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The Journal of Immunology, 2006, 176: 6484-6490.
Copyright © 2006 by The American Association of Immunologists

Cervical Thymus in the Mouse1

James Dooley*, Matthew Erickson*, Geoffrey O. Gillard{dagger} and Andrew G. Farr2,*,{dagger}

* Department of Biological Structure and {dagger} Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although thymic ectopy has long been recognized in humans, the functional activity or potential immunological significance of this thymic tissue is unknown. In this study, we describe murine thymic ectopy, cervical thymic tissue that possesses the same general organization as the thoracic thymus, that is able to support T cell differentiation, and that can export T cells to the periphery. Unexpectedly, the pattern of autoantigen expression by ectopic thymic tissue differs from that of the thoracic thymus, raising the possibility that these two thymic environments may project different versions of self.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The epithelial component of the thymic environment displays considerable heterogeneity and is organized into compartments with discrete functional, morphological, and phenotypic characteristics (reviewed in Ref. 1). Epithelial cells of the cortical compartment, which support positive selection, are phenotypically distinct from medullary epithelial cells, which are thought to contribute to negative selection and may support postselection maturation of single-positive thymocytes.

During thymic organogenesis, endoderm from the third pharyngeal pouch separates from the pharyngeal tube and gives rise to both the thymus and parathyroid glands. Nonoverlapping domains of endoderm that give rise to thymus and parathyroid gland have been identified by the expression pattern of Foxn1 and Gcm2, transcription factors that define thymic and parathyroid domains, respectively (2). An obligate role for Foxn1, a member of the forkhead transcription family, in thymic organogenesis is well-established (3), although the role of Foxn1 in this process is not clear (4, 5). Recently, Aire has been shown to play a critical, but ill-defined role in expression of a spectrum of tissue-restricted Ags (TRA)3 by medullary thymic epithelium (MTEC), which in turn impacts the contribution of the thymus to self-tolerance (6).

Although thymic tissue normally has a mediastinal location, ectopic cervical thymic tissue in humans has been reported (7, 8). This tissue has gained attention clinically because it presents as a cervical tissue mass that can obstruct breathing or can give rise to ectopic thymic tumors. Some investigators have considered cervical thymic tissue to be rare (9), but it has also been suggested that the incidence of cervical thymic tissue in adult humans may be >50% (10).

Histological demonstration of what appeared to be cervical thymic tissue in mice was suggested to explain the failure of some strains of mice to display reductions in peripheral lymphocyte cellularity following neonatal thymectomy (11, 12). More recently, increased incidence of thymic ectopy has been reported to occur in autoimmune-prone NOD (13) and rats subjected to elevated dietary iodine (14). Despite the relevance of ectopic thymic tissue to several issues of thymus biology (organogenesis, thymic epithelium differentiation, "extra-thymic" T cell production, and autoimmunity), and the potential to model human thymic ectopy, murine cervical thymic tissue has received surprisingly little attention. We report here that the cervical thymic tissue in mice resembles the thoracic counterpart in many respects. However, variability in autoantigen expression among individual cervical thymic samples compared with their thoracic counterparts raises the possibility that the range self-Ags that are projected may not be equivalent at these two sites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c, C57BL/6 and BALB/c nude mice were obtained from Charles River Laboratories. RAG-GFP mice (15) were obtained from Dr. P. Fink (University of Washington, Seattle, WA). Foxp3-GFP (16), OT-2 (17), and RipOVA (18) mice were obtained from Dr. A. Rudensky (University of Washington). All mice were used in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Washington. Tissue samples were from neonatal 8-wk-old mice.

Human tissue samples

The use of the human tissue sections was approved by the subject review board at the University of Washington.

Abs and reagents

Primary Abs for immunohistochemistry or flow cytometry have been described previously (5, 19).

Immunohistochemistry

Immunohistology techniques were performed as previously described (5, 19).

Analysis of gene expression

Thoracic and cervical thymic TRA expression survey in pooled samples. Thoracic thymic samples and pools of cervical thymic samples (from two to three mice) were obtained from 3-wk-old BALB/c mice. Samples were homogenized and total RNA extracted using the Absolute RNA Miniprep kit per the manufacturer’s protocol (Stratagene). cDNA was synthesized using the Omniscript RT kit (Qiagen), starting with equal amounts of total RNA from each of the samples. Control samples of cDNA without reverse transcriptase (RT) (no RT) were also made to test for nonspecific real-time PCR products. cDNA samples were mixed with TRA-specific primers and SYBR Green master mix (Applied Biosystems) and the reactions were assayed with a 7300 Real-Time PCR machine (Applied Biosystems). Primers for the following molecules were generated: Aire, Ep-Cam, e-cadherin recoverin, pancreatic polypeptide, insulin, c-reactive protein, rhodopsin, interphotoreceptor retinoid-binding protein (IRBP), H-K ATPase (beta-chain), and green cone opsin (sequences available on request). Gene expression was evaluated by the {Delta}–{Delta} cycle threshold ({Delta}{Delta}Ct) method, where the expression of a TRA by each sample was normalized to Ep-cam, a reliable marker for MTEC. An average {Delta}Ct value was determined for each of the target genes in both thoracic and cervical samples and these values were then used to calculate differences in {Delta}Ct values for the two sample types after normalization to levels of Ep-cam to control for relative abundance of medullary epithelium in the samples. To validate the SYBR Green PCR products, a dissociation step was done to verify the Tm (annealing temperature) of the SYBR Green PCR product after the PCR were run.

Individual thymic and cervical sample preps. Total RNA from individual cervical and thoracic thymi from 3-wk-old BALB/c mice was isolated (RNEasy kit; Qiagen). Only sample pairs from mice that had >200 ng of cervical total RNA were amplified. Total RNA from both sample types was then amplified according to Ref. 20 , starting with 200 ng of cervical RNA, 200 ng of thoracic RNA, and a third sample of 2 µg of thoracic RNA. cDNA was synthesized from the amplified RNA and used as a template for SYBR Green real-time relative quantitation as described above, using equivalent amounts of cDNA. Because "No RT" controls were not generated from amplified RNA, the SYBR-Green RT-PCR results were validated by the dissociation temperatures (Tm) of the amplicons.

To confirm the linearity of mRNA amplification by the protocol used here, we amplified different amounts of thoracic thymic RNA and interrogated the resulting cDNA with SYBR Green real-time PCR. Selected target genes showed <1-fold variation among the samples after one or two rounds of amplification (data not shown).

Grafting procedures. Although under ketamine/xylazine anesthesia, mice were laparotomized to expose the left kidney and thoracic or cervical thymic tissue was implanted under the kidney capsule, using aseptic technique. Four weeks after grafting, graft and host tissues were evaluated with immunohistochemistry and flow cytometry.

In vitro mitogenesis assay. In 96-well flat-bottom plates, 1 x 105 thymocytes from thoracic or cervical BALB/c thymi were cultured with 2 x 105 irradiated syngeneic spleen cells in the presence or absence of anti-CD28 (1 µg/ml; eBiosciences) and graded concentrations of anti-CD3 Abs (15–500 ng/ml; eBiosciences). Medium was RPMI 1640 with 10% FBS. Cells were cultured for 72 h at 37°C in humidified air containing 5% CO2. 1 µCi of tritiated thymidine was present for the last 16 h of culture. Cells were harvested and thymidine incorporation was measured with a scintillation counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ectopic thymic tissue occurs frequently in the cervical region

We performed histological evaluation of tissues in the cervical region to identify "islands" of thymic tissue encapsulated by connective tissue. We also used immunohistochemistry to screen serial sections of cervical tissue blocks for CD3 expression and sections of tissue from RAG-GFP mice to locate sites of RAG expression in this region. These initial experiments focused attention to areas lateral to the groove formed by the juxtaposition of the trachea and esophagus (Fig. 1a). Cervical thymic tissue was rarely bilateral and there was considerable variability in localization as to left or right side location and in cephalic-caudal positioning, which ranged from immediately above the sternum to association with the thyroid (Fig. 1b) and parathyroid (Fig. 1c) glands. Additional discrete lymphatic tissue located lateral to these structures were identified as either additional ectopic thymic tissue or lymph nodes. Because of this heterogeneity, lateral structures were omitted from subsequent analyses.


Figure 1
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  		FIGURE 1. a, Cervical thymic tissue is often associated with the trachea and esophagus. The most reproducible location for cervical thymic tissue (3 ) was lateral to the groove between the trachea (1 ) and the underlying esophagus. There was considerable variation in the relative cephalocaudal axis; ectopic thymic tissue could be found adjacent to the sterum (2 ) or toward the larynx (4 ) and associated thyroid and parathyroid tissue. b, Thymic tissue more cephalic, surrounded by thyroid tissue. c, Thymic tissue more cephalic, adjacent to parathyroid tissue and surrounded by thyroid tissue. d–i, Cervical and thoracic thymic tissue have similar stromal organization. Thoracic (d, f, and h) and cervical (e, g, and i) thymi were reacted with Abs that identify cortical epithelium, ER-TR4, (d and e), medullary epithelium, ER-TR5, (f and g) and the fibroblastic component, ER-TR7 (h and i). C, cortex; M, medulla; T, trachea. Arrow in g and i indicates a discrete medullary region that is enriched in ER-TR7+ components. j, RT-PCR demonstration that cervical thymic tissue expresses Foxn1 and pre-T{alpha}, two hallmark features of functional thymic tissue. k, Cellularity of cervical thymic tissue. Incidence of cervical thymic tissue detection (as defined in the text) is indicated below the x-axis label. l and m, One of two archival histology slides of human parathyroid gland (PT) adjacent to cervical thymic tissue (T). A Hassall’s body is shown in m.

 
In agreement with Ref. 13 , immunohistochemical analyses of the cervical tissue showed stromal organization that was very similar to thoracic thymus, with discrete cortical and medullary compartments. Expression patterns of CD40, CD80, CD86, and MHC class II in the cervical thymus were unremarkable (data not shown). However, the mesenchymal fibroblastic component of the cervical thymus (as defined by ER-TR7) was less integrated with the MTEC compartment than in the thoracic thymus. As shown in Fig. 1i, ER-TR7 reactivity in the cervical thymus occupied a central region of the tissue that was relatively devoid of ER-TR5+ cells (Fig. 1g). This is contrasted by the ER-TR7+ cells that were typically investing blood vessels and scattered throughout the thoracic thymus (Fig. 1h).

Consistent with their thymic character, cervical thymic tissue also expressed Foxn1 and pre-T{alpha} (Fig. 1j). Approximately 50% of the BALB/c mice (25 of 47) examined displayed cervical thymic tissue, while the incidence was reduced in C57BL/6 mice (33%; 8 of 24), with cellularity ranging from around 105 to upwards of 5 x 105 cells/organoid (Fig. 1k). The incidence of cervical thymic tissue reported here is a conservative estimate because mice were considered positive only if medial thymic tissue was identified.

The frequency of cervical thymic tissue juxtaposed with thyroid and parathyroid tissue in the mouse prompted an evaluation of normal human parathyroid in the medical histology collection at the University of Washington. Two of three of human parathyroid tissue samples contained lymphatic tissue judged to be thymus (lacked lymphatic sinuses, afferent lymphatic vessels, or cortical follicular structures, and displayed multicellular structures considered to be Hassall’s bodies) and resembled the human sample described by Wu et al. (21). One of the samples is demonstrated in Fig. 1, l and m. The frequency of occurrence in this small unselected sample population suggests that the incidence of "occult" thymic ectopy in humans may be significant.

Cervical thymic tissue supports thymocyte differentiation

Because previous characterization of cervical thymus was based on morphology, we wanted to formally assess the capacity of this tissue to support thymocyte development. Initial examination of thymocyte development in cervical thymic tissue, as assessed by the relative sizes of double-negative, double-positive, and single-positive thymocyte populations, was very similar to that of thoracic thymus (Fig. 2a). Furthermore, thymocytes recovered from pooled thoracic or cervical thymic tissue responded equivalently to mitogenic stimulation by anti-CD28 and anti-CD3 Abs (Fig. 2b), indicating that comparable programs of {alpha}beta thymocyte development were supported at both sites.


Figure 2
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  		FIGURE 2. Cervical thymic tissue support the development of {alpha}beta-lineage T cells. a, Thoracic and cervical thymic tissue display comparable representation of thymocyte subsets. Upper two panels, Expression of CD4 and CD8 by thoracic and cervical thymocytes; lower two panels, the expression of CD3. Numbers indicate percentages in indicated quadrant or portion of histogram. b, Thymocytes from thoracic and cervical thymus tissue respond equivalently to ligand-induced proliferation. Thoracic and cervical thymocyte proliferation in the presence of anti-CD28 Abs alone was 155 and 204 cpm, respectively. c, Selection efficiency is comparable in thoracic and cervical thymus. Pools of cervical and thoracic tissue from OT-2 or OT-2/RIPOVA mice were processed for flow cytometric analyses. Panels on the left depict the representation of thymocytes that are V{alpha}2+. d, Cervical thymic tissue contains Foxp3 cells in the appropriate location, indicating a mature organization of the medulla. Ctx, cortex; M, medulla.  

 
To evaluate thymocyte selection in the cervical thymus in more detail, we analyzed transgenic mice bearing the MHC class II-restricted, OVA-specific OTII TCR transgene (17). Fig. 2c demonstrates that the accumulation of mature single-positive thymocytes was similar, although the frequency of double-positive thymocytes was reduced in this pool of cervical thymocytes. The extent of negative selection in mice coexpressing the OTII TCR with an OVA-encoding a transgene driven by the rat insulin promoter directing the expression of the cognate Ag, OVA (18) was similar in cervical and thymic tissue. These data indicate that the cervical thymic environment can support positive selection and is permissive for negative selection if the selecting ligand is present.

As another assessment of the organization of the cervical thymus, we used Foxp3-GFP mice to compare the distribution of regulatory Foxp3+CD4+ cells within the cervical and thoracic thymus. We have previously shown that thymocytes expressing Foxp3 are localized to the medullary compartment in the thoracic thymus (16). As shown in Fig. 2d, Foxp3+ thymocytes were highly restricted to the medullary compartment in both cervical and thoracic environments. These data indicate that the medullary compartment of the cervical thymic provides the necessary environment for the development of regulatory T cells and that these cells occupy the same thymic compartment in cervical and thoracic thymi.

The cervical thymus can contribute to the peripheral T cell pool

To formally demonstrate that the cervical thymic tissue can support the export of T cells to peripheral lymphatic tissue, we grafted cervical thymic tissue under the kidney capsule of nude mice. Fig. 3, a–f, depicts one of six cervical thymic grafts processed to demonstrate that normal thymic organization persisted in the grafts for at least one month after transplant, with appropriate cortical and medullary epithelial compartmentalization and typical distribution of thymocytes and dendritic cells. Furthermore, the peripheral lymph nodes (Fig. 3g) or spleen (data not shown) from nude mice bearing these grafts clearly contain T cells one month after initiation of the grafts. These data indicate that thymocytes from the cervical thymus can contribute to the peripheral T cell pool, although at this time we do not know the relative contribution of steady-state output from the grafted tissue and peripheral homeostatic proliferation.


Figure 3
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  		FIGURE 3. Cervical thymic tissue can be transplanted and exports T cells to the periphery. a–f, Typical thymic architecture is maintained a month after transplanting under the kidney capsule. a, E-cadherin (ECCD-2); b, Ep-CAM (G8.8); c, 6C3; d, MHC class II (M5114); e, N418; f, CD3 (500A2). g, The output of thoracic and cervical thymic grafts in nude mice recipients.

 
The representation of self-Ags in cervical and thoracic thymic tissue is not equivalent

To assess the expression of TRA by cervical thymus, we performed real-time PCR on cDNA from paired cervical and thoracic thymus samples from individual mice. Because it was not feasible to isolate MTEC from individual samples, TRA expression by whole thymus samples was normalized to expression levels of Ep-cam (preferentially expressed by MTEC (22). As depicted in Fig. 4, the average values for Aire and TRA expression by at least 6 independent thoracic (T) and cervical (C) thymic samples were approximately equivalent. In contrast to the modest SD of the thoracic samples, the range of {Delta}Ct values of TRA expression by individual cervical samples was rather large, on the order of six cycles.


Figure 4
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  		FIGURE 4. Expression of TRAs by cervical and thoracic thymi. Expression of TRA by cervical (C) and thoracic (T) thymi. Horizontal bars are average values; vertical bars represent the SD of at least six samples. A, Aire; B, recoverin; C, pancreatic polypeptide, D, rhodopsin; E, IRBP; F, H/K ATPase beta; G, green cone opsin.

 
To assess this variability more directly, equivalent amounts of cervical and thoracic thymic RNA from a second set of five individual mice were amplified and processed identically, then subjected to the same type of analysis. Table I displays the relative quantity values obtained, where TRA expression was normalized to Ep-cam expression and differences in {Delta}Ct values ({Delta}{Delta}Ct) among cervical or thoracic thymic samples was used as an index of variability of target gene expression. Consistent with the data presented in Fig. 4, 2 of 32 thoracic and 21 of 39 cervical thymic samples displayed a 2-fold or greater variance from their cohorts. Comparison of values obtained with different amounts of thoracic thymic RNA indicated the starting amount of RNA was not a significant contribution to the variability (1 of 30 samples with a 2-µg sample vs 2 of 32 samples with 200-ng samples had a 2-fold or greater variance from their cohorts), further supporting the conclusion that increased variability of TRA expression reflects the biology of cervical thymic tissue.


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  		Table I. Relative quantity of gene expression in thoracic and cervical thymus samples

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we have revisited cervical thymic tissue in the mouse and provide formal demonstration that it functionally resembles the thoracic thymus in terms of stromal cell composition and organization, and the abilities to support thymocyte development, to export T cells to the periphery, and to contribute to central tolerance. In contrast to a previous report that correlated thymic ectopy and autoimmune disease in the NOD mice (13), we found that thymic ectopy was a feature of commonly used strains of mice that do not display elevated incidence of autoimmunity.

The occurrence of thymic ectopy is significant in several respects. First, the presence of extrathoracic sites of "thymic" T cell development represents a potential confounder for studies of extrathymic T cell development, particularly those based on thoracic thymectomy models (23, 24). The contribution of ectopic thymic tissue to the peripheral T cell pool could be affected by strain-dependent prevalence of thymic ectopy and relative contribution of these two sources of T cells to subsequent peripheral homeostatic proliferation.

The occurrence of cervical thymic tissue in mice is also important in the context of thymic organogenesis. Ectopic thymic tissue in humans has been widely considered to reflect third pharyngeal pouch endoderm that is specified to a thymic fate but fails to migrate to the appropriate location (7). If that is the basis for cervical thymic tissue in mice, commingling of epithelial populations destined to have thymic and parathyroid fates would be expected to occur, because the ectopic thymic tissue is found along the path likely to be taken by derivatives of pharyngeal pouch endoderm during development. However, the distribution of markers of thymic and parathyroid epithelium, Foxn1 and Gcm2, respectively, do not appear to overlap during murine embryogenesis (2). Although this may simply reflect assay sensitivity, the lack of Foxn1 expression in cervical regions of the developing embryo where Foxn1+ thymic tissue later develops raises the possibility that progenitor epithelial cells could become specified to a thymic fate (and express Foxn1) some time after their migration to a cervical location. The mechanism underlying the organogenesis of cervical thymic tissue is an important question that warrants examination. The reproducible thymic ectopy in mice represents an opportunity to follow the organogenesis of ectopic thymus and to clarify its embryological origins, issues that are not readily addressed in humans.

Finally, ectopic thymic tissue may have a bearing on the issue of self-tolerance. It is now generally recognized that medullary epithelial cells express a remarkable spectrum of self-Ags that are normally considered to be unique or specific to a particular tissue or organ and that their expression represents an important mechanism to effect self-tolerance. We have shown here that cervical thymic tissue collectively expresses a spectrum of TRA that appears similar to the thoracic thymus. However, assessment of individual cervical thymic "lobes" revealed considerable heterogeneity in levels of TRA expression. Because individual TRAs are expressed by relatively rare medullary epithelial cells (25) or discrete subsets of epithelial cells (19), scaling thymic size (and the number of MTEC) could have an impact on the range or levels of TRA expression.

Analyses of TRA expression by isolated MTEC from the thoracic thymus has been based on relatively large pools of cells and represent a profile average of many cells. Although we have compared equivalent amounts of cervical and thoracic thymic RNA, the thoracic sample represents the activity of many more MTEC and thus also presents an average expression of TRAs by many cells. Consistent with the immunohistochemical analysis of TRA expression, we have found that a low frequency of MTEC express TRA when individual or small numbers of thoracic MTEC was assessed by RT-PCR (26). These data suggest that the variability of TRA expression observed in the cervical thymic samples could reflect the small size of this tissue and the low numbers of MTEC that they contain. It is likely that the smaller absolute number of MTEC in the cervical thymus cannot provide a significant averaging effect in terms of TRA expression. This interpretation is consistent with the data in Fig. 4, where the average levels of individual TRA expression by multiple cervical thymi approximate those of thoracic thymus. These differences may reflect temporal oscillations in the production of these TRAs by MTEC or may be due to intrinsic differences in the epithelial composition of individual cervical thymic lobes that could in turn affect the spectrum of TRAs they express. Takase et al. (27) recently reported that expression of a subset of TRA in the human thymus displayed considerable individual variation. The relative contributions of genetic polymorphism and the number of MTEC analyzed to the variability of TRA expression in the human thymus is presently unclear.

Based on the premise that perturbed expression of TRAs can contribute to autoimmunity (28, 29, 30), a smaller ectopic thymus with fewer MTEC may display a spectrum of TRAs that does not totally overlap the TRA profile of the thoracic thymus. This may lead to export of T cells by the cervical thymus that have been vetted by a different projection of self than that of the majority of the peripheral T cell pool derived from the thoracic thymus. Although not necessarily sufficient to initiate autoimmune disease, by placing additional pressure on peripheral tolerance mechanisms, T cells exported from ectopic thymic tissue may represent a previously unrecognized susceptibility factor for autoimmunity. In this context, it is interesting that human thymic ectopy has been shown to modify some of the clinical parameters of myasthenia gravis and is correlated with lower remission rates following treatment (8).

It remains to be determined whether the differences in TRA expression between cervical and thoracic thymus reflect intrinsic differences in these two thymic environments or whether they reflect a common property of thymic epithelium that becomes evident as the thymic environment is scaled down. In either case, these subtle differences in the cervical thymic environment may have significant immunological consequences.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 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 work was supported by National Institutes of Health (NIH) Grants AI 24137 and AI50957. G.O.G. was supported in part by training grants from the NIH and the Cancer Research Institute. Back

2 Address correspondence and reprint requests to Dr. Andrew G. Farr, Department of Biological Structure, University of Washington, Box 357420, Seattle, WA 98195-7420. E-mail address: farr{at}u.washington.edu Back

3 Abbreviations used in this paper: TRA, tissue-restricted Ag; MTEC, medullary thymic epithelial cell; Ct, cycle threshold; RT, reverse transcriptase; IRBP, interphotoreceptor retinoid-binding protein Back

Received for publication February 8, 2006. Accepted for publication March 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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G. O. Gillard, J. Dooley, M. Erickson, L. Peltonen, and A. G. Farr
Aire-Dependent Alterations in Medullary Thymic Epithelium Indicate a Role for Aire in Thymic Epithelial Differentiation
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