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The Journal of Immunology, 2007, 178: 3544-3550.
Copyright © 2007 by The American Association of Immunologists, Inc.

Discrete T Cell Populations with Specificity for a Neo-Self-Antigen Bear Distinct Imprints of Tolerance1

Nathan E. Standifer2,*, Sue Stacy{dagger}, Ellen Kraig*,{dagger} and Anthony J. Infante3,*,{ddagger}

* Department of Microbiology and Immunology, {dagger} Department of Cellular and Structural Biology, and {ddagger} Department of Pediatrics, University of Texas Health Science Center, San Antonio, TX 78229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mice expressing the Torpedo acetylcholine receptor {alpha}-chain as a neo-self-Ag exhibit a reduced frequency of T cells responding to the immunodominant epitope T{alpha}146–162 indicating a degree of tolerance. We characterized tolerance induction in these animals by analyzing the residual T{alpha}146–162-responsive T cell population and comparing it to that of nontransgenic littermates. Using CD4high sorting, we isolated the vast majority of Ag-reactive T cells from both strains of mice. Quantitative studies of the CD4high populations in transgenic mice following immunization with T{alpha}146–162 revealed a diminished expansion of cells expressing the canonical TCRBV6 but not other TCRBV gene segments when compared with nontransgenic littermates. In addition, CD4high cells from transgenic mice were functionally hyporesponsive to T{alpha}146–162 in terms of proliferation and cytokine secretion regardless of TCRBV gene segment use. TCR sequence analysis of transgenic Vbeta6+CD4high cells revealed a reduced frequency of cells expressing a conserved motif within the TCRbeta CDR3. Thus, the canonical T{alpha}146–162 responsive, Vbeta6+ population demonstrates both quantitative and qualitative deficits that correlate with an altered TCR repertoire whereas the non-Vbeta6 population in transgenic mice exhibits only a reduction in peptide responsiveness, a qualitative defect. These data demonstrate that discrete autoreactive T cell populations with identical peptide/MHC specificity in Torpedo acetylcholine receptor-{alpha}-transgenic animals bear distinct tolerance imprints.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell receptors are generated by random combinatorial events yielding a diverse repertoire of Ag specificities some of which are reactive to self-peptides. These autoreactive T cells are rendered tolerant to self-tissues either by deletion during thymic development or later by numerous mechanisms functioning in the periphery (1, 2, 3). Traditionally, tolerance mechanisms have been studied using doubly transgenic animals in which both a neo-self-Ag and its cognate TCR are coexpressed (4). In these model systems, all T cells have an identical AgR thereby enabling the identification of the tolerance mechanism acting on a single T cell clone. Tolerance in nonengineered animals with diverse TCR repertoires, however, is presumed capable of being mediated by multiple mechanisms acting simultaneously, but this has seldom been demonstrated experimentally.

Although studies with transgenic neo-self-Ag/cognate TCR models have enabled the analysis of tolerance mechanisms acting on a single T cell population, the characterization of tolerance mechanisms acting on distinct T cell populations has been hindered due to the experimental limitations in methods of isolating Ag-reactive T cells. Several technologies now allow for the isolation of specific T cells from a heterogenous population, including one based on increased expression levels of the CD4 coreceptor (5, 6). These CD4high cells have been shown to comprise the vast majority of activated autoreactive cells in models of multiple sclerosis and type I diabetes. Thus, CD4high sorting can be used to isolate and analyze autoreactive cells from heterogenous T cell populations.

We characterized tolerance mechanisms acting on discrete T cell populations in a neo-self-Ag system of experimental autoimmune myasthenia gravis (EAMG),4 the rodent model of myasthenia gravis (7). These animals expressed the {alpha}-chain of the eliciting Ag Torpedo acetylcholine receptor (TAChR) at levels and locations similar to those of the endogenous AChR (8). Furthermore, transgenic animals demonstrated a substantial degree of T cell tolerance to the H-2b-restricted immunodominant peptide comprised of amino acid residues 146–162 (T{alpha}146–162; Ref. 9). The transgenic mice still made substantial amounts of anti-AChR Ab when immunized and while their incidence of clinical signs of EAMG (43%) was less than nontransgenic littermates (66%), the difference was not statistically significant (8).

The canonical T cell population in C57BL/6 mice responding to T{alpha}146–162 used Vbeta6 and expressed one or more acidic amino acids (Asp or Glu) in residues 96–100 of the TCRbeta CDR3 (10, 11, 12). Modeling of TCR-peptide-MHC interactions suggested that negatively charged CDR3beta residues contact a critical lysine of T{alpha}146–162 (13). However, there were also noncanonical T cell populations that responded to T{alpha}146–162 (14, 15). Using CD4high sorting, we compared the canonical and noncanonical T{alpha}146–162-reactive T cells from TAChR-{alpha} transgenic mice to populations from nontransgenic littermates. Parameters assessed included quantity of responding cells following immunization, responsiveness to restimulation with T{alpha}146–162, TCRBV gene segment expression and CDR3 sequence. This type of analysis allowed for the characterization of population-specific tolerance induction.


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

The TAChR-{alpha}-transgenic animals and nontransgenic littermates were generated as detailed previously (8). To confirm that the genetic background of the transgenic animals was appropriate for this analysis (see Fig. 9 of Ref. 8), we tested individual mice for the presence of the TAChR-{alpha} transgene and endogenous I-Abeta gene by PCR. Female C57BL/6 (B6) animals purchased from The Jackson Laboratory were used as a source of APCs for experiments. All mice used in experiments were sex matched and under 24 wk of age. Experiments with animals were approved by the University of Texas Health Science Center Institutional Animal Care and Use Committee.

Peptides and immunization

The T{alpha}146–162 (LGIWTYDGTKVSISPES) and p61–76 (IDVRLRWNPADYGGIK) peptides were synthesized by Dr. K. Wall (Ref. 9 ; University of Toledo, Toledo, OH) or by the University of Texas Health Science Center at San Antonio Protein Core Facility. Peptide p61–76 represents a well-characterized AChR B cell epitope (16). It does not stimulate T cells and serves as a negative control. Peptides were purified by HPLC and the composition of each peptide was confirmed by amino acid analysis. Mice were immunized s.c. at the base of the tail with 100 µl of a solution containing 3 µg of T{alpha}146–162 dissolved in HBSS and emulsified with an equal volume of CFA (H37Ra; Sigma-Aldrich).

Cellular assays

T cell proliferation assays were performed using 1 x 104 sorted T cells incubated in 96-well plates with 2.5 x 105 irradiated (3500 Gy) spleen cells and either p61–76 at 0.05 µM or T{alpha}146–162 at concentrations indicated. All cells and peptides were diluted in T cell medium consisting of RPMI 1640 supplemented with 12 mM HEPES buffer (Mediatech), 2 x 10–3 M L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Life Technologies), 3 x 10–5 M 2-ME (Sigma-Aldrich), and 10% FCS. Plates were incubated for 72 h at 37°C in 5% CO2. Plates were pulsed with 1 µCi of [3H]TdR (ICN Pharmaceuticals) per well during the last 18 h of incubation and then harvested onto glass fiber strips for analysis with a beta-scintillation counter. Cell proliferation was recorded as the average cpm of triplicate wells less the background values obtained from cultures containing the nonstimulatory p61–76 peptide.

Cytokine assays

Cytokine secretion levels were quantified using a Th1/Th2 Cytometric Bead Array kit (BD Pharmingen). In brief, Vbeta6+ or non-Vbeta6 CD4high cells were sorted from 4-day lymph node cell (LNC) cultures from peptide-immunized transgenic or nontransgenic mice. Sorted cells (5 x 103) were cultured in the presence of irradiated B6 splenocytes (1.25 x 105) and either 0.0184 µM T{alpha}146–162 or 0.05 µM p61–76. Following a 24-h incubation period, cell supernatants from triplicate wells were harvested, combined, and diluted at 1/2, 1/10, or 1/100 before analysis.

Flow cytometry

T cells from immunized and restimulated lymph nodes were used for CD4high sorting by FACS. In brief, draining periaortic and inguinal lymph nodes were harvested on day 7 postimmunization and single-cell suspensions were prepared. LNC suspensions were aliquoted in 12-well plates at 5 x 106 cells/well with 0.0184 µM T{alpha}146–162 for 4 days. A total of 1.5 x 107 cells were stained with 4 µg of PE-conjugated rat anti-mouse CD4 Ab (clone L3T4) and 4 µg of FITC-conjugated rat anti-mouse Vbeta6 Ab (clone RR4.7; BD Pharmingen) for 30 min on ice. Cells were washed once with FACS buffer (PBS and 1% BSA) and once with PBS alone. The stained cells were resuspended in 3 ml of PBS and 15 µl of propidium iodide (PI) were added to each tube. Cells were gated for forward scatter and PI exclusion. The gate separating CD4normal and CD4high populations was set at the upper limit of CD4 expression of spleen cells from normal, unimmunized animals. Cells were sorted into 2 ml of T cell medium and counted on a hemocytometer before setting up T cell assays. Small aliquots of sorted T cells were analyzed for purity immediately following the sort. On average, the CD4+ populations were found to be 85–90% pure with regard to Vbeta gene segment usage. There were ~6% CD4normal T cells contaminating the CD4high populations.

TCR CDR3 sequencing

A total of 2–5 x 105 cells from each FACS-isolated population were used for analysis of TCR CDR3 sequences. Cells were suspended in 1 ml of TRIzol reagent (Invitrogen Life Technologies) and total RNA was extracted from the cells by adding 200 µl of chloroform to the lysates, spinning for 15 min at 10 x g at 4°C, and transferring the aqueous phase to a fresh tube containing 1 µl of glycogen (20 mg/ml; Roche Applied Science). The RNA was precipitated by adding 0.5 ml of isopropyl alcohol and spinning at 10 x g for 10 min. The resulting pellet was washed once with 75% ethanol, dried in a vacuum dessicator for 5 min, and then diluted in 20 µl of DEPC-treated water. The RNA was then denatured at 65°C for 10 min and immediately placed on ice.

Denatured RNA was used as a template for the synthesis of cDNA using the First-Strand cDNA Synthesis kit (Amersham Biosciences) according to the manufacturer’s instructions. Briefly, 11 µl of Bulk First-Strand Reaction Mix (containing FPLCpure Moloney murine leukemia virus reverse transcriptase, porcine RNAguard, RNase/DNase-Free BSA, dATP, dCTP, dGTP, and dTTP in aqueous buffer), 1 µl of 200 mM DTT, and 1 µl of a 0.2 µg/µl NotI-(dT)18 oligonucleotide were added to the denatured RNA. The reaction mix was incubated at 37°C for 1 h and then frozen at –70°C.

The TCRBV-BC gene segments were amplified from cDNA samples by PCR with region-specific oligonucleotides (11). For cDNA synthesized from non-Vbeta6 expressing T cells, four pools comprised of five TCRBV region oligonucleotides each were used with a single TCRBC oligonucleotide. Oligonucleotides with specificity for the TCRBV 6 and TCRBC gene segments were used in PCR with cDNA synthesized from Vbeta6+ T cells. PCR products were isolated on a 1.5% agarose gel, excised, and purified using a QIAquick Gel Extraction kit (Qiagen) according to the manufacturer’s protocol. Products generated from reactions with cDNA of FACS-isolated bulk T cell populations were cloned into pCR TOPO 2.1 using the TOPO-TA kit (Invitrogen Life Technologies). The resulting positive transformants were inoculated into 2 ml of Luria Bertani broth and incubated overnight at 37°C with shaking at 200 rpm. Plasmids were purified from overnight cultures using a QIAprep Miniprep kit (Qiagen) according to the manufacturer’s protocol. Plasmid inserts were directly sequenced using a 3100 genetic analyzer (Applied Biosystems) with M13 forward and M13 reverse oligonucleotides. Sequencing reactions were performed at the University of Texas Health Science Center for Advanced Nucleic Acids Core Facility. In cases where overnight cultures failed to yield ample amounts of plasmids for sequencing reactions, the plasmid inserts were PCR amplified using TCRBC- and BV-region specific oligonucleotides and the resulting PCR products were sequenced.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4high T cells are the Ag-specific population in immunized transgenic animals

Mice expressing the TAChR-{alpha} chain as a neo-self protein exhibit a degree of tolerance to the immunodominant epitope T{alpha}146–162, although a definite response is observed at moderate to high Ag concentrations (Ref. 8 ; Fig. 1). We sought to isolate the responding cells to characterize tolerance induction in transgenic animals. Ag-reactive Th cells have been demonstrated to up-regulate CD4 levels in autoimmune disease models (5, 6). Therefore, we analyzed CD4 expression on LNC from immunized transgenic animals that were cultured with T{alpha}146–162. Increased numbers of CD4high cells arose when transgenic LNC were restimulated with T{alpha}146–162 compared with p61–76 (Fig. 2A, populations above dashed line). As the expansion of a CD4high population correlated with a recall response to the immunizing Ag, we postulated that these were the autoreactive cells with specificity for T{alpha}146–162. To test this hypothesis, we sorted Vbeta6+CD4high or Vbeta6+ CD4normal cells from peptide-stimulated LNC cultures and compared the proliferative responses of each population to T{alpha}146–162. The CD4high cells exhibited a 22-fold increase in proliferation compared with the CD4normal population when stimulated with T{alpha}146–162 (Fig. 2B). Thus, the vast majority of Vbeta6+ cells with specificity for T{alpha}146–162 are found within the CD4high population.


Figure 1
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  		FIGURE 1. LNC of transgenic animals exhibit reduced proliferative responses to T{alpha}146–162. Error bars represent the SD of the mean cpm of triplicate wells. For each data point, the mean cpm of a triplicate determination in the absence of Ag (background control) was subtracted, yielding {Delta}cpm. Data are representative of five independent experiments.

 

Figure 2
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  		FIGURE 2. CD4high cells represent the Ag-reactive populations in transgenic animals. Ag-stimulated LNCs were double-stained with anti-CD4 and anti-Vbeta6 mAbs. The populations above the dashed lines indicate the CD4high populations present in cells restimulated with either T{alpha}146–162 or the nonstimulatory peptide, p61–76 (A). In the experiment shown, the percentage of Vbeta6+,CD4high cells increased from 0.33% of gated events with p61–76 to 3.44% with T{alpha}146–162. The Vbeta6+, CD4high, and CD4normal populations from T{alpha}146–162 restimulated cells were sorted and tested for proliferative responses against T{alpha}146–162 (B).

 
Transgenic animals expand fewer canonical Vbeta6+ T cells in response to T{alpha}146–162 immunization

Limiting dilution analysis of LNC from immunized transgenic mice demonstrated a 6-fold reduction in the frequency of T{alpha}146–162-specific T cells (8). Therefore, we hypothesized that peptide-immunized transgenic animals would expand a smaller Ag-reactive, CD4high population. LNC from immunized transgenic and nontransgenic mice were expanded in culture for 4 days, stained for CD4 and Vbeta6 expression, and analyzed by FACS. The CD4high populations were quantified by calculating the proportion of total CD4+ cells that were CD4high in each strain. We termed this the CD4high index. The CD4high index of transgenic mice was statistically significantly lower than that of nontransgenic littermates (Fig. 3, no overlap of 95% confidence intervals). We next determined whether the reduction in CD4high cells observed in transgenic animals was the result of fewer responding canonical cells expressing TCRBV6 or noncanonical cells expressing other TCRBV genes. To this end, we calculated the CD4high indices of both Vbeta6 and non-Vbeta6 populations from expanded LNC of immunized transgenic or nontransgenic animals (seven and five animals used, respectively). In all experiments, the CD4high indices of Vbeta6+ T cells in transgenic animals were reduced compared with the same populations isolated from nontransgenic animals (Fig. 4). By comparison, there was no difference between the CD4high indices of non-Vbeta6 T cells from transgenic and nontransgenic animals. Taken together, these data show a correlation between the reduced frequency of Ag-responsive Vbeta6+CD4high cells and the tolerance observed in transgenic animals.


Figure 3
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  		FIGURE 3. Transgenic animals expanded fewer CD4high cells following immunization and restimulation with T{alpha}146–162; the reduction involves primarily cells expressing Vbeta6. Pooled T{alpha}146–162-stimulated lymph node cells were analyzed by flow cytometry for expression of Vbeta6 and CD4. Cells above the horizontal line in each histogram represent the total CD4+ cells, and the boxes delineate the CD4high cells. The values in the four corners represent the percentages in the entire quadrant. The values in italics are the percentages within the indicated boxes. The populations were quantitated as described in Materials and Methods. The CD4high indices were compared by assessing the overlap of 95% confidence intervals and the value denoted by the asterisk (*) was significantly lower than that of the other mouse strain.

 

Figure 4
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  		FIGURE 4. The reduced expansion of CD4high T cells in transgenic mice is due to a reduction in the Vbeta6+ population but not cells expressing other TCRBV gene segments. CD4high indices were calculated for Vbeta6+ and non-Vbeta6 cell populations from immunized transgenic and nontransgenic animals (seven and five, respectively).

 
T{alpha}146–162-specific T cells from transgenic animals are functionally hyporesponsive

We next wished to ascertain whether tolerance observed in transgenic animals was wholly the result of the reduced frequency of responding CD4high T cells or whether the CD4high populations were functionally hyporesponsive to Ag. To test this, we analyzed the proliferative responses of Vbeta6+ and non-Vbeta6 CD4high cells isolated from transgenic or nontransgenic, peptide-immunized littermates. LNC were harvested and expanded in culture with peptide for 4 days before analysis and sorting by FACS. Equal numbers of sorted cells were tested for proliferation against T{alpha}146–162 presented by irradiated B6 splenocytes. Both Vbeta6+ and non-Vbeta6 T cells from transgenic mice exhibited significant reductions in peptide responsiveness compared with nontransgenic littermates (Fig. 5). These data indicate that both canonical and noncanonical T cell populations in transgenic animals are hyporesponsive on a per cell basis as measured by proliferation even though only the former population exhibits a reduction in frequency.


Figure 5
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  		FIGURE 5. CD4high cells from transgenic animals exhibit a reduced recall response to T{alpha}146–162. Primed LNC were expanded for 4 days in vitro before sorting into Vbeta6 or non-Vbeta6 expressing CD4high populations by flow cytometry. Proliferation assays were performed on each population. Error bars indicate the SD of the mean.

 
Several investigators have demonstrated that Th1 but not Th2 cytokines are necessary for the induction of EAMG (17, 18, 19, 20, 21, 22, 23, 24, 25). We compared cytokine secretion by CD4high T cells from transgenic mice to that of nontransgenic littermates. We hypothesized that T cells from transgenic mice would secrete less IFN-{gamma} as these mice exhibit a degree of tolerance to T{alpha}146–162. Sorted, transgenic CD4high T cells secreted reduced levels of the Th1 cytokines IFN-{gamma} and TNF-{alpha} compared with nontransgenic animals (Fig. 6). Significant levels of cytokines were detected only in samples diluted at 1/2 indicating that the recorded values were within the linear range of detection. No Th2 cytokines were observed in either group (data not shown). These data demonstrate that T{alpha}146–162-specific T cells from transgenic animals are hyporesponsive as measured by cytokine expression regardless of TCRBV expression.


Figure 6
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  		FIGURE 6. CD4high T cells from transgenic animals secrete reduced levels of Th1 cytokines. Supernatants were collected from 24-h cultures of sorted CD4high cells from the indicated populations incubated with Ag and APC. Cytokine secretion was quantified using the Th1/Th2 Cytometric Bead Array kit and the dotted line denotes the limit of detection.

 
A reduced proportion of transgenic Vbeta6+ T cells express an acidic CDR3 motif

The Ag hyporesponsiveness observed in T cells from transgenic mice could reflect the expression of an altered TCR repertoire. The canonical Vbeta6+ T cells responding to T{alpha}146–162 express an acidic amino acid (Glu or Asp) within residues 96–100 of the CDR3 (7, 8, 10). We hypothesized that fewer T{alpha}146–162-specific TCR from transgenic animals would express this CDR3 motif compared with nontransgenic T cells. Analysis of TCR sequences from CD4highVbeta6+ T cells from nontransgenic mice demonstrated that 100% (13 of 13) of the CDR3 contained acidic amino acids within residues 96–100 (Table I, nontransgenic). However, only 58.8% (20 of 34) of the CDR3 regions derived from TAChR-{alpha} transgenic, Vbeta6+ T cells used this canonical motif, a statistically significant difference (Table I, transgenic). These sequencing results associate the reduced frequency of Vbeta6+ T cells with expression of the canonical CDR3 motif. TCR sequences of the non-Vbeta6 populations demonstrated no substantial difference in usage of the CDR3 motif (Table II).


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  		Table I. Canonical CD4high T cells from transgenic mice express proportionally fewer TCR bearing the conserved TCRbeta CDR3 motif of acidic amino acidsa

 

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  		Table II. Noncanonical CD4high T cells from transgenic and nontransgenic animals express the conserved CDR3 motif at similar frequencies

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The characterization of tolerance induction to self Ags and its breakdown is imperative to the understanding of autoimmunity and the derivation of effective targeted therapeutics for autoimmune diseases. We sought to analyze the effects of tolerance induction on discrete autoreactive T cell populations in a model relevant to myasthenia gravis. A novel aspect of the present study is that our approach allowed us to analyze immunity to a neo-self Ag in mice with a diverse, native TCR repertoire.

Transgenic expression of the TAChR-{alpha} chain, the eliciting Ag of EAMG, has been shown to induce tolerance to the immunodominant epitope T{alpha}146–162, yet a small responding population of T cells was observed upon immunization and restimulation with this peptide (Ref. 8 ; Fig. 1). Using CD4high sorting, we isolated these Ag-responsive cells and observed that transgenic animals expanded fewer CD4high cells following in vitro stimulation compared with nontransgenic animals (Fig. 3). The diminishment was correlated with a reduction in the number of CD4high cells from transgenic animals expressing TCRBV6 but not other TCRBV genes (Fig. 4). Biological analyses of transgenic Vbeta6+ and non-Vbeta6, CD4high cells demonstrated that both populations exhibited reduced proliferation and secretion of Th1 cytokines in response to T{alpha}146–162 (Figs. 5 and 6). Thus, in transgenic animals, the T{alpha}146–162-specific, non-Vbeta6 population expands normally but exhibits reduced proliferation and cytokine production in response to culture with T{alpha}146–162. However, the transgenic Vbeta6+ population exhibits reduced expansion following immunization as well as deficits in both proliferation and secretion of pathological cytokines when activated with Ag in vitro. These results could reflect the deletion of a portion of the Vbeta6+ population that is highly reactive to the neo-self Ag and/or the alteration of the population fine specificity. In an effort to assess which of these mechanisms are acting on the T cells from transgenic animals, we quantified the number of T cells expressing acidic amino acids within the TCR beta-chain CDR3, a motif we previously associated with high avidity for peptide/MHC is this experimental system (10, 11, 14). Sequence analyses of TCR beta-chain CDR3 from Vbeta6+ T cells revealed a 40% reduction in the number of TCR from transgenic mice expressing this motif (Table I, transgenic). Thus, in our neo-self Ag model of tolerance, the high-avidity cells exhibit decreased expansion coupled with overall hyporesponsiveness to Ag (proliferation and cytokine production). Moreover, these alterations in biologic function are correlated with expression of altered TCR resulting in differences in T cell fine specificity.

Several studies have demonstrated similar findings in other models of tolerance induction. In one study using mice that express a high-affinity TCR beta-chain with specificity for the endogenous Smcy3-peptide, Ag-specific CD8 cells that escaped negative selection expressed altered TCR sequences (26). Furthermore, these cells were demonstrated to bind weakly to MHC/peptide tetramers and responded to high doses of peptide in vitro demonstrating that they were low-avidity T cells. The authors concluded that the primary tolerance mechanism operable in this model is likely the deletion of high-avidity T cells. Our data are very similar in that the Vbeta6+ high-avidity cells were greatly reduced in number and the remaining responded poorly to Ag. In another model wherein beta-galactosidase was expressed at high levels in the thymus, practically all Ag-specific T cells were deleted. However, by shortening the promoter, thymic expression levels were reduced resulting in the functional inactivation of only Th1 cells (27). Although these results correlate differential tolerance levels with thymic Ag expression levels, they are applicable in that the neo-self Ag is expressed at low levels within the thymus in our model (8), and we observed a similar induction of tolerance of only the high-avidity T cell subset.

Sequence comparisons of transgenic and nontransgenic T cell populations that did not express TCRBV6 failed to demonstrate a significant difference in use of the CDR3 motif. We are uncertain whether this reflects the low number of T cells recovered from transgenic animals available for sequencing or whether the mechanisms of tolerance acting on these cells do so in a way such that the TCR repertoire is not altered. One possibility is that the non-Vbeta6-transgenic T cells escape deletion mechanisms but are rendered unresponsive to Ag in the periphery by anergy. This is a likely explanation because the immunodominant T cell population in EAMG has been clearly shown to express TCRBV6 and higher MHC/peptide avidity, whereas the non-Vbeta6 cells represent a secondary population of lower avidity (14). As such, it would be likely that these lower avidity T cells undergo anergy induction rather than deletion.

We did not attempt to directly identify the tolerance mechanisms acting on Ag-specific cells of transgenic animals in the present study. It is plausible that both deletional and nondeletional tolerance mechanisms (e.g., anergy induction or immunological suppression) act on T{alpha}146–162 specific T cells. Akkaraju et al. (28) postulated that the mechanism of tolerance induction is a function of the amount of autoantigen presentation and the receptor affinity for the autoantigen. In their model, T cells bearing high-affinity TCR are deleted either in the thymus or periphery whereas cells with lower affinity receptors are rendered functionally inactive or ignorant. More recently, Lerman et al. (29) showed that high-affinity, self-reactive T cells that escaped deletion in the thymus function as CD25+ regulatory T cells in the periphery. We did not directly analyze expression levels of CD25 on the surface of T{alpha}146–162-specific T cells from transgenic animals. However, several T cell clones were generated from these mice and were found to be responsive to peptide (data not shown). We propose that the reduction in Vbeta6+ T cells bearing the acidic CDR3 motif in transgenic animals represents an "imprint" of the deletion of the highest affinity T cells. We postulate that the non-Vbeta6 cells are rendered tolerant by one or more nondeletional mechanisms. The imprints of this mechanism are the Ag hyporesponsiveness of non-Vbeta6 cells from transgenic animals coupled with the similar expansion levels and frequency of CDR3 motif expression between both strains. Thus, the tolerance to T{alpha}146–162 observed in transgenic animals likely results from both a reduction in the number of high-avidity T cells and hyporesponsiveness of the remaining Ag-reactive cells.

Taken together, these data imply that different tolerance mechanisms are simultaneously operable on discrete cell populations with the same Ag specificity. This could not have been observed in most other models of tolerance because these systems generally do not use polyclonal T cell populations. Our system holds great promise for future analyses of tolerance induction and evasion mechanisms operable in myasthenia gravis.


   Acknowledgments
 
We thank Charles A. Thomas, III, of the University of Texas Health Science Center Institutional Flow Cytometry Core Facility for technical expertise in cell-sorting experiments, Steve Mouton for amino acid analyses of peptides, Keith Krolick for TAChR, Kathy Wall for peptide synthesis, Ester Coronado-Heinsohn, Patricia Currier, and Daren Stephens. We gratefully acknowledge Dr. John A. Gebe for critical comments on the manuscript.


   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 the following National Institutes of Health Grants: AI43061 (to A.J.I.), AG14557 (to E.K.), T32 Grants AG00165 and AG00205 (to S.S.), and the Nathan Shock Aging Center. Additional support was obtained from the Howard Hughes Medical Institute Research Resources Program and a fellowship from the Myasthenia Gravis Foundation of America (to S.S.). Back

2 Current address: Benaroya Research Institute at Virginia Mason, Diabetes Program, 1201 Ninth Avenue, Seattle, WA 98101-2795. Back

3 Address correspondence and reprint requests to Dr. Anthony J. Infante, University of Texas Health Science Center MC7802, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail address: infantea{at}uthscsa.edu Back

4 Abbreviations used in this paper: EAMG, experimental autoimmune myasthenia gravis; AChR, acetylcholine receptor; TAChR, Torpedo AChR; LNC, lymph node cell. Back

Received for publication August 29, 2006. Accepted for publication December 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Liston, A., S. Lesage, D. H. Gray, R. L. Boyd, C. C. Goodnow. 2005. Genetic lesions in T-cell tolerance and thresholds for autoimmunity. Immunol. Rev. 204: 87-101. [Medline]
  2. Goodnow, C. C., J. Sprent, B. Fazekas de St. Groth, C. G. Vinuesa. 2005. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature 435: 590-597. [Medline]
  3. Macian, F., S. H. Im, F. J. Garcia-Cozar, A. Rao. 2004. T-cell anergy. Curr. Opin. Immunol. 16: 209-216. [Medline]
  4. Miller, J. F., R. A. Flavell. 1994. T-cell tolerance and autoimmunity in transgenic models of central and peripheral tolerance. Curr. Opin. Immunol. 6: 892-899. [Medline]
  5. Ridgway, W., M. Fasso, C. G. Fathman. 1998. Following antigen challenge, T cells up-regulate cell surface expression of CD4 in vitro and in vivo. J. Immunol. 161: 714-720. [Abstract/Free Full Text]
  6. Lejon, K., C. G. Fathman. 1999. Isolation of self antigen-reactive cells from inflamed islets of nonobese diabetic mice using CD4high expression as a marker. J. Immunol. 163: 5708-5714. [Abstract/Free Full Text]
  7. Christadoss, P., M. Poussin, C. Deng. 2000. Animal models of myasthenia gravis. [Published erratum appears in 2000 Clin. Immunol. 95: 170]. Clin. Immunol. 94: 75-87. [Medline]
  8. Stacy, S., B. E. Gelb, B. A. Koop, J. J. Windle, K. A. Wall, K. A. Krolick, A. J. Infante, E. Kraig. 2002. Split tolerance in a novel transgenic model of autoimmune myasthenia gravis. J. Immunol. 169: 6570-6579. [Abstract/Free Full Text]
  9. Wall, K. A., J. Y. Hu, P. Currier, S. Southwood, A. Sette, A. J. Infante. 1994. A disease-related epitope of Torpedo acetylcholine receptor: residues involved in I-Ab binding, self-nonself discrimination, and TCR antagonism. J. Immunol. 152: 4526-4536. [Abstract]
  10. Pierce, J. L., K. A. Zborowski, E. Kraig, A. J. Infante. 1994. Highly conserved TCR beta chain CDR3 sequences among immunodominant acetylcholine receptor-reactive T cells in murine myasthenia gravis. Int. Immunol. 6: 775-783. [Abstract/Free Full Text]
  11. Kraig, E., J. L. Pierce, K. Z. Clarkin, N. E. Standifer, P. Currier, K. A. Wall, A. J. Infante. 1996. Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis. J. Neuroimmunol. 71: 87-95. [Medline]
  12. Yang, B., K. R. McIntosh, D. B. Drachman. 1998. How subtle differences in MHC class II affect the severity of experimental myasthenia gravis. Clin. Immunol. Immunopathol. 86: 45-58. [Medline]
  13. Infante, A. J., E. Kraig. 1999. Myasthenia gravis and its animal model: T cell receptor expression in an antibody mediated autoimmune disease. Int. Rev. Immunol. 18: 83-109. [Medline]
  14. Standifer, N. E., E. Kraig, A. J. Infante. 2003. A hierarchy of T cell receptor motifs determines responsiveness to the immunodominant epitope in experimental autoimmune myasthenia gravis. J. Neuroimmunol. 145: 68-76. [Medline]
  15. Wu, B., M. Shenoy, E. Goluszko, R. Kaul, P. Christadoss. 1995. TCR gene usage in experimental autoimmune myasthenia gravis pathogenesis: usage of multiple TCRBV genes in the H-2b strains. J. Immunol. 154: 3603-3610. [Abstract]
  16. Tzartos, S. J., A. Kokla, S. L. Walgrave, B. M. Conti-Tronconi. 1988. Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67–76 of the {alpha} subunit. Proc. Natl. Acad. Sci. USA 85: 2899-2903. [Abstract/Free Full Text]
  17. Balasa, B., C. Deng, J. Lee, L. M. Bradley, D. K. Dalton, P. Christadoss, N. Sarvetnick. 1997. Interferon {gamma} (IFN-{gamma}) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice. J. Exp. Med. 186: 385-391. [Abstract/Free Full Text]
  18. Balasa, B., C. Deng, J. Lee, P. Christadoss, N. Sarvetnick. 1998. The Th2 cytokine IL-4 is not required for the progression of antibody-dependent autoimmune myasthenia gravis. J. Immunol. 161: 2856-2862. [Abstract/Free Full Text]
  19. Karachunski, P. I., N. S. Ostlie, C. Monfardini, B. M. Conti-Fine. 2000. Absence of IFN-{gamma} or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice. J. Immunol. 164: 5236-5244. [Abstract/Free Full Text]
  20. Moiola, L., F. Galbiati, G. Martino, S. Amadio, E. Brambilla, G. Comi, A. Vincent, L. M. Grimaldi, L. Adorini. 1998. IL-12 is involved in the induction of experimental autoimmune myasthenia gravis, an antibody-mediated disease. Eur. J. Immunol. 28: 2487-2497. [Medline]
  21. Ostlie, N., M. Milani, W. Wang, D. Okita, B. M. Conti-Fine. 2003. Absence of IL-4 facilitates the development of chronic autoimmune myasthenia gravis in C57BL/6 mice. J. Immunol. 170: 604-612. [Abstract/Free Full Text]
  22. Sitaraman, S., D. W. Metzger, R. J. Belloto, A. J. Infante, K. A. Wall. 2000. Interleukin-12 enhances clinical experimental autoimmune myasthenia gravis in susceptible but not resistant mice. J. Neuroimmunol. 107: 73-82. [Medline]
  23. Wang, H. B., F. D. Shi, H. Li, P. H. van der Meide, H. G. Ljunggren, H. Link. 2000. Role for interferon-{gamma} in rat strains with different susceptibility to experimental autoimmune myasthenia gravis. Clin. Immunol. 95: 156-162. [Medline]
  24. Zhang, G. X., B. G. Xiao, X. F. Bai, A. Orn, P. H. van der Meide, H. Link. 1998. IFN-{gamma} is required to induce experimental autoimmune myasthenia gravis. Ann. NY Acad. Sci. 841: 576-579. [Medline]
  25. Zhang, G. X., B. G. Xiao, X. F. Bai, P. H. van der Meide, A. Orn, H. Link. 1999. Mice with IFN-{gamma} receptor deficiency are less susceptible to experimental autoimmune myasthenia gravis. J. Immunol. 162: 3775-3781. [Abstract/Free Full Text]
  26. Bouneaud, C., P. Kourilsky, P. Bousso. 2000. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Immunity 13: 829-840. [Medline]
  27. Salmon, A. M., C. Bruand, A. Cardona, J. P. Changeux, S. Berrih-Aknin. 1998. An acetylcholine receptor {alpha} subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis. J. Clin. Invest. 101: 2340-2350. [Medline]
  28. 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. Immunity 7: 255-271. [Medline]
  29. Lerman, M. A., J. Larkin, 3rd, C. Cozzo, M. S. Jordan, A. J. Caton. 2004. CD4+CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen. J. Immunol. 173: 236-244. [Abstract/Free Full Text]




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