Pervasive and stochastic changes in the TCR repertoire of regulatory T-cell-deficient mice

Abstract

We hypothesize that regulatory T-cell (Treg)-deficient strains have an altered TCR repertoire in part due to the expansion of autoimmune repertoire by self-antigen. We compared the Vβ family expression profile between B6 and Treg-lacking B6.Cg-Foxp3^sf/Y (B6.sf) mice using fluorescent anti-Vβ mAbs and observed no changes. However, while the spectratypes of 20 Vβ families among B6 mice were highly similar, the Vβ family spectratypes of B6.sf mice were remarkably different from B6 mice and from each other. Significant spectratype changes in many Vβ families were also observed in Treg-deficient IL-2 knockout (KO) and IL-2Rα KO mice. Such changes were not observed with anti-CD3 mAb-treated B6 mice or B6 CD4⁺CD25⁻ T cells. TCR transgenic (OT-II.sf) mice displayed dramatic reduction of clonotypic TCR with concomitant increase in T cells bearing non-transgenic Vβ and Vα families, including T cells with dual receptors expressing reduced levels of transgenic Vα and endogenous Vα. Collectively, the data demonstrate that Treg deficiency allows polyclonal expansion of T cells in a stochastic manner, resulting in widespread changes in the TCR repertoire.

Introduction

T-cell tolerance to self-antigen is acquired during thymic selection which eliminates high affinity autoreactive clones and allows the maturation and emigration of selected clones (1, 2). Autoreactive T cells with moderate affinity for self-antigen could escape thymic negative selection and exit to the periphery (2, 3, 4). Thymic education also selects CD4⁺CD25⁺Foxp3⁺ (fork-head box P3) regulatory T cells (Treg) that constitute ∼5–10% of the total CD4⁺ T cells (5, 6). The repertoire of Treg is large and overlaps significantly with that of CD4⁺CD25⁻ T cells but a sizable fraction of it is skewed toward self-antigen with high affinity (7, 8, 9). Treg suppress immune responses by a mechanism in which the induction phase is antigen-specific and the effector phase is antigen-non-specific (10). Autoreactive T cells, allo-antigen-specific T cells and T-cell-dependent foreign antigen responses are suppressed by Treg (11). Treg-based suppression provides a major control mechanism for limiting T-cell expansion in response to various forms of antigen including self-antigen.

As normal constituents of the lymphocyte repertoire in the periphery, autoreactive (autoimmune) T cells are suppressed by Treg; hence, how widespread are the autoimmune T cells in the TCR family is difficult to determine. One approach is to study the TCR repertoire in Treg-deficient mice. We hypothesize that autoimmune T-cell expansion by self-antigen in mice lacking Treg such as B6.Cg-Foxp3^sf/Y (B6.sf) will result in a significant change in the TCR repertoire. Indeed, when compared with B6 or anti-CD3 mAb-treated B6 mice, significant changes in the spectratypes of 20 Vβ families were observed for individual B6.sf mice. Similarly, changes in spectratypes were found in many Vβ families of the Treg-deficient IL-2 knockout (KO) and IL-2Rα KO mice. Because TCR transgenic mice always contain a small fraction of non-transgenic TCR, such a wide change in spectratypes in Vβ families predicts that TCR transgenic B6.sf mice will display multiorgan autoimmune syndrome and that T cells bearing non-transgenic Vβ paired with transgenic Vα and/or endogenous Vα be expanded to become the dominant population. Here, we demonstrated that this is indeed the case. Our findings suggest that the repertoire of autoimmune T cells is large and widespread across V-region families. Furthermore, the spontaneous immune response is unabated and stochastic, resulting in selective T-cell expansion and significant changes in TCR repertoire.

Methods

Mice

Breeding pairs for B6, B6.129S4-Il2ra^tm1Dw/J (IL-2Rα KO), B6.129P2-Il2^tm1Hor/J (IL-2 KO), B6-Tg (TcrαTcrβ) 425Cbn/J [OT-II B6, Vβ5.1/5.2^highVα2^high, ovalbumin peptide (323–339)-specific, I-A^b-restricted] and B6.Foxp3^sf/Y (B6.sf) mice were purchased from The Jackson Laboratories, Bar Harbor, ME, USA. The production, genotyping and maintenance of these strains have been described (12). TCR transgenic B6.sf (OT-II.sf) mice were generated by breeding OT-II B6 males with B6.Foxp3^sf/x mice. Mice were housed at the University of Virginia animal facility and all experiments were conducted in accordance with an IACUC approved protocol.

Flow cytometry

Single-cell suspensions were prepared from blood or lymph nodes. Erythrocytes were lysed with Tris-buffered NH₄Cl, pH 7.2. Approximately 10⁶ cells were stained with 0.2 μg each of PE-anti-CD4 (GK1.5, eBioscience, San Diego, CA, USA), PE-Cy5-anti-CD8 (53–6.7, eBioscience) and a panel of 15 FITC-anti-Vβ family mAbs (BD Pharmingen, San Diego, CA, USA) in 50 μl of PBS containing 1 μg anti-FcγRII/RIII mAb (2.4G2) and 4% BSA at 4°C for 30 min. At least 10⁴ stained cells were analyzed using a FACScan equipped with CellQuest (BD Biosciences, Santa Jose, CA, USA). Purified B20.1 anti-Vα2 (13), RR3-16 anti-Vα3.2 (14), C6 anti-Vα8 (15) and RR8-1 anti-Vα11 (16) mAbs were prepared and conjugated with fluorescent probes as previously described (17). Post-acquisition analyses were carried out using FlowJo™ software (Tree Star, Inc., OR, USA). The proportion and the total number of T-cell populations were determined from flow cytometric analyses.

Preparation of CD4⁺ T cells

Single-cell suspensions were prepared from pooled inguinal, axillary, brachial and cervical lymph nodes. CD4⁺ T cells were purified using immunomagnetic beads as described by the manufacturer (Miltenyi Biotec, Auburn, CA, USA). The CD4⁺CD25⁻ population was obtained by sorting using FACS (BD and Company, Franklin Lakes, NJ, USA). The purity of the isolated populations was ≥97% as determined by flow cytometry.

CDR3 size spectratyping

Total RNA, extracted from 2 × 10⁶ purified CD4⁺ T cells with Trizol reagent (Life Technologies, Grand Island, NY, USA), was reverse transcribed using oligo-dT primer and M-MuLV reverse transcriptase (New England Biolab, Ipswich, MA, USA). TCR Vβ segments were amplified by PCR with each of the 20 Vβ family specific primers and a constant beta (Cβ) primer end-labeled with 6-carboxyfluorescein (18). Vβ12, Vβ17 and Vβ19 were not studied because the former displays a single-peak spectratype and B6 mice do not express Vβ17 and Vβ19 (18). A standard containing fluorescently labeled DNA fragments of known sizes (400 HD [ROX]™; Applied Biosystems, Foster City, CA, USA) was added to the PCR products and the mixtures were analyzed with a 3130 Xl Genetic Analyzer using GeneMapper software (Applied Biosystems; version 4.0).

Comparison between spectratype patterns

In a normal population of T cells, spectratyping of individual Vβ families usually reveals 5–10 identifiable peaks and a profile showing an approximate Gaussian distribution (19). When the profile deviates from the bell-shaped Gaussian distribution, it is indicative of a TCR repertoire composition change in that Vβ family. To determine the magnitude of deviation, each spectratype was scored according to the method described by Haegert et al. (20) with modifications. The intensity of an individual peak reflects the relative representation of a particular CDR3 size within the Vβ family. The fluorescence intensity for individual CDR3 sizes (peaks) was quantified by the area under each peak using Gene-Mapper software. These values were used to determine the Pearson product moment correlation coefficient (r) for each Vβ family member between two samples by Excel software according to the formula: Σ(X − X_m) − (Y − Y_m)/{Σ(X − X_m)²Σ(Y − Y_m)²}^1/2 in which X and Y are areas of individual peaks of the two samples and X_m and Y_m are the mean of the areas of total peaks for sample X and sample Y, respectively (21). The r² value (coefficient of determinations) is used so that both positive and negative correlation is treated the same way. It represents the percent of similarity in sample X (e.g. a B6.sf mouse) that is accounted for by the spectratype of sample Y (e.g. a B6 mouse). The closer the r² value is to 1, the greater the similarity between the two Vβ family spectratypes. Generally, absolute r values ≥0.8 or r² values ≥0.64 are considered acceptable statistical correlation. Because our controls (among B6 mice) showed high and narrow ranged r² values, the r² values of the experimental groups were interpreted as relative deviation from the control spectratypes. The Student's t-test and F-test analyses were carried out with Microsoft Excel software version 2002. The Student's t-test was used to determine the significance of r² values between B6 and B6.sf mice. The F-test was used to determine whether two samples have different variances. The Wilcoxon rank test was used for the comparison between paired non-parametric data. A P value of ≤0.05 between compared groups is considered statistically significant.

Results

Vβ family expression profiles of various T-cell populations

We first determined the expression levels of TCR Vβ family members of lymph node lymphocytes obtained from individual B6 mice aged between 4 and 25 weeks. The distributions of 14 Vβ families were analyzed for CD4⁺ T cells, CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells by flow cytometry (Fig. 1). The expression profiles of Vβ families were similar among the three T-cell populations, suggesting that the CD4⁺CD25⁺ Treg population, which constitutes 5–10% of the total CD4⁺ T cells, does not cause a major change in the CD4⁺ T-cell Vβ family expression levels. Interestingly, there were also no significant changes in the T-cell Vβ family expression profile of CD4⁺ T cells of B6.sf mice despite the fact that they lacked Treg and had severe polyclonal autoimmune response (12) (Fig. 1).

Fig. 1.

TCR Vβ expression profiles of B6 CD4⁺, B6 CD4⁺CD25⁺, B6 CD4⁺CD25⁻ and B6.sf CD4⁺ T cells do not show significant changes as determined by staining with fluorescent anti-Vβ mAb. Lymph node samples (n ≥ 3) were stained and analyzed as described in Methods. The total Vβ families examined represented 76–86% of the total CD4⁺ T cells. The percentage of individual Vβ family was normalized based on these values.

TCR Vβ repertoire analysis based on spectratyping

Spectratyping based on TCR CDR3 lengths and their proportions in a defined Vβ family provides better resolution for repertoire changes than anti-Vβ staining. We compared the spectratypes among B6 CD4⁺ T cells, B6 CD4⁺CD25⁻ T cells and B6.sf CD4⁺ T cells (Fig. 2). Two representative samples from each group were presented. The TCR Vβ spectratypes of CD4⁺ T cells of individual B6 mice did not differ significantly and all maintained a semi-Gaussian distribution. In contrast, marked variability in the Vβ spectratypes of CD4⁺ T cells of individual B6.sf mice was observed. Most often, variability involved the proportion of a specific peak, resulting in loss of a semi-Gaussian distribution. A spectratype profile lacking a particular peak was found occasionally. These differences were not caused by the mere absence of Treg because they were not observed with the B6 CD4⁺CD25⁻ T cells (Figs 2 and 3).

Fig. 2.

Spectratypes of Vβ families in the CD4⁺ T-cell populations of individual B6 and B6.sf mice. Samples (n ≥ 3) were obtained from lymph nodes of 4-week-old male individuals of indicated groups. Two representative samples from each group are shown. The spectratypes of individual B6 Vβ families (B6#1 and B6#2) are similar, whereas the spectratypes of individual B6.sf mice (B6.sf#1 and B6.sf#2) are variable. The depletion of CD4⁺CD25⁺ Treg from normal B6 males (B6#5 and B6#6) did not change the spectratypes as compared with CD4⁺ T cells from B6#1 and B6#2.

Fig. 3.

Quantitative measurements of changes in the CD4⁺ T-cell Vβ spectratypes among Treg-deficient mice. The similarity between Vβ spectratypes of CD4⁺ T cells is presented based on r² values. (A) Three B6 mice, three anti-CD3ε (αCD3) mAb-treated B6 mice and three B6.sf mice were used in this presentation. Each symbol represents the comparison between one individual (B6, αCD3-treated B6 or B6.sf) with a B6 control. Each mark represents a Vβ family and 20 Vβ families are compared. Comparisons among B6 mice are as follows: B6#1 versus B6#2, B6#1 versus B6#3 and B6#2 versus B6 #3. For presentation purpose, comparisons between B6 and B6.sf mice (or αCD3-treated B6) are as follows: B6#1 versus B6.sf#1 (or αCD3-treated B6#8), B6#2 versus B6.sf#2 (or αCD3-treated B6#9) and B6#3 versus B6.sf#3 (or αCD3-treated B6#10), although similar results were obtained when comparisons were made in other combinations between B6 and B6.sf (or αCD3-treated B6) mice. Student's t-test for all Vβ families between individual B6 and B6.sf mice were significant (P < 0.05) in all cases. Significant differences (P < 0.05) were also obtained using F-test for variance between two groups and Wilcoxon rank test for paired non-parametric data comparison. Non-significant differences in r² values were obtained between B6#1 and αCD3-treated B6 groups (P = 0.11, 0.13 and 0.69, respectively, for the three paired comparisons). (B) The comparisons of r² values between the Vβ spectratypes of Treg-depleted CD4⁺ T cells of B6 mice (n = 3) and CD4⁺ T cells of B6.sf mice (n = 3) and between B6 CD4⁺ T cells and B6 CD4⁺CD25⁻ T cells (n = 3) are presented. (C) Each CD4⁺ T-cell Vβ family spectratype of a B6 individual was compared with that of another B6, B6.sf, IL-2 KO or IL-2Rα KO individual. In addition, comparison of each Vβ family between individual B6.sf mice is also presented. For both (B) and (C), the intra-group differences using Wilcoxon rank test were highly significant (P < 0.05 in all cases).

The extent of change in the spectratype of a particular CD4⁺ T-cell Vβ family of B6.sf mice relative to its B6 counterpart can be semiquantified by calculating the r² value as described in Methods (Fig. 3A). The Vβ family spectratypes of CD4⁺ T cells were highly similar among individual B6 mice and this is true for each of the Vβ families as demonstrated by the high r² values (0.936–0.999) in this comparison. In contrast, Vβ family spectratypes of CD4⁺ T cells of B6.sf mice were markedly different from B6 mice as demonstrated by the many low r² values in most although not all Vβ families. A significant difference in r² values based on Student's t-test was observed when all Vβ families of B6.sf mice were compared with B6 control (Fig. 3A). Similarly, significant differences were observed between Treg-depleted CD4⁺CD25⁻ T cells of B6 mice and CD4⁺ T cells of B6.sf mice (Fig. 3B). As controls, no significant differences were observed when comparisons were made in B6 mice either between CD4⁺ and CD4⁺CD25⁻ T cells of the same mouse or between CD4⁺CD25⁻ T cells from two different mice. The same conclusion was obtained with additional statistical analyses using F-test and Wilcoxon rank test.

To determine if the changes in TCR Vβ spectratypes are simply caused by a ‘global’ polyclonal activation, we treated 10- to 12-week-old B6 mice with purified 145-2C11 anti-CD3ε mAb (40 μg in 0.2 ml PBS, intraperitoneally) twice, spaced 1 week apart and determined the Vβ spectratype profiles of CD4⁺ T cells 10 days later. This treatment induced polyclonal activation of T cells as evidenced by the increase in lymph node T-cell numbers (∼2-fold) and the high expression of CD44 (>80%) on CD4⁺ T cells. Importantly, no significant difference between untreated and treated mice in the Vβ family spectratypes was observed (Fig. 3A). The result suggests strongly that the extensive changes in the Vβ spectratypes of B6.sf mice were due to, at least in part, the expansion of autoimmune T-cell repertoire by self-antigen.

We also compared the Vβ family spectratypes of CD4⁺ T cells for IL-2 KO and IL-2Rα KO mice of 8–12 weeks old (Fig. 3C). Unlike B6.sf mice that totally lack Treg, these mice contain residual Treg and their autoimmune response develops less rapidly and is less severe. Nonetheless, moderate changes in the spectratypes of the Vβ families were observed as indicated by many of their r² values in the range between 0.6 and 0.9. Moreover, changes were observed often in some but not all Vβ family members of individual mice. The Vβ family members that displayed changes in spectratypes varied among individuals, indicating that the expansion of T cells occurred in a stochastic manner. This interpretation is supported by the observation that even among B6.sf mice, their TCR Vβ spectratypes were markedly different as shown by the many low r² values when Vβ family members between any two B6.sf mice were compared (Fig. 3C).

Expansion of non-transgenic Vα repertoire in B6.sf mice bearing a foreign antigen-specific transgenic TCR

Because we observed significant changes in many Vβ spectratypes, we hypothesize that similar changes should be observed for Vα repertoire; in any randomly selected TCR transgenic B6.sf mice, the great majority of their thymic CD4⁺ T cells bear the fixed transgenic Vβ. We addressed this issue at the clonal level by flow cytometric analyses. We compared the expression levels of Vα families between OT-II B6 mice and OT-II.sf mice using various anti-Vα mAbs. The latter, but not the former, group spontaneously developed multiorgan autoimmune inflammation, although the manifestation of disease was delayed a few more weeks than B6.sf mice. Similar to B6.sf mice, inflammation was observed in ear, tail, skin, lung and liver as determined by hematoxylin and eosin stain (data not shown). Expression of the transgenic TCR (Vβ5.1/5.2^highVα2^high) on blood CD4⁺ T cells was high in OT-II B6 mice (∼90%). Importantly, ∼2% of the CD4⁺ T cells bear non-transgenic Vβ paired with non-transgenic Vα2 and 3% paired with transgenic Vα2^intermediate (Fig. 4A, middle panel). In sharp contrast to OT-II B6 mice, the transgenic TCR population was dramatically reduced in OT-II.sf mice (7%) due to the expansion of T cells bearing non-transgenic Vβ (Fig. 4A, lower panel). The expanded T-cell populations contained various Vβ family members (data not shown) as well as T cells bearing Vα2^intermediate (57%) and T cells expressing mainly non-transgenic Vα (22%, Fig. 4A, lower panel). We determined whether the Vα2^intermediate expressed dual-TCR in the second experiment (Fig. 4B). In this experiment, the CD4⁺ T cells bearing the Vα2^intermediate and non-transgenic Vα represented 73–76% and 11–12% of the total OT-II.sf CD4⁺ T cells, respectively. The former population contains T cells bearing dual-TCR expressing Vα2^intermediate Vα3.2^high, Vα2^intermediate Vα8^high or Vα2^intermediateVα11^high as demonstrated by double staining with anti-Vα2 plus anti-Vα3.2, anti-Vα2 plus anti-Vα8 and anti-Vα2 plus anti-Vα11 mAbs, respectively (Fig. 4B, lower panels). The low frequency of a specific Vα family reflects the large number of potential Vα genes (∼70) in the mouse T-cell repertoire. The data extended the conclusion obtained with Vβ spectratype analyses and suggested that autoimmune T-cell repertoire is pervasive and stochastically expanded in B6.sf mice, contributing to changes in both Vα and Vβ repertoire as compared with normal control.

Fig. 4.

Reduction of transgenic TCR and expansion of non-transgenic Vα repertoire in OT-II.sf mice. (A) Peripheral blood lymphocytes from B6, OT-II B6 and OT-II.sf mice were stained with anti-CD4, anti-Vβ5.1/5.2 and anti-Vα2 mAbs and analyzed on CD4-gated cells. (B) Cells were stained with anti-CD4, anti-Vα2 and anti-Vα3.2 (or anti-Vα8 or anti-Vα11) and analyzed on CD4-gated cells. Expression of Vα2 was artificially grouped into Vα2^high, Vα2^intermediate and Vα2^low, based on staining with anti-Vα2 and isotype control (data not shown). Note that Vα2^low and Vβ5.1/5.2^low are equivalent to undetectable level of expression based on staining of a normal B6 control, which contains 83% non-Vα2 populations and 96% non-Vβ5.1/5.2 (top panels in A). Numbers in the panels indicate the percentage of a defined population represented in the total CD4⁺ T cells. OT-II.sf but not OT-II B6 contained expanded populations of Vβ5.1/5.2^lowVα2^intermediate and Vβ5.1/5.2^lowVα2^low CD4⁺ T cells (lower panels in A and B). In the expanded populations, co-expression of Vα2 with Vα3.2, Vα8 or Vα11 on individual CD4⁺ T cells was observed (B). A and B were the results obtained from individual mice.

Discussion

The present study addresses a very important issue of autoimmunity, i.e. how widespread are the potential autoimmune T cells among the CD4⁺ T-cell population and in what manner are they regulated. We hypothesize that in the absence of Treg, autoimmune T cells will expand in response to self-antigen, contributing to a significant and magnified change of T-cell repertoire. By using Vβ spectratyping and TCR transgenic mice, we are able to demonstrate that (i) polyclonal activation of CD4⁺ T cells in Treg-sufficient mice by anti-CD3ε mAb does not induce apparent changes of CD4⁺ TCR Vβ spectratypes, (ii) Treg deficiency allows polyclonal activation of the CD4⁺ T cells in which autoimmune T cells are preferentially activated, resulting in extensive changes of T-cell repertoire, (iii) the CD4⁺ autoimmune T-cell repertoire is potentially large, widespread across many or all Vβ families and occurs in a stochastic manner and (iv) TCR transgenic OTII.sf mice displayed multiorgan autoimmune syndrome with concomitant changes in their Vβ and Vα repertoires in which the non-transgenic Vβ was paired with the transgenic Vα and/or endogenous Vα family members.

Using TCR (Vα18 and Vβ6) transgenic mice and based on Vα2 CDR3 sequence analysis, Hsieh et al. (7) have shown a high frequency of TCR clones with self-reactivity and an overrepresentation of an autoreactive Treg cell clone (R19) in the CD25⁺ T cells of Foxp3⁻ mice. In addition, several individual TCRs from Foxp3⁻ mice were found in the wild-type CD25⁺ Treg repertoire (7). This and their subsequent study suggest that the TCR repertoire in the thymus of Foxp3⁻ mice contains those destined to Treg in normal mice and these cells are autoreactive (7, 9). However, sorted GFP⁺ cells lacking functional Foxp3 from Foxp3-green fluorescent protein (GFP) knock-in mice did not transfer multiorgan autoimmune disease in another study (22). In addition, B6.sf mice have normal thymic negative selection mechanism (23). Consequently, clones with high affinity against self-antigen should be deleted when the Treg differentiation pathway is blocked, resulting in low affinity autoreactive T cells in the periphery (1, 2). Whether and to what extent do Treg divert to CD4⁺ autoimmune T-cell pool in B6.sf mice remains to be determined (7, 9, 22). Our Vβ family profiling and spectratyping could not determine this issue. Our data, however, showed that the major point regarding autoimmune T-cell repertoire regulation is not the Treg diversion but the self-antigen-mediated autoimmune T-cell expansion consequent to Treg deficiency. The mere diversion of Treg repertoire into autoimmune T-cell pool could not affect Vβ family expression profiles and Vβ spectratyping (Figs 2 and 3B). Only in the periphery where T cells are activated and expanded due to the absence of Treg will the pervasive changes in TCR Vβ repertoire be observed by spectratyping.

Comparing sequences among various Vβ and Vα families between normal B6 and B6.sf mice could provide a more definitive biochemical evidence for the expansion of autoimmune T-cell repertoire but this comparison also requires the determination whether the individual TCR is autoimmune, either by adoptive transfer or by stimulation with autologus antigen-presenting cells using T cells transfected with the specific TCR gene pair (7, 9). This tedious and laborious approach is limited to mice with restricted TCR repertoire such as TCR transgenic mice. In addition, the study is restricted to a Vα family (7, 9). Analysis of Vβ repertoire will be difficult by this approach. Our study is the first to demonstrate that an extensive change in the naturally derived TCR repertoire of CD4⁺ T cells had occurred in 20 Vβ families examined in B6.sf mice in <4 weeks after birth and this change correlated with the expression of multiorgan autoimmune inflammation. This expanded population indeed contains autoimmune and is highly diversified because as few as 1.25 × 10⁶ CD4⁺ B6.sf T cells could transfer severe autoimmune disease against 12 organs/tissues in RAG-1 KO recipients (24). Thus, these studies linked the molecular evidence for a large and expanded autoimmune repertoire in B6.sf mice to the functional induction of multiorgan autoimmune syndrome due to the absence of Treg.

Foreign antigen-specific TCR transgenic mice display fixed Vβ and allow rearrangement of the non-transgenic Vα. Despite the overwhelming power of transgenic Vβ-mediated freezing of Vβ rearrangement, a small fraction of T cells express non-transgenic Vβ (Fig. 4). Thus, the autoimmune T cells can be derived by pairing the fixed Vβ with the non-transgenic Vα or by pairing the non-transgenic Vβ with either transgenic Vα or endogenous Vα. According to our observation, these non-clonotypic TCR in TCR transgenic male mice bearing the sf mutation should contain autoimmune T cells and should be preferentially expanded. Indeed, all foreign antigen-specific TCR transgenic male mice bearing the sf mutation displayed the scurfy-like autoimmune syndrome (7, 23, 25, this study). In addition, these cells should have reduced expression of the transgenic TCR. In the OT-II.sf mice, a dramatic reduction of the clonotypic T cells is accompanied by the expansion of CD4⁺ T cells bearing the dual-TCR (non-transgenic Vβ paired with non-transgenic Vα and transgenic Vα) and CD4⁺ T cells that express non-transgenic Vβ and non-transgenic Vα. Study is in progress to determine if these subpopulations could transfer multiorgan autoimmune syndrome in RAG-1 KO recipients.

The stochastic and pervasive nature of changes in multiple Vβ families in Treg-deficient mice are in contrast to several organ-specific autoimmune disease models in which a restricted and recurrent Vβ spectratype was often observed (26, 27). In addition, despite extensive Vβ spectratype changes in B6.sf mice, the percentile of each Vβ family presented in the CD4⁺ T-cell population remained essentially the same as that of normal B6 mice (Fig. 1). These observations suggest that autoimmune response occurrence is proportional to the frequency of the Vβ family in the CD4⁺ T-cell repertoire, an interpretation consistent with the stochastic and pervasive nature of the autoimmune T-cell development. In normal mice, these autoimmune T cells are kept in check by the Treg, despite the presence of a large repertoire. By losing such a critical control of autoimmune response and autoimmune repertoire, Treg-deficient mice, unlike other organ-specific autoimmune disease mouse models, will rapidly develop severe multiorgan autoimmune syndrome and die at an early age.

Abbreviations

Foxp3

fork-head box p3

GFP

green fluorescent protein

KO

knockout

Treg

regulatory T cells