbrenner, K., L. M. D’Cruz, E. H. Vollmann, M. Hinterberger, CD40–CD40L and CD28–CD80/86 interactions in the thymus are J. Emmerich, L. K. Swee, A. Rolink, and L. Klein. 2007. Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ bidirectional and influence both CD28- and CD40L-expressing medullary thymic epithelial cells. Nat. Immunol. 8: 351–358. T cells and the CD80/86- and CD40-expressing cells with which 18. Lei, Y., A. M. Ripen, N. Ishimaru, I. Ohigashi, T. Nagasawa, L. T. Jeker, M. R. Bo¨sl, G. A. Holla¨nder, Y. Hayashi, Rde. W. Malefyt, et al. 2011. Aire- these T cells interact; in the absence of these costimulatory path- dependent production of XCL1 mediates medullary accumulation of thymic ways, a normal mTEC compartment fails to be formed and auto- dendritic cells and contributes to regulatory T cell development. J. Exp. Med. reactive thymocytes are allowed to persist. Thus, these costimulatory 208: 383–394. 19. Noel, P. J., M. L. Alegre, S. L. Reiner, and C. B. Thompson. 1998. Impaired molecules that are vital components in the dialogue that occurs negative selection in CD28-deficient mice. Cell. Immunol. 187: 131–138. between T cells and the APCs with which they interact in the pe- 20. Vacchio, M. S., J. A. Williams, and R. J. Hodes. 2005. A novel role for CD28 in riphery are also critical elements of the crosstalk that shapes the thymic selection: elimination of CD28/B7 interactions increases positive selection. Eur. J. Immunol. 35: 418–427. development of thymocytes and the epithelial cells of the thymus. 21. Xu, J., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Dunn, T. J. Waldschmidt, J. Elsemore, R. J. Noelle, and R. A. Flavell. 1994. Mice deficient for the CD40 ligand. Immunity 1: 423–431. Acknowledgments 22. Mathis, D., and C. Benoist. 2009. Aire. Annu. Rev. Immunol. 27: 287–312. We dedicate this paper to the memory of Dr. David Klug. David’s intense 23. Nitta, T., I. Ohigashi, Y. Nakagawa, and Y. Takahama. 2011. Cytokine crosstalk intellectual curiosity, keen insight, and infectious enthusiasm were an for thymic medulla formation. Curr. Opin. Immunol. 23: 190–197. inspiration to all colleagues. We thank Dr. Alfred Singer and Karen 24. Seach, N., T. Ueno, A. L. Fletcher, T. Lowen, M. Mattesich, C. R. Engwerda, H. S. Scott, C. F. Ware, A. P. Chidgey, D. H. Gray, and R. L. Boyd. 2008. The Hathcock for critical reading of the manuscript. lymphotoxin pathway regulates Aire-independent expression of ectopic genes and chemokines in thymic stromal cells. J. Immunol. 180: 5384–5392. 25. Boehm, T., S. Scheu, K. Pfeffer, and C. C. Bleul. 2003. Thymic medullary epithelial Disclosures cell differentiation, thymocyte emigration, and the control of autoimmunity re- The authors have no financial conflicts of interest. quire lympho-epithelial cross talk via LTbetaR. J. Exp. Med. 198: 757–769. 640 THYMIC mTEC AND SELF-TOLERANCE REQUIRE CD80/86 AND CD40

26. Mouri, Y., M. Yano, M. Shinzawa, Y. Shimo, F. Hirota, Y. Nishikawa, T. Nii, 41. White, A. J., D. R. Withers, S. M. Parnell, H. S. Scott, D. Finke, P. J. Lane, H. Kiyonari, T. Abe, H. Uehara, et al. 2011. Lymphotoxin signal promotes E. J. Jenkinson, and G. Anderson. 2008. Sequential phases in the development of thymic organogenesis by eliciting RANK expression in the embryonic thymic Aire-expressing medullary thymic epithelial cells involve distinct cellular input. stroma. J. Immunol. 186: 5047–5057. Eur. J. Immunol. 38: 942–947. 27. Viola, A., and A. Lanzavecchia. 1996. T cell activation determined by T cell 42. Williams, J. A., K. S. Hathcock, D. Klug, Y. Harada, B. Choudhury, J. P. Allison, receptor number and tunable thresholds. Science 273: 104–106. R. Abe, and R. J. Hodes. 2005. Regulated costimulation in the thymus is critical 28. Lumsden, J. M., S. J. Prasad, R. J. Peach, and F. Ronchese. 2003. The effects of for T cell development: dysregulated CD28 costimulation can bypass the pre- B7-dependent costimulation on T cell division and survival in vivo and in vitro TCR checkpoint. J. Immunol. 175: 4199–4207. are dependent on antigen concentration. Eur. J. Immunol. 33: 2074–2082. 43.Ueno,T.,F.Saito,D.H.Gray,S.Kuse,K.Hieshima,H.Nakano, 29. Love, P. E., J. Lee, and E. W. Shores. 2000. Critical relationship between TCR T. Kakiuchi, M. Lipp, R. L. Boyd, and Y. Takahama. 2004. CCR7 signals are signaling potential and TCR afﬁnity during thymocyte selection. J. Immunol. essential for cortex-medulla migration of developing thymocytes. J. Exp. 165: 3080–3087. Med. 200: 493–505. 30. Liu, C. P., J. W. Kappler, and P. Marrack. 1996. Thymocytes can become mature 44. Coope, H. J., P. G. Atkinson, B. Huhse, M. Belich, J. Janzen, M. J. Holman, T cells without passing through the CD4+ CD8+, double-positive stage. J. Exp. G. G. Klaus, L. H. Johnston, and S. C. Ley. 2002. CD40 regulates the processing Med. 184: 1619–1630. of NF-kappaB2 p100 to p52. EMBO J. 21: 5375–5385. 31. Zheng, X., H. Zhang, L. Yin, C. R. Wang, Y. Liu, and P. Zheng. 2008. Modu- 45. Novack, D. V., L. Yin, A. Hagen-Stapleton, R. D. Schreiber, D. V. Goeddel, lation of NKT cell development by B7-CD28 interaction: an expanding horizon F. P. Ross, and S. L. Teitelbaum. 2003. The IkappaB function of NF-kappaB2 for costimulation. PLoS One 3: e2703. p100 controls stimulated osteoclastogenesis. J. Exp. Med. 198: 771–781. 32. Williams, J. A., J. M. Lumsden, X. Yu, L. Feigenbaum, J. Zhang, 46. Dejardin, E., N. M. Droin, M. Delhase, E. Haas, Y. Cao, C. Makris, Z. W. Li, S. M. Steinberg, and R. J. Hodes. 2008. Regulation of thymic NKT cell devel- M. Karin, C. F. Ware, and D. R. Green. 2002. The lymphotoxin-beta receptor opment by the B7-CD28 costimulatory pathway. J. Immunol. 181: 907–917. induces different patterns of gene expression via two NF-kappaB pathways. 33. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, and Immunity 17: 525–535. J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of 47. Burkly, L., C. Hession, L. Ogata, C. Reilly, L. A. Marconi, D. Olson, R. Tizard, the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. R. Cate, and D. Lo. 1995. Expression of relB is required for the development of Immunity 12: 431–440. thymic medulla and dendritic cells. Nature 373: 531–536. 34. Tai, X., M. Cowan, L. Feigenbaum, and A. Singer. 2005. CD28 costimulation of 48. Weih, F., D. Carrasco, S. K. Durham, D. S. Barton, C. A. Rizzo, R. P. Ryseck, Downloaded from developing thymocytes induces Foxp3 expression and regulatory T cell differ- S. A. Lira, and R. Bravo. 1995. Multiorgan inﬂammation and hematopoietic entiation independently of interleukin 2. Nat. Immunol. 6: 152–162. abnormalities in mice with a targeted disruption of RelB, a member of the NF- 35. Guiducci, C., B. Valzasina, H. Dislich, and M. P. Colombo. 2005. CD40/CD40L kappa B/Rel family. Cell 80: 331–340. interaction regulates CD4+CD25+ T reg homeostasis through dendritic cell- 49. Lomada, D., B. Liu, L. Coghlan, Y. Hu, and E. R. Richie. 2007. Thymus medulla produced IL-2. Eur. J. Immunol. 35: 557–567. formation and central tolerance are restored in IKKalpha2/2 mice that express an 36. Spence, P. J., and E. A. Green. 2008. Foxp3+ regulatory T cells promiscuously IKKalpha transgene in keratin 5+ thymic epithelial cells. J. Immunol. 178: 829– accept thymic signals critical for their development. Proc. Natl. Acad. Sci. USA 837.

105: 973–978. 50. Kinoshita, D., F. Hirota, T. Kaisho, M. Kasai, K. Izumi, Y. Bando, Y. Mouri, http://www.jimmunol.org/ 37. Irla, M., J. Guenot, G. Sealy, W. Reith, B. A. Imhof, and A. Serge´. 2013. Three- A. Matsushima, S. Niki, H. Han, et al. 2006. Essential role of IkappaB kinase dimensional visualization of the mouse thymus organization in health and im- alpha in thymic organogenesis required for the establishment of self-tolerance. J. munodeﬁciency. J. Immunol. 190: 586–596. Immunol. 176: 3995–4002. 38. Rossi, S. W., M. Y. Kim, A. Leibbrandt, S. M. Parnell, W. E. Jenkinson, 51. Kajiura, F., S. Sun, T. Nomura, K. Izumi, T. Ueno, Y. Bando, N. Kuroda, S. H. Glanville, F. M. McConnell, H. S. Scott, J. M. Penninger, E. J. Jenkinson, H. Han, Y. Li, A. Matsushima, et al. 2004. NF-kappa B-inducing kinase et al. 2007. RANK signals from CD4(+)3(2) inducer cells regulate development establishes self-tolerance in a thymic stroma-dependent manner. J. Immunol. of Aire-expressing epithelial cells in the thymic medulla. J. Exp. Med. 204: 172: 2067–2075. 1267–1272. 52. Irla, M., L. Guerri, J. Guenot, A. Serge´, O. Lantz, A. Liston, B. A. Imhof, 39. Roberts, N. A., A. J. White, W. E. Jenkinson, G. Turchinovich, K. Nakamura, E. Palmer, and W. Reith. 2012. Antigen recognition by autoreactive CD4+ thy- D. R. Withers, F. M. McConnell, G. E. Desanti, C. Benezech, S. M. Parnell, et al. mocytes drives homeostasis of the thymic medulla. PLoS One 7: e52591. 2012. Rank signaling links the development of invariant gd T cell progenitors 53. Akiyama, T., S. Maeda, S. Yamane, K. Ogino, M. Kasai, F. Kajiura,

and Aire(+) medullary epithelium. Immunity 36: 427–437. M. Matsumoto, and J. Inoue. 2005. Dependence of self-tolerance on TRAF6- by guest on September 28, 2021 40. Akiyama, T., Y. Shimo, H. Yanai, J. Qin, D. Ohshima, Y. Maruyama, Y. Asaumi, directed development of thymic stroma. Science 308: 248–251. J. Kitazawa, H. Takayanagi, J. M. Penninger, et al. 2008. The tumor necrosis 54. Zhang, X., H. Wang, E. Claudio, K. Brown, and U. Siebenlist. 2007. A role for factor family receptors RANK and CD40 cooperatively establish the thymic the IkappaB family member Bcl-3 in the control of central immunologic toler- medullary microenvironment and self-tolerance. Immunity 29: 423–437. ance. Immunity 27: 438–452.