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The Journal of Immunology, 2007, 179, 8069 -8075
Copyright © 2007 by The American Association of Immunologists, Inc.
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Lymphotoxin β Receptor Is Required for the Migration and Selection of Autoreactive T Cells in Thymic Medulla1

Mingzhao Zhu, Robert K. Chin, Alexei V. Tumanov, Xiaojuan Liu and Yang-Xin Fu2

Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
 Disclosures
 References
 
How organ-specific central tolerance is established and regulated has been an intriguing question. Lymphotoxin β receptor (LTβR) deficiency is associated with autoimmune phenotypes characterized by humoral and cellular autoreactivity to peripheral organs. Whether this results from defective negative selection of T cells directed at tissue-restricted Ags has not been well understood. By tracing the development of OT-I thymocytes in rat insulin 2 promoter-mOVA transgenic mice on either Ltbr+/+ or Ltbr–/– background, we demonstrate that LTβR is necessary for thymic negative selection. LTβR deficiency resulted in a dramatic escape of "neo-self" specific OT-I cells that persist in circulation and lead to development of peri-insulitis. When the underlying mechanism was further explored, we found interestingly that LTβR deficiency did not result in reduced thymic expression of mOVA. Instead, LTβR was revealed to control the expression of thymic medullary chemokines (secondary lymphoid tissue chemokine (SLC) and EBV-induced molecule 1 ligand chemokine (ELC)) which are required for thymocytes migration and selection in medulla. Furthermore, RIP-mOVA transgenic mice on SLC/ELC deficient background (plt) demonstrated significant impaired negative selection of OT-I cells, suggesting that the dysregulation of SLC/ELC- expression alone in Ltbr–/– thymi can be sufficient to impair thymic negative selection. Thus, LTβR has been revealed to play an important role in thymic negative selection of organ-specific thymocytes through thymic medullary chemokines regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note added in proof.
 Disclosures
 References
 
The thymus plays an essential role in facilitating T cell self-tolerance by organizing negative selection and generating regulatory T cells (Treg).3 Although it is easy to understand how central tolerance to ubiquitous self-Ags is established in the thymus, how these same mechanisms might forestall autoimmunity against peripheral tissue-specific Ags remained a mystery, until the appearance of recent clues (1, 2). A lengthening list of tissue-restricted self-Ags (TRAs), including insulin, have been found ectopically expressed in medullary thymic epithelial cells (mTECs). The appropriate expression of TRAs and interaction between developing thymocytes and thymic APCs in thymic medulla are regarded important for the efficient negative selection of autoreactive T cells and the generation of regulatory T cells. Autoimmune regulator (Aire) was found clearly involved in the regulation of thymic expression of TRAs (2, 3, 4, 5). A few other molecules have also been suggested to take part in the regulation of central tolerance, including lymphotoxin β receptor (LTβR), NF-{kappa}B-induding kinase, TNFR-associated factor 6 (TRAF6), v-rel reticuloendotheliosis viral oncogene homolog B (Rel B), NF-{kappa}B2, and chemokine (c-c motif) receptor 7 (6, 7, 8, 9, 10, 11), although their roles have not been directly demonstrated. Clear definition of their functions in the organ-specific central tolerance will not only help to uncover the complex regulation of organ-specific autoimmune diseases, but also provide new targets for future diagnostic and therapeutic intervention of organ-specific autoimmune diseases.

LTβR-deficient mice harbor an autoimmune phenotype characterized by increased lymphocytic infiltration of peripheral organs and humoral autoreactivity (8, 12, 13). LTβR was recently proposed to be involved in the control of negative selection of organ-specific autoreactive thymocytes in both Aire-dependent and independent pathways (8, 12, 13). However, this hypothesis has not been formally tested and little is known about the underlying mechanisms. This issue becomes even more intriguing given the controversial data available at present. Although one study showed that LTβR signaling may be directly involved in the regulation of Aire expression (12), another study suggested an indirect mechanism via the development and differentiation of thymic medulla epithelial cells (13). A third study demonstrated apparently normal Aire expression in Lta–/– mice, in which the ligand for LTβR is deficient, raising the possibility that increased peripheral tissue infiltration is not related to the defect in Aire-dependent negative selection (11). Whether and how LTβR is involved in organ-specific thymic negative selection remains to be determined. In this study, we clarify and confirm the essential role of LTβR in the negative selection of organ-specific autoreactive T cells. Our data further suggests that LTβR controls this process by regulating medullary chemokine expression for cortex-to-medulla thymocyte migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note added in proof.
 Disclosures
 References
 
Mice

Ltbr–/– mice have been previously described (8). Aire–/– mice were generated with a mixed genetic background as described (14) and back-crossed to C57BL/6 for at least six generations in our laboratory. OT-I TCR transgenic mice on C57BL/6 background were purchased from The Jackson Laboratory. RIP-mOVA transgenic mice on C57BL/6 background were obtained from Dr. W. R. Heath, Division of Immunology (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). The plt mice were obtained from J. G. Cyster (University of California, San Francisco, CA). Animal care and experiments were performed in accordance with the institution and National Institutes of Health guidelines and approved by the animal use committee at the University of Chicago.

Bone marrow reconstitution and splenocytes adoptive transfer

Mice were lethally irradiated with 1000 rads and adoptively transferred i.v. with 3 x 106 bone marrow cells the next day. Bactrim was added to the drinking water for 3 wk following irradiation. Thymocytes and splenocytes were analyzed 8–9 wk after transfer and in some cases 12 wk after transfer. For splenocyte adoptive transfer experiments, splenocytes from bone marrow chimeric mice were recovered when mice were sacrificed and 20 x 106 splenocytes were transferred to sublethally (400 rad) irradiated rat insulin 2 promoter (RIP)-mOVA mice. Bactrim was applied for 3 wk following irradiation. Mice were sacrificed for pancreas histopathology analysis 4 wk after splenocytes adoptive transfer.

Immunofluoresence staining

Six-micron frozen sections were fixed in acetone at 4°C for 10 min, rehydrated in PBS/saponin (0.1%), and blocked with goat serum (5%) or BSA (1%) in PBS at room temperature for 1 h. Primary Abs include anti-Ep-CAM (G8.8; BD Biosciences), UEA-1-bio (Sigma-Aldrich), anti-CD8 (53-6.7; BD Biosciences), anti-CD11c (N418; BD Biosciences), anti-CD19 (1D3; BD Biosciences), anti-F4/80 (BM8; eBiosciences), and anti-insulin (abCAM). Tissue sections were incubated with primary Abs at 4°C overnight and visualized with appropriate fluorescence reagents.

Thymi morphometrics analysis

Mice were sublethally irradiated with 400 rad. Thymi sectioning and immunocluoresence staining were performed as described above. ImageJ 1.34 (National Institutes of Health) software was used to calculate the cortex and medulla areas. The medulla was identified as a continuous area with UEA1 positive staining. Because only rare CD4CD8 DP thymocytes were present in sublethally irradiated mice, CD8 SP thymocytes distribution and quantity were visualized by FITC-conjugated anti-CD8 Ab (53-6.7; BD Biosciences). CD8 SP cells in medulla/cortex were counted and their distribution density was calculated.

Flow cytometry analysis

Single cell suspensions from the thymus and spleen were stained with anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD24 (30-F1), anti-CD69 (H1.2F3) mAbs (BD Biosciences), and OVA Tetramer H-2 Kb SIINFEKL-PE (Beckman Coulter) in PBS containing 0.2% BSA and 0.09% sodium azide. Before staining, the cells were preincubated with anti-Fc{gamma}III/II receptor (2.4G2; BD Biosciences). Foxp3 intracellular staining was conducted using Mouse Regulatory T Cell Staining Kit following the manufacturer’s instructions (eBioscience). Stained cells were analyzed on FACSCanto (BD Biosciences). mTECs sorting was performed as previously described (6). The purity was routinely higher than 90%.

Real-time PCR

Real-time PCR was conducted on cDNA prepared from DNase I-treated RNA extracted from whole thymus or purified mTECs from 4- to 6-wk-old mice. The primers and probes used are as follows. For Insulin2: Forward, 5'-CTTCAGACCTTGGCGTTGGA-3', Reverse, 5'-ATGCTGGTGCAGCACTGATC-3', Probe, 5'-FAM-CCCGGCAGAAGCGTGGCATT-TAMRA-3. For mOVA: Forward, 5'-ATCTCAAGCTGTCCATGCAG-3', Reverse, 5'-TGCGATGTGCTTGATACAGA-3', Probe, 5'-FAM-CGCTTGCAGCATCCACTCCA-TAMRA-3'. For secondary lymphoid tissue chemokine (SLC): Forward, 5'-AGACTCAGGAGCCCAAAGCA-3', Reverse, 5'-GTTGAAGCAGGGCAAGGGT-3', Probe, 5'-FAM-CCACCTCATGCTGGCCTCCGTC-TAMRA-3'. For EBV-induced molecule 1 ligand chemokine (ELC): Forward, 5'-ATGCGGAAGACTGCTGCCT-3', Reverse, 5'-GGCTTTCACGATGTTCCCAG-3', Probe, 5'-FAM-TCTGTGACCCAGCGCCCCATC-TAMRA-3'. For GAPDH: Forward, 5'-TTCACCACCATGGAGAAGGC-3', Reverse, 5'-GGCATGGACTGTGGTCATGA-3' Probe, 5'-FAM-TGCATCCTGCACCACCAACTGCTTAG-TAMRA-3'. Reactions were run on the ABI/Prism 7300 (Applied Biosystems), in a final volume of 25 µl with 900 nM of the forward and reverse primers and 200 nM of the probe using 2x Taqman Master Mix (Applied Biosystems) containing AmpliTaq Gold polymerase. Cycling conditions were a single denaturing step at 95°C for 15 min followed by 45 cycles of 94°C for 15 s and 60°C for 1 min. Analysis of Ins2, mOVA, SLC, ELC, and Gapdh gene expression were performed with a standard curve, and then normalized to sample Gapdh. The standard curves had R2 values >0.99.

Histopathology

Tissues for histological examination were fixed in 10% buffered formalin and embedded in paraffin. Four- to five-micron sections were obtained from the paraffin blocks and stained using H&E stain methods. All sections were then examined by a pathologist in a blinded fashion.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note added in proof.
 Disclosures
 References
 
LTβR deficiency rescues autoreactive T cells from thymic negative selection

In mice with a native polyclonal TCR repertoire, the fate of autoreactive thymocytes can be difficult to trace. We thus took advantage of the OT-I/RIP-mOVA transgenic system to uncover the role of LTβR in regulating negative selection. In this system, mOVA expression is driven by RIP, mimicking an organ-specific self-Ag. OT-I thymocytes are routinely negatively selected in the thymi of RIP-mOVA transgenic mice (4, 15, 16). To determine whether LTβR controls the negative selection of organ-specific autoreactive thymocytes, we made bone marrow chimera with OT-I+ donor bone marrow transplanted into either RIP-mOVA+ or RIP-mOVA+Ltbr–/– hosts. The development of OT-I+ T cells was analyzed 8–9 wks later by flow cytometric staining with OT-I clonotype-specific tetramer and thymocyte maturation markers.

Following OT-I bone marrow transplantation, thymocytes in recipients wild type (WT) (Ltbr+/+) and Ltbr–/– developed normally and CD8+OT-I+ T cells were predominantly positively selected (Fig. 1A). In RIP-mOVA/Ltbr+/+ recipients of OT-I bone marrow transplantation, the CD8+ single positive (SP) population in the thymus was significantly reduced by 3-fold. Within this reduced CD8+ SP population, the percentage of OT-I+ clonotypic cells was even more severely decreased from around 77% in WT to 21% in RIP-mOVA+ mice (Fig. 1, A and B). This led to, in absolute terms, about a 10-fold reduction of CD8+ OT-I clonotypic T cells in the presence of thymic mOVA expression in the RIP-mOVA recipients, suggesting an efficient thymic negative selective process (Fig. 1, A and B and Refs. 4, 16). The further addition of LTβR deficiency (RIP-mOVA/Ltbr–/–), however, rescued 2.5-fold of OT-I+ clonotypic cells (Fig. 1, A and B). When CD24 was added as a marker to further define the postselection, fully mature OT-I cells, few CD24– OT-I cells were seen in the RIP-mOVA+Ltbr+/+ recipients. In contrast, analysis of RIP-mOVA+Ltbr–/– thymi revealed a 40-fold increase in postselection mature OT-I+ population (CD24 negative), apparently rescued by LTβR deficiency (Fig. 1, A and B). Dramatic rescue of mature OT-I+ cells by LTβR deficiency was also seen when CD69 was used as a maturation marker (Fig. 1, A and B). These data thus strongly suggested an important role of LTβR in the thymic negative selection of OT-I+ thymocytes.


Figure 1
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  		FIGURE 1. Autoreactive T cells are rescued in Ltbr–/– thymus. A, OT-I TCR transgenic bone marrow cells were depleted of mature T cells and transferred into lethally irradiated recipients mice as indicated in the figure. Eight to nine weeks later, OT-I thymocytes development was analyzed by flow cytometry. Thymocytes were stained with CD4, CD8, OT-I tetramer, and T cell maturation markers. Representative profiles from mice of indicated genotypes were shown. Data shown are mean ± SD for each group (n = 4–8). B, Total CD8 SP thymocyte numbers and proportion of indicated clonotypic thymocytes were analyzed. Mean ± SD (n = 4–8). **, p < 0.01; *, p < 0.05; NS, no significance. At least three independent experiments were conducted with similar results.

 
Normal CD4+Foxp3+ Treg development in Ltbr–/– thymus

Development of CD4+Foxp3+ Treg is another important mechanism in the maintenance of central tolerance. To determine whether Treg development was impaired in the absence of LTβR, thymocytes from WT and Ltbr–/– mice were first intracellular stained with Foxp3 and analyzed by flow cytometry. No differences in the percentage or the absolute number of CD4+Foxp3+ cells were found between WT and Ltbr–/– mice (Fig. 2, A and B). To confirm this finding, Foxp3 expression level was also analyzed by quantitative real-time PCR and found unchanged in the thymi of Ltbr–/– mice (Fig. 2C). The expression level of Foxp3 also found no difference by comparing the mean fluorescence intensity between WT and Ltbr–/– Treg cells (data not shown), indicating a normal Treg function (17). Therefore, it appears that LTβR deficiency does not significantly influence the development of conventional CD4+Foxp3+ Treg cells. Whether other regulatory T cell populations, e.g., Th1, Tr1, or even CD8+ regulatory T cells (18), are generated during thymic development and influenced by LTβR remains to be determined.


Figure 2
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  		FIGURE 2. Normal Treg development in Ltbr–/– thymus. A, Regulatory thymocytes were intracellularly stained with Foxp3. Representative profiles from indicated mice are shown. B, Percentage of CD4+Foxp3+ Tregs among CD4 SP thymocytes. Mean ± SEM (n = 3) p > 0.05. C, Real-time PCR analysis of relative Foxp3 gene expression in the Ltbr–/– thymus. Expression values are shown in arbitrary units and normalized relative to Gapdh. Data are mean ± SEM of duplicate, p > 0.05. Representative result from two independent experiments is shown.

 
LTβR deficiency leads to preserved ectopic mOVA expression but reduced medullary chemokine expression and defective migration

To further dissect how LTβR deficiency impinges on thymic negative selection, we first analyzed the ectopic thymic expression level of the neo-self Ag, as this is a crucial factor for thymocytes selection (4, 19, 20). Thymic expression levels of the neo-Ag mOVA were determined by quantitative real-time PCR. LTβR was shown to be necessary for thymic Aire expression, and Aire shown necessary for thymic insulin 2 expression. Given that the RIP-mOVA gene is driven by insulin 2 promoter, we expected reduced thymic mOVA expression in the absence of LTβR signaling. We found rather that expression levels of mOVA in the thymi of RIP-mOVA+Ltbr+/+ and RIP-mOVA+Ltbr–/– were equivalent (Fig. 3A). This is consistent with a recent study demonstrating that mOVA thymic expression is not reduced on Aire–/– background (4, 19, 20). However, the native insulin 2 expression was indeed reduced in RIP-mOVA+Ltbr–/– thymi (Fig. 3A). The RIP promoter’s independence from Aire control may be due to its deficiency in necessary cis-elements, either due to species specific differences or loss during cloning. Chromosomal integration-site effect on the RIP-driven expression of transgenes is a further possible explanation (4, 21).


Figure 3
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  		FIGURE 3. Cellular mechanisms of LTβR control thymic negative selection. A, Real-time PCR analysis of relative mOVA and Ins2 gene expression in RIP-mOVA+Ltbr–/– thymus. Expression values are shown in arbitrary units and normalized relative to Gapdh. Mean ± SD (n = 3). **, p < 0.01; *, p < 0.05; NS, no significance. B, Real-time PCR analysis of relative thymic SLC and ELC expression in Ltbr–/– mice. Expression values are shown in arbitrary units and normalized relative to Gapdh. Data shown are mean ± SD (n = 3). *, p < 0.05. C, Real-time PCR analysis of SLC and ELC expression in purified mTECs from WT and Ltbr–/– mice. Expression values are shown in arbitrary units and normalized relative to Gapdh. Data shown are mean ± SD (n = 3). *, p < 0.05. D, CD8 SP thymocytes localization in 400 rad irradiated mice. Red, UEA-1; Green, CD8. M, medulla; C, cortex. E, Mean ± SD (n = 3) of the numbers of CD8 SP cells preunit area (0.1 mm2) in indicated thymic regions from different mice. *, p < 0.05, compared with WT.

 
Given that OT-I thymocytes in RIP-mOVA+Ltbr–/– mice are prominently rescued without a significant decrease in thymic expression of mOVA, we wondered whether LTβR acts via other mechanisms in organizing thymic negative selection. Thymic medulla is the major site for negative selection of TSA-specific autoreactive T cells. Appropriate cortex-to-medulla migration of thymocytes is critical for their differentiation development and maturation (22, 23). Mice with severely diminished thymic medulla, as in Relb–/– mice, demonstrate significant autoimmune phenotype (24, 25, 26). Because LTβR has been known to regulate the expression of chemokines in secondary lymphoid organs (27, 28), we hypothesized that chemokine expression might be defective in the Ltbr–/– thymus and the defective negative selection in Ltbr–/– mice might result from impaired thymocytes cortex-to-medulla migration. We therefore examined the thymic expression of CCL21 (SLC) and CCL19 (ELC) in WT and Ltbr–/– mice. These two chemokines are produced predominantly in the medulla of thymus. Their receptor, CCR7, is expressed on semimature thymocytes, and has been recently shown to be important for the thymocytes migration from cortex to medulla (29, 30). By quantitative real-time PCR, we found both SLC and ELC were significantly reduced in the Ltbr–/– thymi compared with those in WT mice (Fig. 3B). Because the mTECs population is slightly reduced in Ltbr–/– thymi, to more specifically determine the role of LTβR on intrinsic regulation of SLC/ELC expression within the mTECs population, we purified mTECs (CD45–G8.8+B7.1+) by FACS sorting and reanalyzed SLC/ELC gene expression in this purified population. SLC/ELC gene expression was again found reduced significantly in Ltbr–/– mTECs compared with WT mTECs on a per cell basis (Fig. 3C). Thus, LTβR controls thymic SLC/ELC expression by controlling both the number of mTECs and the level of expression on a per-cell basis. Because on a per cell basis level, the level of chemokine expression was near identical to that found globally, it is likely that chemokine expression by mTECs is predominantly transcriptionally controlled by LTβR. What downstream signaling pathways are used by LTβR for controlling thymic SLC/ELC requires further investigation.

To further determine whether the reduced expression of medullary chemokines will disturb the migration of thymocytes into the medulla, we adopted the following approach (29). Mice were sublethally (400 rad) irradiated to deplete the majority of their immature double positive (DP) thymocytes. The thymic localization of SP thymocytes of WT, Ltbr–/–, and plt were determined by immunofluoresence staining. Sublethal irradiation has been shown to induce apoptosis of immature cortical DP thymocytes, while sparing mature medullary SP thymocytes (31). In fact, when immunofluoresence staining was performed to check CD4/CD8 expression, only rare DP cells were found in the irradiated thymi (data not shown). Because CD8 staining was better visualized than that of CD4, we compared CD8 SP intrathymic localization in the thymi from different genotypes. In sublethally irradiated WT mice, most CD8 SP cells were found to be located in the medulla (Fig. 3, D and E). plt mice have significantly reduced SLC expression and no ELC expression (32, 33) and significant arrest of cortex-to-medulla migration of SP cells was reported in plt mice due to lack of SLC/ELC thymic expression (29). Similar to that in plt thymi, in the medulla of Ltbr–/– thymi, the CD8SP cell numbers were found also significantly reduced compared with that in WT thymi (Fig. 3, D and E). As a consequence, the CD8SP cells in the cortex of both Ltbr–/– and plt thymi are significantly increased (Fig. 3, D and E). Thus, LTβR deficiency not only results in down-regulation of thymic SLC/ELC expression, but also impairs the cortex-to-medulla migration of CD8SP thymocytes.

Impaired thymic negative selection leads to peri-insulitis

To characterize the impact of this defect in negative selection on the peripheral T cell repertoire, splenocytes from bone marrow chimeras were first analyzed by flow cytometric staining with OT-I specific tetramer. We found a significantly increased peripheral population of OT-I+ T cells in RIP-mOVA+Ltbr–/– recipients relative to RIP-mOVA+Ltbr+/+ recipients (Fig. 4, A and B), suggesting that this thymic defect may have real peripheral consequences.


Figure 4
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  		FIGURE 4. Impaired thymic negative selection results in peri-insulitis. A, Islet-specific T cells in the peripheral repertoire of Ltbr–/– mice. Splenocytes of OT-I Tg mice in various backgrounds were stained with CD4, CD8, and OT-I tetramer. Representative profiles are shown. Numbers shown are mean ± SD (n = 4–8) for each group. B, Total splenocytes and proportion of indicated clonotypic splenocytes were analyzed. Statistic analysis was performed using Student t test. Mean ± SD (n = 4–8). ***, p < 0.001; NS, no significance. C, Impaired thymic negative selection leads to peri-insulitis. Total splenocytes from bone marrow chimeric mice were secondarily adoptively transferred to sublethally irradiated RIP-mOVA+ mice and pancreatic infiltration was checked 4 wks later by H&E staining. Representative results from two experiments (n = 2 in each group) are shown.

 
To determine the functional impact of these rescued OT-I+ T cells, we first checked whether this selection defect will culminate in insulitis or hyperglycemia in RIP-mOVA+Ltbr–/– mice. However, we found neither insulitis nor hyperglycemia in RIP-mOVA+Ltbr–/– mice, even at 12 wks after bone marrow transfer (data not shown). With the consideration that this may represent a falsely negative result, given the absence of draining lymph nodes in Ltbr–/– mice for priming, and congenitally disturbed spleen microarchitecture, we designed adoptive transfer experiments. Splenocytes from OT-I ->RIP-mOVA+Ltbr+/+ and OT-I ->RIP-mOVA+Ltbr–/– bone marrow chimeric mice were secondarily adoptively transferred to sublethally irradiated RIP-mOVA+ mice to test whether they lead to autoimmune response targeting to the pancreas. Four weeks after secondary transfer, mice were sacrificed and pancreata were checked for lymphocyte infiltration by histostaining. Although there was only occasional peri-islet infiltration noted in the RIP-mOVA+ recipients of OT-I -> RIP-mOVA+Ltbr+/+ splenocytes, significant infiltration was seen in >75% of the islets analyzed in RIP-mOVA+ recipients OT-I ->RIP-mOVA+Ltbr–/– chimeric mice (Fig. 4C). Immunofluoresence staining revealed that the infiltrates were composed of CD8 T cells, B cells, macrophages, and dendritic cells (data not shown), a similar composition to previously reported insulitis (34). Thus, not only do more OT-I+ cells escape thymic negative selection in the absence of LTβR, but also they carry a functional impact, leading to infiltration of the islets and development of peri-insulitis.

Thymic chemotactic defect alone is sufficient to impair the negative selection

To evaluate whether the dysregulation of chemokines in the Ltbr–/– thymus are in themselves sufficient to produce the witnessed defects in thymic negative selection, we used RIP-mOVA+/plt mice as OT-I BM transfer recipients. plt thymi harbor an isolated defect in the expression of thymic SLC/ELC and were earlier shown to have impaired medullary migration of SP thymocytes. Similarly to RIP-mOVA+Ltbr–/– recipients, we found significantly increased populations of clonotypic OT-I+ T cells in RIP-mOVA+/plt recipients (Fig. 5, A and B). This result confirms regulation of thymocyte chemotaxis as a significant component in the schema of LTβR control of negative selection. However, our current data do not exclude other mechanisms used by LTβR for control of thymic negative selection (see Discussion).


Figure 5
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  		FIGURE 5. Impaired thymic negative selection in plt mice. OT-I TCR transgenic bone marrow cells were depleted of mature T cells and transferred to lethally irradiated recipients mice as indicated in the figure. Eight to nine weeks later, OT-I thymocytes development was analyzed by flow cytometry. Thymocytes (A) or splenocytes (B) were stained with CD4, CD8, or OT-I tetramer. Upper panel, Representative profiles from mice of indicated genotypes. Experiment was repeated with similar results. Lower panel, Data shown are mean ± SEM (n = 2). ***, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note added in proof.
 Disclosures
 References
 
Thymic medullary epithelial cells are known to play an important role for organ-specific central tolerance, but how these cells are regulated and which ligand or receptors are involved in this process has not been well defined. Aire is considered a major transcriptional factor for the deletion of organ-specific T cells in thymus, but how Aire itself is regulated is also poorly defined. LTβR-deficient mice have peripheral organ lymphocytic infiltration that may be associated with defects in negative selection. Some studies have proposed that the LTβR pathway controls negative selection in an Aire-dependent fashion (12, 13), while others have shown no change in Aire expression in Lta–/–, challenging this hypothesis (11). In this study, we present direct evidence for impaired negative selection in Ltbr–/– mice. Furthermore, the underlying mechanisms of LTβR signaling that regulate organ-specific central tolerance remained unclear. Over many studies, molecules other than Aire have been noted to be important for negative selection, including LTβR, NF-{kappa}B-induding kinase, TRAF6, RelB, NF-{kappa}B2, and CCR7 (6, 7, 8, 9, 10, 11, 12). In mice genetically deficient in these factors, there is clear lymphocytic infiltration targeting multiple peripheral organs. However, the mechanism of their contribution to organ-specific central tolerance has not been directly demonstrated. In this study, by taking advantage of OT-I and RIP-mOVA transgenic system, we present direct evidence that LTβR is critical in the thymic negative selection of organ-specific T cells. We further revealed that LTβR controls thymic expression of SLC and ELC, key chemokines controlling the migration of developing thymocytes from the thymic cortex to the medulla (9, 29, 30, 35). Dysregulation of the CCR7-CCR7L (SLC/ELC) axis has been proposed to be associated with impaired central tolerance (9), but it is not clear whether negative selection, Treg generation, or anergy is affected. We have now provided the first direct evidence that SLC/ELC, dependent on LTβR signaling, is required for proper thymic negative selection.

Because it is difficult to trace the fate of organ-specific autoreactive T cells in a native polyclonal repertoire, the OT-I/RIP-mOVA transgenic system was used in this study. Although the mOVA gene expression is under control of RIP in these mice and is supposed to mimic the regulation of insulin 2 expression, a recent study has shown clear evidence that significant differences remain (4). In our system, mice deficient in LTβR fail to demonstrate reductions in thymic RIP-mOVA expression, even while native thymic Ins2 expression is reduced (4). Although RIP-mOVA is an imperfect mimic of native insulin expression, the OT-I—RIP-mOVA system was valuable in dissecting other mechanisms essential for negative selection. As presented earlier, LTβR also controls several key medullary chemokines required for proper thymocyte migration, in a manner similar to what has been found in Aire–/– thymi (4). Given the reduced Aire expression in Ltbr–/– thymi, it therefore becomes an interesting question of whether these defects in Ltbr–/– thymi are dependent on Aire function. To address this question, we compared several genes regulated by Aire and/or LTβR. SLC and ELC are two key chemokines known to be involved in the regulation of thymocyte migration into the thymic medulla. Although both are significantly reduced in Ltbr–/– thymi, neither of them was reduced in Aire–/– thymi (data not shown). This difference suggests that, although both LTβR and Aire are involved in the negative selection process, they may have both overlapping and distinct mechanisms.

Although our data suggest that reduced expression of medullary chemokine (SLC and ELC) expression in Ltbr–/– thymus is in itself sufficient to disturb thymic negative selection, other mechanisms may also be involved. This is indicated by the partial rescue of OT-I T cells in RIP-mOVA+plt mice relative to RIP-mOVA+Ltbr–/– mice. Boehm et al. (13), for example, reported disorganized thymus medulla in Ltbr–/– mice. Other potential mechanisms of LTβR control of thymic negative selection might reside in function of mTECs or other thymic APCs. Their relative contributions need to be determined in further studies.

Dendritic cells (DC), upon activation, also express CCR7 and become responsive to SLC/ELC (36). Their migration and localization were also found defective in the secondary lymphoid organs of plt mice (37). Because DCs have been shown to mediate thymic negative selection in the OT-I/RIP-mOVA system (16), the impaired negative selection in our Ltbr–/– mice could be also due to improper DCs localization in the thymus. However, tissue staining failed to show any differences in thymic localization of DCs among WT, Ltbr–/–, and plt mice. Most DCs were properly located into the medulla region (data not shown and Ref. 9). Thus, thymic migration and localization of DCs may not rely on the CCR7-CCR7L chemotaxis and is unlikely to be a major contributor to impaired thymic negative selection in our Ltbr–/– mice. Whether our Ltbr–/– DCs have an intrinsic functional defect that contributes to the Ltbr–/– phenotype, as was suggested recently (38), remains an interesting question for future study.

Although significantly more OT-I T cells escaped thymic negative in the RIP-mOVA+Ltbr–/– than in RIP-mOVA+Ltbr+/+ hosts, these autoreactive T cells were held in check by the absence of lymph nodes necessary for priming in their Ltbr–/– hosts. Florid autoimmunity developed upon secondary transfer, when this restriction was relieved. The absence of hyperglycemia in these mice, which could be due to the limited number of autoreactive T cells transferred. This is consistent with previous findings that transfer of small numbers of OT-I T cells (< 1 x 106, which is the case in our experiments) into RIP-mOVA+ mice fails to induce diabetes (34).

In conclusion, we have now clearly demonstrated that LTβR is required for the thymic negative selection of organ-specific T cells. Medullary chemokine regulation has been revealed to be an important pathway used by LTβR to control organ-specific negative selection. Our study defines the novel role of LTβR in establishing organ-specific central tolerance, helps to unravel the complex regulation of organ-specific autoimmunity, and provides new targets for future diagnostic and therapeutic intervention of organ-specific autoimmune diseases.


   Note added in proof.
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
Disclosures
 References
 
During the proofreading of this manuscript, the Mathis group had just published their study about the indirect role of LTβR in thymic Aire expression via regulation of mTECs (39). It appears that LTβR controls the development/structural organization of mTECs rather than direct control of Aire expression cell-intrinsically. But the blocking LTβR signaling could reduce the number of mTECs, suggesting LTβR is required for constantly maintenance of organization. Furthermore, the administration of agonistic Ab to LTβR can enhance the expression of Aire within a few hours. While LTβR signaling is certainly required for development/organization of mTECs, the authors revealed insignificant influence of LTβR deficiency on clonal deletion using OT-II/RIP-mOVAtg system. But our present study has revealed a significant influence of LTβR on thymic clonal deletion of OT-I thymocytes.


   Acknowledgment
 
We thank Shihong Li from the Immunohistochemistry Facility for H&E staining and analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by Grants from National Institutes of Health AI062026 and DK58897 (to Y.X.F.). Back

2 Address correspondence and reprint requests to Dr. Yang-Xin Fu, Department of Pathology and Committee on Immunology, The University of Chicago, 5841 S. Maryland, Room J541, MC3083, Chicago, IL 60637. E-mail address: yfu{at}uchicago.edu Back

3 Abbreviations used in this paper used in this manuscript: Treg, regulatory T cell; TRA, tissue-restricted self-Ag; SLC, secondary lymphoid tissue chemokine; ELC, EBV-induced molecule 1 ligand chemokine; mTEC, medullary thymic epithelial cell; Aire, autoimmune regulator; LTβR, lymphotoxin β receptor; RIP, rat insulin 2 promoter; WT, wild type; DC, dendritic cell; TRAF6, TNF receptor-associated factor 6; RelB, v-rel reticuloendotheliosis viral oncogenehomolog B; CCR7, chemokine (C-C motif) receptor 7; SP, single positive; DP, double positive. Back

Received for publication June 14, 2007. Accepted for publication October 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
 Disclosures
 References
 

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