The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haribhai, D.
Right arrow Articles by Williams, C. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haribhai, D.
Right arrow Articles by Williams, C. B.
The Journal of Immunology, 2003, 170: 3007-3014.
Copyright © 2003 by The American Association of Immunologists

A Threshold for Central T Cell Tolerance to an Inducible Serum Protein1

Dipica Haribhai*, Deborah Engle2,{dagger}, Michelle Meyer{dagger}, David Donermeyer{ddagger}, J. Michael White{ddagger} and Calvin B. Williams3,*

* Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226; and Departments of {dagger} Pediatrics and {ddagger} Pathology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report an inducible system of self Ag expression that examines the relationship between serum protein levels and central T cell tolerance. This transgenic approach is based on tetracycline-regulated expression of a secreted form of hen egg lysozyme, tagged with a murine hemoglobin (Hb) epitope. In the absence of the tetracycline-regulated transactivator, serum levels of the chimeric protein are extremely low (<=0.1 ng/ml) and the mice show partial tolerance to both Hb(64–76) and lysozyme epitopes. In the presence of the transactivator, expression increases to 1.5 ng/ml and the mice are completely tolerant. Partial tolerance was further investigated by crossing these mice to strains expressing transgenic TCRs. At the lowest Ag levels, 3.L2tg T cells (specific for Hb(64–76)/I-Ek) escape the thymus and ~10% of CD4+ splenocytes express the 3.L2 TCR. In contrast, 3A9 T cells (specific for hen egg lysozyme(46–61)/I-Ak) are completely eliminated by negative selection. These data define a tolerogenic threshold for negative selection of Ag-specific T cells by circulating self proteins that are 100-fold more sensitive than previously demonstrated. They suggest that partial tolerance at extremely low levels of self Ag exposure is the result of a restricted repertoire of responding T cells, rather than a simple reduction in precursor frequency; tolerogenic thresholds are T cell specific.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell tolerance to self Ags is a dynamic process, involving central and peripheral mechanisms (1, 2). These processes must eliminate or render inactive all T cells with receptors that are activated by self peptide/MHC complexes. Many proteins, such as those generated during an inflammatory response (acute phase proteins), have rapidly changing levels, posing a special challenge (3, 4). Developmentally regulated proteins may also fluctuate between tolerogenic and nontolerogenic levels. At low levels of circulating self protein, the relevant self peptide/MHC complexes in the thymus could be limiting, allowing some self-reactive T cells to escape negative selection. When protein levels are induced, the number of peripheral self peptide/MHC complexes could become sufficient to activate these T cells, resulting in autoimmunity (5). In general, there are two central mechanisms that are thought to prevent autoimmunity under these conditions. First, the increased sensitivity of thymocytes relative to mature T cells may provide a sufficient margin of safety by eliminating self-reactive T cells when the number of selecting complexes is low (6). Second, there is some evidence to suggest that ectopic thymic expression of tissue-specific and acute phase proteins may increase the number of relevant peptide/MHC complexes in the thymus and play an important role in T cell tolerance (reviewed in Refs. 7 and 8). Tolerance to inducible, circulating proteins is therefore likely to involve many factors, making it difficult to correlate serum levels with tolerance and to dissect the relative contributions of the mechanisms involved.

Several experimental systems have addressed these important issues with varying results. One estimate for the minimum serum concentration of any self protein required for negative selection comes from data generated by immunizing metallothionein-hen egg lysozyme (MT-HEL)4 mice. These transgenic animals express HEL under the control of the metallothionein promoter, resulting in a serum concentration of HEL between 13 and 20 ng/ml (~1 x 10-9 M). Immunization studies in these animals demonstrate that T cells are partially tolerant to HEL (9). However, when MT-HEL mice were bred to 3A9 TCR transgenic mice (specific for HEL(46–61)), complete negative selection of 3A9 T cells was observed (5). It has been estimated that as few as three peptide/MHC complexes are needed for this to occur, highlighting the sensitivity of this process for some T cells (10). Tolerance in the MT-HEL system contrasts sharply with studies using an Ig L chain as the self Ag (11). Although clonal deletion of specific T cells was achieved, the serum concentration of Ig needed was greater than 100 µg/ml (>10-6 M). In another system, negative selection of CD4+ T cells specific for C5 was observed at the physiologic concentration of 50 µg/ml (10-7 M) (4). Tolerance to an inducible serum protein was examined in transgenic mice expressing human C-reactive protein (hCRP) under autologous regulatory region control. It was determined that female mice were tolerant at basal hCRP levels (10-9 M), but were also found to express the hCRP transgene in the thymic medullary epithelium (3). Indeed, ectopic or targeted thymic expression leading to tolerance has been reported in several transgenic systems (12, 13, 14, 15, 16). In one report, this expression was localized to rare medullary cells that also contained transcripts for tissue-restricted genes. This finding led the authors to postulate that these rare thymic peripheral Ag-expressing cells played an important role in central tolerance (17).

We have designed an inducible system to test the hypothesis that there is a threshold for central tolerance to secreted self proteins and to examine the consequences of a subthreshold stimulus. This system uses a tetracycline (tet)-regulated transactivator (tTA) (18) to control expression of an epitope-tagged version of HEL (secreted HEL (sHEL)/hemoglobin (Hb)). This secreted, chimeric protein circulates at levels ranging from <=0.1 ng/ml (<=1 x 10-11 M) to 1.5 ng/ml (~1 x 10-10 M) and contains two well-characterized H-2k-restricted epitopes, HEL(46–61)/I-Ak and Hb(64–76)/I-Ek. The transgenic protein is widely expressed in many tissues, including the thymus. We find that mice expressing the lowest levels of protein are only partially tolerant. When immunized with HEL protein or Hb(64–76) peptide, these mice make a diminished T cell response. The mechanism of partial tolerance was investigated at the clonal level by breeding sHEL/Hb mice to TCR transgenic mice. These later studies demonstrate that partial tolerance results from the complete elimination of some clones and partial elimination of others. Thymic transplantation studies confirm that the circulating self protein alone is sufficient for tolerance induction. Together these data define a lower limit for central tolerance to an inducible, secreted protein. They suggest that these limits are likely to be T cell specific and that the repertoire of responding T cells in partially tolerant animals is restricted.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic mice

We have used tet-regulated expression of a neo-self Ag to study T cell tolerance in transgenic mice (18, 19). For this system, we produced two new lines of mice. The tTA line was generated from a 2.7-kb XhoI/PflMI fragment derived from plasmid pUGH 15-1. The fragment contains the human CMV promoter and enhancer (bases -675 to +75), followed by a chimeric gene encoding the tet repressor/VP16 fusion protein. The transactivator coding region is followed by a portion of the rabbit {beta} globin gene, which provides signals for RNA splicing and polyadenylation. Other transgenes using this human CMV promoter and enhancer are ubiquitously expressed in tissues of epithelial origin (20, 21).

The second line was created from the plasmid pJB1.7 (Fig. 1A). This plasmid was constructed by removing a 1.7-kb BglII to XbaI fragment containing the luciferase gene from plasmid pUHC 13-6 and replacing it with a 500-bp chimeric construct composed of the HEL cDNA and a Hb epitope tag (22, 23). The Hb tag encodes aa 64–76 of Hb{beta}d minor, and was added to the HEL cDNA between codons 43 and 44 by PCR, as previously described (24). The construct uses a minimal HSV thymidine kinase promoter, bases -81 to +52, and contains seven tandem repeats of the 42-bp tet operator sequence as 5' regulatory elements proximal to the promoter. The 3' untranslated region contains the SV40 small-t Ag splice site and SV40 polyadenylation site. This chimeric transgene (Fig. 1) is designed to express a secreted protein of ~16 kDa (sHEL/Hb) under the control of the tet-off transactivator. For pronuclei injection, we removed a 3.2-kb BamHI to EaeI fragment containing the sHEL/Hb cDNA and all regulatory elements.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 1. Construction of the inducible sHEL/Hb transgene and analysis of the recombinant sHEL/Hb protein. A, The Hb(64–76) epitope tag was inserted between aa 43 and 44 of the HEL cDNA. The 1.7-kb fragment used to make sHEL/Hb mice contains seven tet operator sequences and a 52-bp promoter derived from the HSV thymidine kinase gene. B, Coomassie-stained SDS-PAGE (15%) demonstrating that the recombinant sHEL/Hb protein produced in baculovirus runs at the expected molecular mass of ~15.7 kDa. C, {beta}1–4 glycosidase activity of HEL ({diamond}) compared with rHEL/Hb ({blacksquare}), measured as the ability of equivalent amounts of protein to clear a lyophilized bacterial cell wall suspension. Results are plotted as the absorbance of the suspension at 540 nm at 1-min intervals following the addition of the purified protein. D, The YO1.6 T cell hybridoma, which is specific for Hb(64–76)/I-Ek, responds equivalently to native d-type Hb ({circ}) and the chimeric rHEL/Hb protein ({blacksquare}). This is a standard hybridoma assay with CH27 used as the APC. Results are plotted as the cpm incorporated in the CTLL-2 assay vs the concentration of the Hb{beta}d minor(64–76) epitope in the well. Each data point is the mean of triplicate wells, and the experiment was performed three times with similar results. E, Stimulation of the YO1.6 T cell hybridoma by the rHEL/Hb ({blacksquare}) protein requires Ag processing. This experiment was identical with that in D, with the exception that the APCs were fixed with 1% paraformaldehyde before their use. The Hb(64–76) peptide ({triangleup}) was used as a positive control, and Hb ({circ}) as the negative control.

 
We obtained two tTA founders and three sHEL/Hb founders and set up a breeding matrix. From these experiments, we identified one tTA line and one sHEL/Hb line that together demonstrated tet-regulated expression for further study. Note that all mice used in these studies were between 8 and 16 wk of age.

Production, purification, and analysis of sHEL/Hb

The sHEL/Hb portion of the transgene was cloned into the baculovirus expression plasmid pVL1393 and expressed in sf9 cells using standard techniques (BD PharMingen, San Diego, CA). The protein was purified from culture supernatants by affinity chromatography using the anti-HEL Ab 154.1B12 bound to cyanogen bromide-activated Sepharose 4B. The bound material was eluted with 50 mM glycine and 150 mM NaCl, pH 2.5, directly into neutralization buffer (1 M Tris-HCl, pH 8.6). The fractions containing protein were pooled and dialyzed against PBS. The purified protein obtained was analyzed on a 15% polyacrylamide gel containing SDS. The purified protein was also analyzed for {beta}1–4 glycosidase activity (25). In this assay, a 25 mg% solution of lyophilized Micrococcus lysodeikticus (Sigma-Aldrich, St. Louis, MO) was prepared in 50 mM NaPO4 buffer, pH 6.2. To this suspension, 2 mg of either HEL or sHEL/Hb was added, and the absorption at 540 nm was measured at 1-min intervals. The rate of decrease in A540, as bacterial cell walls are enzymatically digested, is a measure of enzyme activity. The sHEL/Hb protein was also analyzed for its ability to stimulate T cell hybridomas, as outlined below.

T cell hybridomas and hybridoma assays

T cell hybridomas were used to detect the presence of stimulatory ligands, as previously described (24). Briefly, 1 x 105 T hybridoma cells were mixed with 5 x 104 APCs in each well of a 96-well plate. To this mixture, increasing amounts of a peptide or protein were added, and the plates were incubated for 24 h at 37°C in 5% CO2. Triplicate wells were performed at each Ag concentration. Stimulation of each hybridoma was then determined by measuring the level of IL-2 produced using the IL-2-dependent cell line CTLL-2. The data shown represent one typical assay, and each assay was repeated at least three times with similar results. The T cell hybridomas used in these assays were YO1.6, which is specific for Hb(64–76)/I-Ek (26), and 3A9 (27), which is specific for HEL(46–61)/I-Ak. CH27 cells were used for Ag processing and presentation. In some assays (Fig. 1E), the CH27 cells were lightly fixed with 1% paraformaldehyde in HBSS for 15 min at room temperature. The cells were then washed three times in HBSS to remove the paraformaldehyde before use.

Transgenic T cell proliferation assays

Primary T cells were stimulated with Hb(64–76) peptide, as described (28). Briefly, 5 x 105 splenocytes/well were incubated with increasing amounts of peptide for 48 h, pulsed with 0.4 µCi of [3H]TdR for 18 h, and harvested. Proliferation was measured as cpm incorporated (mean of triplicate wells). Results from several experiments were then averaged to obtain the data shown in Fig. 4.



View larger version (51K):
[in this window]
[in a new window]
  		FIGURE 4. Negative selection of 3.L2tg T cells correlates with the level of circulating self protein. Top four panels, Show the effect on thymic development when 3.L2tg mice were bred to mice expressing either the sHEL/Hb protein at 0.1 ng/ml, the sHEL/Hb protein at 1.5 ng/ml (induced), or the native Hb{beta}d protein. FACS analysis for CD4 and CD8 demonstrates a reduction in CD4-SP and double-positive compartments as the circulating concentration of sHEL/Hb is increased (top right compared with middle left). The CD4-SP compartment is reduced even further when 3.L2tg mice are bred to Hb{beta}d mice. These mice express the native Hb upon which the system is based (middle right panel). These results should be compared with 3A9 x sHEL/Hb mice (lower right panel), which show complete elimination of all Ag-specific 3A9 T cells at the lowest circulating concentration of sHEL/Hb. The 3.L2tg mouse bred to a membrane form of the HEL/Hb protein expressed in all class II+ APCs (lower left panel) is also shown for comparison. The percentage of thymocytes in each quadrant is indicated, and the numbers represent the mean values derived from 8–20 mice per group. The average thymus size is as follows: 3.L2tg, 108 ± 9 x 106; 3.L2tg x sHEL/Hb, 56 ± 6 x 106; 3.L2tg x sHEL/Hb x tTA, 38 ± 7 x 106; 3A9 x sHEL/Hb, 68 ± 3 x 106.

 
Flow cytometry

Single cell suspensions of thymocytes or splenocytes were stained in FACS buffer (PBS supplemented with 0.5% BSA and 0.1% sodium azide) using the following protocol. Aliquots of cells (1 x 106/sample in 100 µl FACS buffer) were placed in polypropylene culture tubes (12 x 75 mm; VWR, West Chester, PA) and incubated on ice for 1 h with the biotinylated or directly labeled Abs. Cells were then washed once with 3 ml of FACS buffer and incubated for 30 min on ice with the streptavidin-fluorochrome conjugate where appropriate. Cells were washed again, fixed for 18–24 h in PBS-azide (0.1%) plus 1% paraformaldehyde, and analyzed on a FACScan (BD Biosciences, Mountain View, CA) analytical flow cytofluorometer using CellQuest (BD Biosciences) software. Samples were gated on live cells, and 100,000 live cell events per sample were collected.

Peptides

The peptides used in this study were synthesized, purified, and analyzed, as previously described (29). The peptide sequences, in single-letter amino acid code, are: GKKVITAFNEGLK (N72(wild type)); NTDGSTDYGILQINSR (HEL(46–61)).

Antibodies

The Abs used were PE-conjugated anti-mouse CD4 (BD PharMingen); FITC-conjugated anti-mouse CD8a (BD PharMingen); Fc block (BD PharMingen); biotinylated 3.L2 clonotypic Ab (CAB); biotinylated 3A9 CAB; and 154.1B12 anti-HEL Ab (9). Cells stained with biotinylated Abs were subsequently incubated with Tricolor streptavidin (Caltag, San Francisco, CA).

Lymph node proliferation assays

Mice were immunized s.c. with 20 nmol of either HEL or Hb(64–76) emulsified in CFA. After 10 days, the draining lymph nodes were removed and a single cell suspension was prepared. The suspension was washed, and 5 x 105 lymph node cells in a volume of 100 µl were added to wells in a 96-well plate. Appropriate dilutions of HEL protein or Hb(64–76) peptide were then added in a volume of 100 µl. RPMI 1640 with 0.5% normal mouse serum was used as the culture medium. The plates were incubated at 37°C for 72 h, pulsed with 0.4 µCi/well of [3H]TdR for 18 h, and harvested. Proliferation was measured as cpm incorporated (mean of triplicate wells). Results from three experiments with three or more mice per group were averaged to obtain the data presented in Fig. 3.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. Tolerance to the circulating sHEL/Hb protein. A, Tolerance to the Hb(64–76) epitope was assessed by immunizing either sHEL/Hb only ({circ}), sHEL/Hb x tTA mice ({triangleup}), or B6.AKR control mice ({diamondsuit}) s.c. with 20 nmol of the Hb(64–76) peptide emulsified in CFA. Draining lymph node cells were removed 10 days after immunization and restimulated in culture, as described in Materials and Methods. Results show the mean of the counts incorporated vs the concentration of stimulating Ag in the culture well for all experiments. B, Tolerance to HEL was assessed by immunizing with HEL protein and restimulating in vitro with HEL, as described.

 
ELISA for sHEL/Hb

Immulon 1b plates were coated overnight at 4°C with 100 µl of the anti-HEL mAb 154.1B12 at a concentration of 5 µg/ml in carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). After coating with the capture Ab, the plates were washed five times with wash buffer (0.01 M Na phosphate, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) using an EL 404 microplate washer (Bio-Tek Instruments, Winooski, VT). Nonspecific binding was blocked by adding 100 µl/well of RPMI plus 10% FCS for 1 h at room temperature, and the plates were washed as above. Standards of known concentration were prepared with HEL (Sigma-Aldrich), and serum samples from the mice were analyzed neat or diluted 1/2 to 1/4. All dilutions were performed with RPMI 1640 plus 10% FCS. Samples and standards were added to triplicate wells in a volume of 100 µl, and the plates were incubated overnight at 4°C. Following Ag capture, the plates were washed as above, and 100 µl of rabbit antisera to HEL diluted 1/1000 was added to each well (9). The plates were then incubated for 1 h at room temperature and washed as above. Next, 100 µl of HRP-coupled goat anti-rabbit antisera (Tago, Burlingame, CA) diluted 1/2000 was added to each well, and the plates were incubated for 1 h at room temperature and washed. Ag was detected with 2.2'azino-di(3-athyl-benethiazolinsulfonate (Roche Diagnostics, Mannheim, Germany) in substrate buffer (0.06 M citric acid, 0.09 M Na2HPO4, pH 4.2). H2O2 to a final concentration of 0.1% was added to the substrate just before use. The plates were incubated in the dark at room temperature for 30 min, and the results were read on a microplate reader (Elx800; Bio-Tek Instruments) at a wavelength of 415 nm.

Thymic transplants

Thymectomy was performed on B6.AKR neonatal mice on day of life 5, as follows. A midline thoracic skin incision was made, and the thoracic cavity was exposed by a bilateral incision of the thorax. The thymus was exposed, and a sterile cotton tip previously wet with cold sterile PBS was placed under the organ. The thymus was then lifted, gently cut from the thoracic cavity, placed in chilled sterile PBS, and kept on ice in PBS until transplanted. One thymic lobe from these day 5 neonatal B6.AKR mice was transplanted under the kidney capsule of either 6-wk-old 3A9+/-, 3A9+/- x sHEL/Hb+/-, 3.L2tg+/-, or 3.L2tg+/- x sHEL/Hb+/- recipient mice. All recipient lines were also on the B6.AKR (H2k) background. Recipient mice were first anesthetized with a mouse cocktail that contained ketamine HCl and xylazine at 0.1 ml/20 g body weight administered i.p. The skin of the back and lumbar region was shaved on the left side. The mouse was placed on its right side on a heating pad for temperature regulation. Mice were immobilized by taping across the upper ribs and lower hind limbs. The shaved area of skin was sterilized with 70% ethanol and povidone-iodine 1%. A longitudinal 1-cm skin incision was made on the left lumbar region. The abdominal muscles were then incised, and the left kidney was exposed and gently removed from the peritoneal cavity. The kidney was immobilized outside the cavity with a curved hemostat placed under the kidney. Using a microdissection microscope, a 3- to 5-mm longitudinal incision was made on the kidney capsule, which was lifted gently with a fine point jewelers forceps. One thymic lobe was placed under the kidney capsule and away from the incision point. The kidney was returned to the abdominal cavity and the abdominal muscles were closed with 4-0 Vycril. The skin was closed with 4-0 Ethilon suture, and Enrofloxacin was administered i.m. Recipient mice were kept warm until recovery. Endogenous and transplanted thymi were examined 30 days after transplantation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation and characterization of a neo-self Ag

Our system is based on regulated expression of a neoprotein carrying an epitope derived from murine Hb, a well-characterized self Ag (26, 28, 29, 30). Briefly, there are two allelic variants of the Hb minor {beta}-chain described in the inbred mouse population, d and s (31). The product of the d allele is recognized by s mice as allo-Ag, with most of the response directed toward Hb(64–76)/I-Ek. To study T cell tolerance to this immunodominant epitope, we created a chimeric gene by inserting the codons for Hb(64–76) into a cDNA for HEL. Creation of an epitope-tagged form of Hb(64–76) was desirable for a number of reasons: 1) expression of free (secreted) Hb requires coordinated expression of both the {alpha}- and {beta}-chain; 2) free Hb is nephrotoxic (32); 3) the well-characterized HEL(46–61) epitope adds versatility to the system. To test stability and immunogenicity of the epitope-tagged protein, the coding region of the construct shown in Fig. 1A was cloned into the baculovirus expression plasmid pVL1393. This construct was then transfected into sf9 cells, which secreted the chimeric protein (rHEL/Hb) into culture supernatants at a concentration of 1 µg/ml. (Note that rHEL/Hb refers to the protein produced in culture by insect cells, and sHEL/Hb refers to the secreted protein made by transgenic mice.) The rHEL/Hb protein was purified by affinity chromatography with an anti-HEL Ab and analyzed by acrylamide gel electrophoresis. Results show a single band running slightly larger than the 14.6-kDa band seen with unmodified HEL (Fig. 1B).

Next, stability, processing, and presentation of the rHEL/Hb protein were assessed. Using a modified muramidase assay (25), the hydrolytic activity of rHEL/Hb was demonstrated by mixing either HEL or rHEL/Hb with a suspension of UV-killed, lyophilized M. lysodeikticus. Addition of either HEL or rHEL/Hb resulted in cleavage of the {beta}-1,4 glycosidic bonds found in the peptidoglycan layer of the bacterial cell walls and clearing of the suspension (Fig. 1C). It is important to note that the substrate-binding site in HEL consists of six subsites that are brought together in the properly folded molecule (33). Based on the x-ray structure (34), the additional amino acids inserted into rHEL/Hb are predicted to form a random-chain external loop, which should permit folding, but could interfere with substrate binding. Although rHEL/Hb is not as active as HEL, this demonstration of enzyme activity strongly implies both proper folding and stability of the rHEL/Hb protein.

Previously, we demonstrated that both HEL(46–61) and Hb(64–76) were processed and presented from a membrane-bound form of HEL/Hb (24, 28). The same is true for the recombinant secreted protein. When varying concentrations of either the native Hb protein or rHEL/Hb were mixed with the hybridoma YO1.6 and APCs, the hybridoma was stimulated and produced IL-2 (Fig. 1D). Furthermore, the proliferative responses to Hb protein and rHEL/Hb were equivalent. The T cell hybridoma 3A9, which recognizes HEL(46–61) in the context of I-Ak, was also stimulated by the rHEL/Hb protein (data not shown). Finally, fixing the APCs prevented Ag processing and eliminated the ability of both Hb protein and rHEL/Hb to stimulate the hybridomas, as in Fig. 1E. In summary, these results clearly demonstrate that the chimeric rHEL/Hb protein is stable, retains enzyme activity, and, when processed by APCs, allows recognition of Hb(64–76)/I-Ek and HEL(46–61)/I-Ak complexes by Ag-specific T cells.

Generation of tet-responsive sHEL/Hb transgenic mice

Tet-controlled expression of the neo-self Ag was achieved using two independently derived transgenes: a tTA and a reporter (secreted or sHEL/Hb). In our modification of this binary system, originally developed by Bujard and colleagues (18), the tTA transgene was generated from the 2.7-kb XhoI-PflMI fragment of pUHG 15-1. This transcriptional activator is a fusion protein containing the tet repressor and the HSV VP16 transactivator (22). Binding of tet to the tet repressor induces a conformational change, eliminating binding at the tet operator and transactivation (35). Expression of the tTA transgene is controlled by the major immediate-early human CMV promoter and enhancer, which together have been shown to efficiently drive expression of transgenes in multiple tissues of epithelial and endothelial origin (20, 21). The sHEL/Hb reporter was derived from a 3.2-kb BamHI-EaeI fragment of pJB1.7 (Fig. 1A). Expression of the sHEL/Hb transgene is controlled by a minimal 52-bp thymidine kinase promoter, preceded by seven tet operator sequences. In double transgenic mice, tTA should bind to the tet operator and drive transcription of the sHEL/Hb transgene. In the presence of tet or in single transgenic (sHEL/Hb only) mice, the minimal 52-bp thymidine kinase promoter alone should be unable to support transcription of sHEL/Hb. Both the tTA line and the sHEL/Hb line were generated independently in B6 mice. Founders were obtained and bred onto the congenic B6.AKR (H2k) background, which is necessary for presentation of Hb(64–76) and HEL(46–61). RT-PCR data from sHEL/Hb mice demonstrated very low levels of transcripts from many tissues, including liver, gut, testes, and thymus (data not shown).

Inducible expression of the sHEL/Hb transgene

The HEL and sHEL/Hb proteins can reliably be detected by ELISA down to a concentration of 0.05–0.10 ng/ml (Fig. 2A). The capture Ab for the ELISA detects both native and denatured forms of the protein (unpublished data). When serum from single transgenic mice (sHEL/Hb only) was tested for the presence of the chimeric protein, most mice expressed levels <= 0.1 ng/ml. Given the sensitivity of the anti-HEL ELISA, the value shown in Fig. 2B represents the upper limit of the range of protein expressed in these mice. Double transgenic mice, produced by crossing the tTA and sHEL/Hb founder lines, express ~1.5 ng/ml. At minimum, this represents a 15-fold induction of sHEL/Hb expression. Placing double transgenic mice on tet in the drinking water resulted in suppression of sHEL/Hb expression to baseline levels by 48 h (data not shown). Removal of tet resulted in protein re-expression with similar kinetics. In any event, the single and double transgenic mice have circulating levels of self protein well below the levels achieved in other models of self tolerance.



View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 2. Expression of sHEL/Hb in transgenic mice. A, Typical ELISA for HEL demonstrating the sensitivity of the assay (0.1 ng/ml). B, This ELISA also detects sHEL/Hb in mouse sera. Mice with the sHEL/Hb transgene express a maximum of ~0.1 ng/ml. Mice with both the tTA and sHEL/Hb transgenes (induced) express 1.5 ± 0.2 ng/ml.

 
The threshold for tolerance to the circulating self protein sHEL/Hb

To determine whether these low levels of circulating sHEL/Hb protein were sufficient for tolerance induction, we performed lymph node proliferation assays. Single and double transgenic mice were immunized s.c. with either 20 nmol of Hb(64–76) peptide or 20 nmol of HEL protein emulsified in CFA. After 10 days, the draining lymph nodes were harvested, and the cells were restimulated in culture with either the Hb(64–76) peptide or HEL protein. The lymph node cultures were incubated for 72 h, and the proliferative response was determined as a measure of tolerance. Results of two experiments, with three mice per group in each experiment, are summarized in Fig. 3. The data show that primed lymphocytes from mice with 1.5 ng/ml of circulating sHEL/Hb protein fail to proliferate under these conditions and are completely tolerant to both the Hb(64–76) peptide and the HEL protein. However, single transgenic mice with <=0.1 ng/ml of circulating self protein are only partially tolerant. After immunization with Hb(64–76), T cells from sHEL/Hb mice are ~30 times less sensitive to restimulation than B6.AKR controls. Similarly, T cells from sHEL/Hb mice immunized with HEL are 30–100 times less sensitive to restimulation with HEL than B6.AKR mice.

Graded tolerance to Hb(64–76) by negative selection

The mechanism for tolerance induction was investigated at the clonal level by crossing the sHEL/Hb mice to mice expressing transgenic TCRs. We selected the 3.L2tg and 3A9 TCR transgenic lines, which are specific for Hb(64–76) and HEL(46–61), respectively. Overall, analysis of thymocyte development in 3.L2tg x sHEL/Hb mice shows a decrease in CD4 single-positive (CD4-SP) thymocytes, a decrease in double-positive thymocytes, and an increase in the double-negative population, all consistent with negative selection (Fig. 4). When the level of circulating protein is increased in 3.L2tg x sHEL/Hb, tTA mice, a similar phenotype is observed. Increasing the level of circulating self protein still further, as in 3.L2tg x Hb{beta}d mice, has little additional effect on the major thymic compartments.

In each case, thymocytes with high levels of the 3.L2 TCR escape negative selection and can be found in the periphery. FACS analysis of splenocytes from 3.L2tg x sHEL/Hb mice with a CAB reveals that 9.2% CD4+ cells fall within the CABhigh gate, in contrast to the 50% seen in normal 3.L2tg mice. The percentage of CD4+, CAB+ splenocytes decreases to 5.2% in induced mice (3.L2tg x sHEL/Hb, tTA), reflecting the increase in circulating levels of sHEL/Hb protein. The relative number of CD4+, CAB+ splenocytes falls still further in 3.L2tg x Hb{beta}d mice, demonstrating the effect of natural levels of the Hb(64–76) epitope achieved by processing of endogenous Hb. Also included in Fig. 5 are 3.L2tg x mHEL/Hb mice. The mHEL/Hb transgene is expressed as an integral membrane protein in all MHC class II+ cells and is likely to provide the greatest number of I-Ek/Hb(64–76) complexes per APC (10, 36). As expected, these mice are the most efficient in eliminating 3.L2tg T cells.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5. The number of transgenic Ag-specific T cells in the periphery reflects the efficiency of negative selection. CD4+ splenocytes from the mice used in Fig. 4 were examined by FACS for expression of the transgenic 3.L2 or 3A9 TCR. The data are shown as histograms, with the heavy filled line representing the experimental condition, the thin filled line the negative control (B6.AKR), and the dashed line the positive control (3.L2tg or 3A9). The marker indicates the percentage of CD4+ splenocytes that express high levels of the clonotypic TCR. In normal 3.L2tg mice, ~50% of CD4+ splenocytes are clonotypehigh.

 
In normal 3.L2tg mice, ~9% of splenocytes are CD4+, and about one-half of these are CAB+. It is not surprising that elimination of 3.L2tg T cells by negative selection also results in an overall decrease in the percentage of splenocytes that are CD4+. The magnitude of this overall decrease reflects the efficiency of negative selection. The average number of CD4+, CAB+ T cells per 105 splenocytes is shown in Fig. 6A. The average size of the spleens was similar (3.L2tg, 159 ± 13 x 106; sHEL/Hb x 3.L2tg, 139 ± 15 x 106; sHEL/Hb x tTA x 3.L2tg, 142 ± 13 x 106), and these estimates therefore accurately reflect the total number of CABhigh cells in each group. Importantly, when these splenocytes are stimulated by Hb(64–76) peptide in vitro, the magnitude of the proliferative responses also reflects the predicted number of CAB+ cells (Fig. 6B). Experiments with plate-bound CAB used to cross-link the 3.L2 TCR yielded similar results (data not shown), implying that the observed Hb(64–76) response is not due to the transgenic 3.L2 TCR {beta}-chain pairing with endogenous TCR {alpha}-chains and creating Hb(64–76)-reactive T cells. Together, these data demonstrate that the remaining CAB+ cells in each cross are functional and that the number of cells correlates with the serum level of the sHEL/Hb protein. This graded negative selection of 3.L2tg T cells stands in contrast to negative selection of 3A9 T cells. Even at the lowest Ag dose achieved in our system, all thymocytes with the 3A9 TCR were eliminated, resulting in an absence of clonotype+ cells in the periphery and in complete tolerance to HEL in 3A9 x sHEL/Hb mice (Figs. 5 and 6B).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. The proliferative response of 3.L2tg T cells upon stimulation with Hb(64–76) peptide correlates with relative number of 3.L2tg T cells/1 x 105 splenocytes and demonstrates that the cells detected by FACS are functional. A, The relative number of clonotypehigh T cells per 1 x 105 splenocytes. B, The proliferative response of splenocytes from the various selecting backgrounds. Results from several experiments were averaged to obtain the data shown: 3.L2tg (n = 20, {blacksquare}), 3.L2tg x sHEL/Hb (n = 19, {circ}), 3.L2tg x sHEL/Hb, tTA (n = 8, {triangleup}), 3.L2tg x Hb{beta}d (n = 6, •), 3.L2tg x mHEL/Hb (n = 8, {square}), and 3A9 x sHEL/Hb (n = 6, {blacktriangledown}).

 
Negative selection is due to the circulating self protein

Because low levels of sHEL/Hb RNA could be detected in the thymi of single transgenic mice by RT PCR (data not shown), it was possible that tolerance in this system was due to local secretion and reuptake of the sHEL/Hb protein within the thymic cortical or medullary epithelium and not reflective of the circulating levels. To evaluate this possiblity, we transplanted B6.AKR thymi under the kidney capsule of 3.L2tg x sHEL/Hb, 3A9 x sHEL/Hb, or control mice. The development of transgenic T cells within the transplanted and native thymi was analyzed by FACS 1 mo after transplantation, and the results are shown in Fig. 7. Importantly, the B6.AKR thymi showed negative selection of transgenic T cells equivalent to that seen in the native thymi. Because the sHEL/Hb protein is not expressed in the donor B6.AKR thymi, negative selection must result from low-level expression of this circulating self protein in recipient mice. The role of cross-presentation of sHEL/Hb and recirculation of recipient dendritic cells in the negative selection observed in donor thymi cannot be determined in these experiments (37). Control 3.L2tg and 3A9 mice demonstrate complete repopulation of transplanted B6.AKR thymi with transgenic T cells after 1 mo, with no difference between the transplanted and native thymi.



View larger version (68K):
[in this window]
[in a new window]
  		FIGURE 7. Transplanted nontransgenic (B6.AKR) thymi show negative selection in 3.L2tg, sHEL/Hb and 3A9, sHEL/Hb recipients. A, Thymic lobes from neonatal B6.AKR mice were transplanted under the kidney capsule of 3.L2tg (left panels) and 3.L2tg, sHEL/Hb (right panels) mice. In the absence of sHEL/Hb (left panels), 3.L2tg T cell development proceeds normally after 4 wk, with 60–70% of CD4-SP thymocytes expressing the 3.L2 TCR. When the 3.L2tg mice also express low circulating levels of the sHEL/Hb protein, equivalent negative selection occurs in both the native and transplanted thymi. Percentages of cells in each quadrant are the averages from 3 experiments and 12 transplant recipients. *, Due to the small number of events, the number of CD4-SP clonotypehigh cells was divided by the number of CD4-SP clonotypehigh cells in the 3.L2tg control to obtain these percentages. B, Thymic lobes from neonatal B6.AKR mice were transplanted under the kidney capsule of 3A9 (left panels) and 3A9, sHEL/Hb (right panels) mice. Analysis was performed as in A above. Percentages of cells in each quadrant are the averages from 2 experiments with 14 transplant recipients.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have developed an inducible transgenic system controlling expression of a neo-self Ag to investigate the mechanism and efficiency of tolerance to low levels of circulating self proteins. In this system, transcription of the neo-self Ag sHEL/Hb is controlled by the tet-responsive transactivator tTA. Serum levels of the sHEL/Hb protein are 0.05–0.1 ng/ml in single transgenic sHEL/Hb mice, and 1.5 ng/ml in bigenic sHEL/Hb, tTA mice. We find that when circulating levels of sHEL/Hb protein are <=0.1 ng/ml (<=1 x 10-11 M), tolerance is incomplete and mice immunized with HEL or Hb(64–76) are capable of a T cell response, albeit a diminished one. These data imply that at low Ag levels, some T cells escape central and peripheral tolerance mechanisms and are available for self-specific T cell responses. At higher levels of circulating self protein (1.5 ng/ml or 1 x 10-10 M), tolerance was complete. As such, these data define a tolerogenic threshold for sHEL/Hb, which correlates with the level of circulating protein.

This concept of a tolerogenic threshold was investigated further at the clonal level by breeding bigenic sHEL/Hb, tTA mice to 3.L2tg TCR transgenic mice, which are specific for Hb(64–76). In the progeny of this cross, negative selection is graded within the range of protein concentrations examined. When circulating sHEL/Hb levels are <=0.1 ng/ml, many 3.L2tg T cells escape negative selection and accumulate in the periphery, where they comprise 9.2% of all CD4+ splenocytes (as opposed to 50% of all CD4+ splenocytes in the absence of negative selection). These surviving 3.L2tg T cells generally have lower levels of the clonotypic receptor than normal 3.L2tg T cells, implying that at low Ag doses negative selection preferentially eliminates cells with the highest levels of the 3.L2tg TCR (12). Alternatively, 3.L2tg thymocytes may actively down-regulate their TCR in response to this level of stimulation. Higher Ag doses in the 1.5 ng/ml (1 x 10-10 M) range eliminate more, but not all, 3.L2tg T cells.

In this transgenic system, we can detect sHEL/Hb transcripts in the thymus by RT-PCR, but have not examined thymi for sHEL/Hb protein expression and do not directly determine the potential contribution of thymic sHEL/Hb to central tolerance. Transgenic and peripheral proteins expressed at very low levels in the thymus do induce central tolerance in other systems, even in the apparent absence of mRNA expression (12, 13, 14, 15). Nevertheless, based on the immunization and thymic transplantation studies presented in this work, we can conclude that the circulating sHEL/Hb is sufficient to induce the observed phenotypes.

Importantly, Ag-specific, clonotype+ 3.L2tg T cells do escape the thymus in mice expressing the lowest levels of sHEL/Hb protein, and are found in the periphery in significant numbers. Some of these are L-selectinlow and CD44high, implying that they are no longer naive and are turning over in the periphery (unpublished data). Presumably, this activation is in response to the endogenous Hb(64–76)/I-Ek complexes found on peripheral APCs, as a direct consequence of the processing and presentation of circulating sHEL/Hb. Peripheral tolerogenic mechanisms must therefore operate to limit expansion of this self-reactive population.

It is also possible that 3.L2tg T cells escape central tolerance by expressing a second TCR, comprised of an endogenous {beta}-chain paired with the transgenic {alpha}-chain. Many, if not all, CABhigh 3.L2tg and 3.L2tg x sHEL/Hb T cells possess a second receptor (38) (data not shown). This observation raises the possibility that some cells could be positively selected on one TCR and activated in the periphery on the second TCR. In one scenario, dual receptor T cells escape normal central tolerance mechanisms and are activated by self Ags in the periphery, predisposing to autoimmunity (39, 40, 41). From another point of view, dual receptor T cells rescue nonfunctional T cells in the thymus and are activated by foreign Ag in the periphery, thereby expanding the T cell repertoire (42). Regardless of their role in immune responses, T cells bearing two receptors may not explain all the data, as non-TCR transgenic mice also showed partial tolerance at low Ag levels. The influence of a potential second receptor on tolerance mechanisms was not directly examined in these investigations.

The 3.L2tg T cell data stand in direct contrast to the experiments with HEL(46–61)-specific 3A9 T cells, which appear more sensitive to the same low level of circulating self protein. The 3A9 T cells are efficiently eliminated in the thymus of sHEL/Hb x 3A9 mice, and these mice are completely tolerant. Taken together, these transgenic T cell studies support the immunization data by illustrating the spectrum of T cell responses available. In other words, they demonstrate that for self proteins circulating in the serum at low levels (1 x 10-11 M), tolerance may be incomplete due to the inability to eliminate all self-reactive T cell clones in the thymus. Clearly, many factors not directly examined in this study are likely to influence the tolerogenic threshold at the clonal level, including TCR ligand kinetics (t1/2 of TCR-MHC/peptide complexes) and the number of MHC/peptide complexes available. In regard to the latter factor, it has been estimated that as few as three MHC/peptide complexes per APC are sufficient for complete negative selection of 3A9 T cells (10). Although no such estimate has been possible for 3.L2tg T cells, the number is likely to be considerably larger. This speculation is based on experiments involving a membrane form of HEL/Hb expressed in all thymic APCs, which failed to completely tolerize 3.L2tg mice (28).

It is important to note that in models of transplantation tolerance, proteins do not need to reach the thymus and thymic APCs via the circulatory system to induce central tolerance. Serum proteins can be picked up by bone marrow-derived dendritic cells in the periphery, which then migrate to the thymus and induce negative selection (43, 44, 45). Furthermore, there is recent evidence to suggest that both myeloid and lymphoid precursors give rise to all the different dendritic cell populations, including those found in the thymus (46). These observations suggest that central tolerance via the recirculation of peripheral dendritic cells through the thymus is at least plausible, although the relevance of these findings to our model remains to be established. The participation of multiple tolerogenic mechanisms would not be surprising, given the essential nature of this process.


   Acknowledgments
 
We are most grateful to Dr. Paul Allen, who supported this project with resources and advice from its inception; Dr. Herman Bujard, who provided the plasmids pUHG 15-1 and pUHC 13-6 used for tet-regulated gene expression; Dr. Emil Unanue, who provided the 3A9 CAB and 3A9 TCR transgenic mice; and Dr. Talal Chatila for advice and review of the manuscript.


   Footnotes
 
1 This work was supported by research grants awarded to C.B.W. from the March of Dimes Birth Defects Foundation (5-FY98-0722) and from the National Institutes of Health (AR01998 and AI47154). Back

2 Current address: Southern Illinois University School of Medicine, Springfield, IL 62702. Back

3 Address correspondence and reprint requests to Dr. Calvin B. Williams, Medical College of Wisconsin, MACC Fund Research Center, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail address: cwilliam{at}mcw.edu Back

4 Abbreviations used in this paper: MT, metallothionein; HEL, hen egg lysozyme; CAB, clonotypic Ab; Hb, hemoglobin; hCRP, human C-reactive protein; sHEL, secreted HEL; SP, single positive; tet, tetracycline; tTA, tet-regulated transactivator. Back

Received for publication October 3, 2002. Accepted for publication January 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Garza, K. M., V. S. Chan, P. S. Ohashi. 2000. T cell tolerance and autoimmunity. Rev. Immunogenet. 2:2.[Medline]
  2. Kamradt, T., N. A. Mitchison. 2001. Tolerance and autoimmunity. N. Engl. J. Med. 344:655.[Free Full Text]
  3. Klein, L., T. Klein, U. Rüther, B. Kyewski. 1998. CD4 T cell tolerance to human C-reactive protein, an inducible serum protein, is mediated by medullary thymic epithelium. J. Exp. Med. 188:5.[Abstract/Free Full Text]
  4. Zal, T., A. Volkmann, B. Stockinger. 1994. Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen. J. Exp. Med. 180:2089.[Abstract/Free Full Text]
  5. Akkaraju, S., W. Y. Ho, D. Leong, K. Canaan, M. M. Davis, C. C. Goodnow. 1997. A range of CD4 T cell tolerance: partial inactivation to organ-specific antigen allows nondestructive thyroiditis or insulitis. Immnunity 7:255.
  6. Davey, G. M., S. L. Schober, B. T. Endrizzi, A. K. Dutcher, S. C. Jameson, K. A. Hogquist. 1998. Preselection thymocytes are more sensitive to T cell receptor stimulation than mature T cells. J. Exp. Med. 188:1867.[Abstract/Free Full Text]
  7. Klein, L., B. Kyewski. 2000. "Promiscuous" expression of tissue antigens in the thymus: a key to T-cell tolerance and autoimmunity?. J. Mol. Med. 78:483.[Medline]
  8. Hanahan, D.. 1998. Peripheral-antigen-expressing cells in thymic medulla: factors in self-tolerance and autoimmunity. Curr. Opin. Immunol. 10:656.[Medline]
  9. Yule, T. D., A. Basten, P. M. Allen. 1993. Hen egg-white lysozyme-specific T cells elicited in hen egg-white lysozyme-transgenic mice retain an imprint of self-tolerance. J. Immunol. 151:3057.[Abstract]
  10. Peterson, D. A., R. J. DiPaolo, O. Kanagawa, E. R. Unanue. 1999. Cutting edge: negative selection of immature thymocytes by a few peptide-MHC complexes: differential sensitivity of immature and mature T cells. J. Immunol. 162:3117.[Abstract/Free Full Text]
  11. Bogen, B., Z. Dembic, S. Weiss. 1993. Clonal deletion of specific thymocytes by an immunoglobulin idiotype. EMBO J. 12:357.[Medline]
  12. Heath, W. R., J. Allison, M. W. Hoffmann, G. Schonrich, G. Hammerling, B. Arnold, J. F. Miller. 1992. Autoimmune diabetes as a consequence of locally produced interleukin-2. Nature 359:547.[Medline]
  13. Von Herrath, M. G., J. Dockter, M. B. Oldstone. 1994. How virus induces a rapid or slow onset insulin-dependent diabetes mellitus in a transgenic model. Immunity 1:231.[Medline]
  14. Jolicoeur, C., D. Hanahan, K. M. Smith. 1994. T-cell tolerance toward a transgenic {beta}-cell antigen and transcription of endogenous pancreatic genes in thymus. Proc. Natl. Acad. Sci. USA 91:6707.[Abstract/Free Full Text]
  15. Antonia, S. J., T. Geiger, J. Miller, R. A. Flavell. 1995. Mechanisms of immune tolerance induction through the thymic expression of a peripheral tissue-specific protein. Int. Immunol. 7:715.[Abstract/Free Full Text]
  16. Oehen, S., L. Feng, Y. Xia, C. D. Surh, S. M. Hedrick. 1996. Antigen compartmentation and T helper cell tolerance induction. J. Exp. Med. 183:2617.[Abstract/Free Full Text]
  17. Smith, K. M., D. C. Olson, R. Hirose, D. Hanahan. 1997. Pancreatic gene expression in rare cells of thymic medulla: evidence for functional contribution to T cell tolerance. Int. Immunol. 9:1355.[Abstract/Free Full Text]
  18. Furth, P. A., L. St Onge, H. Boger, P. Gruss, M. Gossen, A. Kistner, H. Bujard, L. Hennighausen. 1994. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91:9302.[Abstract/Free Full Text]
  19. Kistner, A., M. Gossen, F. Zimmermann, J. Jerecic, C. Ullmer, H. Lubbert, H. Bujard. 1996. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. USA 93:10933.[Abstract/Free Full Text]
  20. Baskar, J. F., P. P. Smith, G. S. Ciment, S. Hoffmann, C. Tucker, D. J. Tenney, A. M. Colberg-Poley, J. A. Nelson, P. Ghazal. 1996. Developmental analysis of the cytomegalovirus enhancer in transgenic animals. J. Virol. 70:3215.[Abstract]
  21. Baskar, J. F., P. P. Smith, G. Nilaver, R. A. Jupp, S. Hoffmann, N. J. Peffer, D. J. Tenney, A. M. Colberg-Poley, P. Ghazal, J. A. Nelson. 1996. The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice. J. Virol. 70:3207.[Abstract]
  22. Gossen, M., H. Bujard. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547.[Abstract/Free Full Text]
  23. Kumagai, I., S. Kojima, E. Tamaki, K. Miura. 1987. Conversion of Trp 62 of hen egg-white lysozyme to Tyr by site-directed mutagenesis. J. Biochem. 102:733.[Abstract/Free Full Text]
  24. Williams, C. B., K. Vidal, D. L. Donermeyer, D. A. Peterson, J. M. White, P. M. Allen. 1998. In vivo expression of a T cell receptor antagonist: T cells escape central tolerance but are antagonized in the periphery. J. Immunol. 161:128.[Abstract/Free Full Text]
  25. Morsky, P.. 1983. Turbidimetric determination of lysozyme with Micrococcus lysodeikticus cells: reexamination of reaction conditions. Anal. Biochem. 128:77.[Medline]
  26. Evavold, B. D., S. G. Williams, B. L. Hsu, S. Buus, P. M. Allen. 1992. Complete dissection of the Hb(64–76) determinant using Th1, Th2 clones, and T cell hybridomas. J. Immunol. 148:347.[Abstract]
  27. Johnson, N. A., F. Carland, P. M. Allen, L. H. Glimcher. 1989. T cell receptor gene segment usage in a panel of hen-egg white lysozyme specific, I-Ak-restricted T helper hybridomas. J. Immunol. 142:3298.[Abstract]
  28. Williams, C. B., D. L. Engle, G. J. Kersh, J. M. White, P. M. Allen. 1999. A kinetic threshold between positive and negative selection based on the longevity of the T cell receptor-ligand complex. J. Exp. Med. 189:1531.[Abstract/Free Full Text]
  29. Kersh, G. J., P. M. Allen. 1996. Structural basis for T cell recognition of altered peptide ligands: a single T cell receptor can productively recognize a large continuum of related ligands. J. Exp. Med. 184:1259.[Abstract/Free Full Text]
  30. Williams, C. B., P. M. Allen. 1997. The study of self-tolerance using murine haemoglobin as a model self antigen. G. R. Bock, and J. A. Goode, eds. Novartis Foundation Symposium 215 Immunological Tolerance 41. John Wiley & Sons, West Sussex.
  31. Russell, E. S., E. C. McFarland. 1974. Genetics of mouse hemoglobins. Ann. NY Acad. Sci. 241:25.[Medline]
  32. Cerda, S., S. K. Oh. 1990. Methods to quantitate human haptoglobin by complexation with hemoglobin. J. Immunol. Methods 134:51.[Medline]
  33. Blake, C. C., L. N. Johnson, G. A. Mair, A. C. North, D. C. Phillips, V. R. Sarma. 1967. Crystallographic studies of the activity of hen egg-white lysozyme. Proc. R. Soc. London Ser. B Biol. Sci. 167:378.[Medline]
  34. Blake, C. C., D. F. Koenig, G. A. Mair, A. C. North, D. C. Phillips, V. R. Sarma. 1965. Structure of hen egg-white lysozyme: a three-dimensional Fourier synthesis at 2 Angstrom resolution. Nature 206:757.[Medline]
  35. Orth, P., D. Schnappinger, W. Hillen, W. Saenger, W. Hinrichs. 2000. Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat. Struct. Biol. 7:215.[Medline]
  36. Dadaglio, G., C. A. Nelson, M. B. Deck, S. J. Petzold, E. R. Unanue. 1997. Characterization and quantitation of peptide-MHC complexes produced from hen egg-white lysozyme using a monoclonal antibody. Immunity 6:727.[Medline]
  37. Viret, C., X. He, C. A. Janeway, Jr.. 2000. On the self-referential nature of naive MHC class II-restricted T cells. J. Immunol. 165:6183.[Abstract/Free Full Text]
  38. Kersh, G. J., D. L. Donermeyer, K. E. Frederick, J. M. White, B. L. Hsu, P. M. Allen. 1998. TCR transgenic mice in which usage of transgenic {alpha}- and {beta}-chains is highly dependent on the level of selecting ligand. J. Immunol. 161:585.[Abstract/Free Full Text]
  39. Sarukhan, A., C. Garcia, A. Lanoue, H. von Boehmer. 1998. Allelic inclusion of T cell receptor {alpha} genes possesses an autoimmune hazard due to low-level expression of autospecific receptors. Immunity 8:563.[Medline]
  40. Zal, T., S. Weiss, A. Mellor, B. Stockinger. 1996. Expression of a second receptor rescues self-specific T cells from thymic deletion and allows activation of autoreactive effector function. Proc. Natl. Acad. Sci. USA 93:9102.[Abstract/Free Full Text]
  41. Elliott, J. I., D. M. Altmann. 1995. Dual T cell receptor {alpha} chain T cells in autoimmunity. J. Exp. Med. 182:953.[Abstract/Free Full Text]
  42. He, X., C. A. Janeway, Jr., M. Levine, E. Robinson, P. Preston-Hurlburt, C. Viret, K. Bottomly. 2002. Dual receptor T cells extend the immune repertoire for foreign antigens. Nat. Immun. 3:127.
  43. Ali, A., M. Garrovillo, O. O. Oluwole, H. A. Depaz, R. Gopinathan, K. Engelstad, M. A. Hardy, S. F. Oluwole. 2001. Mechanisms of acquired thymic tolerance: induction of transplant tolerance by adoptive transfer of in vivo alloMHC peptide activated syngeneic T cells. Transplantation 71:1442.[Medline]
  44. Oluwole, O. O., H. A. Depaz, R. Gopinathan, A. Ali, M. Garrovillo, M. X. Jin, M. A. Hardy, S. F. Oluwole. 2001. Indirect allorecognition in acquired thymic tolerance: induction of donor-specific permanent acceptance of rat islets by adoptive transfer of allopeptide-pulsed host myeloid and thymic dendritic cells. Diabetes 50:1546.[Abstract/Free Full Text]
  45. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767.[Medline]
  46. Wu, L., A. D’Amico, H. Hochrein, M. O’Keeffe, K. Shortman, K. Lucas. 2001. Development of thymic and splenic dendritic cell populations from different hemopoietic precursors. Blood 98:3376.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L. M. Relland, M. K. Mishra, D. Haribhai, B. Edwards, J. Ziegelbauer, and C. B. Williams
Affinity-Based Selection of Regulatory T Cells Occurs Independent of Agonist-Mediated Induction of Foxp3 Expression
J. Immunol., February 1, 2009; 182(3): 1341 - 1350.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Bendiksen, M. Van Ghelue, T. Winkler, U. Moens, and O. P. Rekvig
Autoimmunity to DNA and Nucleosomes in Binary Tetracycline-Regulated Polyomavirus T-Ag Transgenic Mice
J. Immunol., December 15, 2004; 173(12): 7630 - 7640.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haribhai, D.
Right arrow Articles by Williams, C. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haribhai, D.
Right arrow Articles by Williams, C. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS