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The Journal of Immunology, 2008, 180, 4577 -4585
Copyright © 2008 by The American Association of Immunologists, Inc.
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Immunodominance in the TCR Repertoire of {alpha}TCR Peptide-Specific CD4+ Treg Population That Controls Experimental Autoimmune Encephalomyelitis1

Loui Thomas Madakamutil*, Igor Maricic*,{dagger}, Eli E. Sercarz*,{ddagger} and Vipin Kumar2,*,{dagger}

* Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA 92037; and {dagger} Laboratories of Autoimmunity and {ddagger} Immune Regulation, Torrey Pines Institute for Molecular Studies, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunodominance in self-Ag-reactive pathogenic CD4+ T cells has been well established in several experimental models. Although it is clear that regulatory lymphocytes (Treg) play a crucial role in the control of autoreactive cells, it is still not clear whether immunodominant CD4+ Treg clones are also involved in control of autoreactivity. We have shown that TCR-peptide-reactive CD4+ and CD8+ Treg play an important role in the spontaneous recovery and resistance from reinduction of experimental autoimmune encephalomyelitis in B10.PL mice. We report, by sequencing of the TCR {alpha}- and β-chain associated with CD4+ Treg, that the TCR repertoire is limited and the majority of CD4+ Treg use the TCR Vβ14 and V{alpha}4 gene segments. Interestingly, sequencing and spectratyping data of cloned and polyclonal Treg populations revealed that a dominant public CD4+ Treg clonotype expressing Vβ14-Jβ1.2 with a CDR3 length of 7 aa exists in the naive peripheral repertoire and is expanded during the course of recovery from experimental autoimmune encephalomyelitis. Furthermore, a higher frequency of CD4+ Treg clones in the naive repertoire correlates with less severity and more rapid spontaneous recovery from disease in parental B10.PL or PL/J and (B10.PL x PL/J)F1 mice. These findings suggest that unlike the Ag-nonspecific, diverse TCR repertoire among the CD25+CD4+ Treg population, TCR-peptide-reactive CD4+ Treg involved in negative feedback regulation of autoimmunity use a highly limited TCR V-gene repertoire. Thus, a selective set of immunodominant Treg as well as pathogenic T cell clones can be targeted for potential intervention in autoimmune disease conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The requirement for a highly diverse TCR repertoire enabling T cells to react to a broad distribution of environmental Ags, coupled with the degeneracy of TCR specificity, could lead to a cascade of autoimmunity. Furthermore, the peripheral T cell repertoire is, by its selective origin in the thymus, based on self-reactivity and, in fact, T cells demonstrating self-reactivity have been consistently detected in the periphery (1, 2). Nonetheless, the immune system has developed proficient regulatory mechanisms designed to prevent autoimmune disease, including central tolerance and active regulation. During thymic development, and later in the periphery, the TCR repertoire is continuously purged of cells that are too strongly activated by self-antigenic determinants (3). Additionally, thymic and peripheral lymphocytes are recruited to join a network of regulatory T cells that assist in preventing the aberrant activation of destructive autoimmunity (4, 5, 6, 7).

Several mechanisms have been described for the regulation of the autoaggressive T cell repertoire. For example, a subpopulation of CD4+ T cells, constitutively expressing CD25 has gained recent attention as being regulatory in both mice and humans (6, 7). These cells act to prevent the expansion of inflammatory reactions. They are unable to produce IL (IL-2), and are dependent on IL-2 for growth in vitro as well as in vivo. The exact mechanism of their regulation in vivo is unclear, but some studies indicate a role for TGF-β and/or CTLA-4. In vitro, the mechanism of suppression appears to be contact-dependent and independent of soluble factors. Another set of regulatory/suppressive T cells in both human and mouse are CD4+ T cells that are generated by repeated stimulation in the presence of IL-10 (8). These cells, termed T regulatory cell 1, proliferate poorly, secrete high levels of IL-10, and regulate both Th1 and Th2 immune responses in vivo.

We have previously described a different regulatory CD4+ T cell population that arises spontaneously during the recovery phase of MBP3 Ac1–9-induced experimental autoimmune encephalomyelitis (EAE) in B10.PL mice (9, 10). These regulatory CD4+ T cells are reactive to the TCR Vβ8.2 chain framework 3 region peptide B5 (aa 76–101) (10, 11, 12). Staining with TCRVβ-chain-specific mAbs showed that B5-reactive CD4+ T cell clones and hybridomas are predominantly Vβ14+. Furthermore, removal of the Vβ14+ T cell repertoire, by anti-Vβ14 mAb treatment, results in exacerbation of EAE and is associated with poor recovery (13). Additionally we demonstrated that the adoptive transfer of clones of the Vβ14+ Treg in the presence, but not in the absence of CD8+ T cells protected recipients from EAE (9, 12). The spontaneous generation of such regulatory T cells during the course of the disease suggested that they are physiologically involved in the regulation of clinical disease and form part of a mature immune network.

Several investigations have suggested the induction of TCR-peptide-reactive T cells following vaccination with pathogenic T cell lines, clones or peptides derived from the TCR expressed on pathogenic T cells (11, 14, 15, 16, 17, 18, 19, 20). Importantly, the Fr3 region of the TCR Vβ8.2 chain has been shown to contain an immunodominant determinant in mouse, rat and human models (11, 12, 21, 22, 23, 24). Furthermore, the T cell repertoire reactive to this region cannot be tolerized by neonatal or i.v. tolerance-inducing protocols in either mice or rats (11, 13, 23). We and others have used different approaches to induce efficient expansion of TCR-peptide-reactive regulatory T cells in the hope of identifying the optimal protocol for the modulation of autoimmunity. For example, recombinant single chain Vβ8.2 proteins, Vβ8.2 plasmid DNA, or vaccinia, or adenovirus gene delivery systems have been investigated (12, 25, 26, 27, 28, 29). Although these studies have shown that TCR-peptide-reactive T cells can be induced by various immunizing protocols, their TCR V-gene usage as well as their frequency in the naive T cell repertoire has not been investigated.

In this study, we have further examined the TCR repertoire of CD4+ Treg by cloning and sequencing their TCR {alpha}- and β-genes. These CD4+ Treg use a limited set of TCR Vβ genes coupled with a predominant CDR3 gene segment, which has enabled us to use immunoscope analysis to track their presence in naive mice and during the course of EAE. Our studies demonstrate that Vβ14+CD4+ regulatory T cells, with a restricted TCR Vβ usage, are persistently observed at the onset and during recovery from MBPAc1–9-induced EAE. Both the Vβ14-Jβ1.2 and the Vβ14-Jβ2.3 clonotypes are expanded following direct immunization with TCR peptide B5 resulting in prevention of disease. These regulatory CD4+ T cell clones are present in the naive repertoire and their number in the peripheral repertoire of parental strains as well as in F1 mice positively correlates with diminished severity and a truncated disease course.


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

B10.PL and PL/J mice were purchased from The Jackson Laboratory and they, along with (B10.PL x PL/J)F1 mice, were bred in the vivarium at the La Jolla Institute for Allergy and Immunology (La Jolla, CA) and at the Torrey Pines Institute for Molecular Studies (San Diego, CA). All the mice were bred and maintained under specific pathogen-free conditions in our own colony. Female mice from 6 to 14 wk of age were used in all experiments. Experiments involving animals were performed in compliance with federal and institutional guidelines and have been approved by La Jolla Institute for Allergy and Immunology/Torrey Pines Institute for Molecular Studies Animal Care and Use Committee.

Abs and flow cytometry

For staining TCR peptide-specific T cell hybridomas, lines and clones, the following anti-TCR mAbs conjugated to either FITC or PE were purchased from BD Biosciences: anti-Vβ2, anti-Vβ3, anti-Vβ4, anti-Vβ5.1/5.2, anti-Vβ6, anti-Vβ7, anti-Vβ8.1/8.2, anti-Vβ8.3, anti-Vβ9, anti-Vβ10, anti-Vβ11, anti-Vβ13, anti-Vβ14, anti-Vβ17 FITC- and PE- conjugated isotype control Abs. Anti-CD4 and anti-CD3 Abs were conjugated to either FITC or PE. Flow cytometric analysis was conducted using the FACS Calibur and Cell Quest software, as described earlier (30).

Peptides

TCR peptides were synthesized as reported earlier by S. Horvath (California Institute of Technology, Pasadena, CA) (19). TCR Vβ8.2 chain peptides correspond to the sequence predominantly used in the MBP-specific response in B10.PL mice (31, 32) and are as follows: B1(aa 1–31) -EAAVTQSPRNKVAVTGGKVTLSCNQTNNHNL and B5(aa 76–101) -LILELATPSQTSVYFCASGDAGGGYE.

Induction of EAE

Mice were immunized subcutaneously with 150 µg Ac1–9 emulsified in CFA. 0.15 µg of Pertussis toxin (PTx) was injected in PBS 24 h and 72 h later. Mice were observed for EAE daily. Disease was scored on a 5-point scale as described earlier (19): 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, whole body paralysis; 5, death.

Generation of TCR peptide B5-reactive T cell hybridomas and T cell lines

B10.PL mice were immunized in the footpad with TCR peptide B5 in CFA. Day 15 spleen cells were harvested and were stimulated in vitro with TCR peptide B5 and after three days, T cells were fused with the BW 5147 (TCR {alpha}–β–) fusion partner. T cell hybridomas were selected in RPMI 1640 medium plus 10% FBS containing HAT (19). Wells that grew to confluency were subjected to limiting dilution cloning to derive single hybridoma cells in 96-well flat-bottom plates. Wells with expansions were tested for their reactivity to TCR peptide B5 in a standard IL-2 sensitive HT-2 cell line assay that measures IL-2 secretion in response to the TCR peptide B5. TCR peptide-reactive T cell hybridomas were selected and weaned from HAT medium into RPMI 10% FBS medium. These T cell hybridomas were screened for their TCR β and {alpha} usage with the available set of anti-TCR mAbs (BD Pharmingen).

T cell lines reactive to TCR peptide B5 were generated from naive B10.PL mice by stimulating splenocytes with TCR peptide B5 (40 µg/ml) in RPMI containing 10% FBS. Cultures were stimulated with rIL-2 (10 U/ml) or TCR peptide B5 and irradiated autologous spleen cells (2–5 x 106 spleen cells/well) in alternate weekly cycles. Flow cytometric analysis showed that a majority of the T cells displayed Vβ14 TCRs (line no. 1: 64% Vβ14+ T cells; line no. 2: 51% Vβ14+ T cells).

Immunoscope analysis

TCR CDR3 length spectratyping or "immunoscope" analysis was performed with a protocol modified from that described by Pannetier et al. (33). Total mRNA was extracted from B5 immunized or Ac1–9 immunized mice (RNeasy mini kit; Qiagen). Spleen cells were cultured with the immunizing peptide for 3 days before extracting total mRNA. The total mRNA was subjected to cDNA synthesis with an oligo(dT)16–18 primer. Equal amounts of cDNA were subjected to PCR using TCR Vβ14 (Vβ14, 5'-ACGACCAATTCATCCTAAGCAC-3') and Cβ (Cβ145, 5'-CACTGATGTTCTGTGTGACA-3') primers; Vβ8.2 (5'-CATTATTCATATGGTGCTGGC-3') and Cβ primers; or Vβ13 (5'-AGGCCTAAAGGAACTAACTCCCAC-3') and Cβ primers. After run-off extensions with 12 Jβ fluoresceinated primers, the run-off products were separated on the basis of their length on an automated 310 genetic analyzer ABI PRISM with POP-4 polymer and a 5–47 cm x 50 µm capillary (Applied Biosystems). The relative index of stimulation (RIS) value is an estimation of the clonal expansion of T cells in a bulk T cell sample (30). The RIS was calculated using the following formula: RIS = (area under the expanded peak/area under the remaining peaks in the profile)/(area under the peak corresponding to the expanded peak in the normal nonimmunized profile/area under the remaining peaks in the normal profile).

DNA sequencing

RNA from TCR peptide B5-reactive CD4+ T cell lines, clones and hybridomas generated from either immunized or naive B10.PL mice was prepared using the RNeasy kit (Qiagen). The mRNA was reverse transcribed to cDNA using Superscript and an oligo(dT)16–18 primer. TCRVβ14-Cβ and V{alpha}-C{alpha} primers were used to amplify the respective cDNA using PCR. (V{alpha}, 5'-ATGATTGTGATGCTCCTCATATTC-3' and C{alpha}, 5'-AGACCGAGGATCTTTTAACTG-3'). The PCR products were cloned into pCR2.1 TOPO vector (Invitrogen), and plasmid DNA was purified using the Qiagen plasmid mini prep kit (Qiagen). Plasmid DNA containing the relevant DNA was selected by using EcoR1 digestion and sequenced using M13 forward and reverse primers. The sequencing reaction was analyzed on the 310 genetic analyzer ABI PRISM with the POP-6 polymer using a 3.61 cm x 75 µm capillary (Applied Biosystems).

Limiting dilution analysis

MACS purified CD4+ T cells from B10.PL and PL/J spleens were plated into 96 U-bottom plates and 7 doubling dilutions were made from 105 cells/well down to 781 cells/well. T cell proliferation to TCR peptides B1 or B5, in the presence of irradiated spleen cells, was assessed in a typical proliferation assay. [3H]Thymidine was added 72 h before harvesting. The fraction of responding cells in each dilution was calculated and the frequency of cells responding to each TCR peptide was calculated using Poisson distribution analysis, as described earlier (34).

Cellular ELISPOT analysis

IFN-{gamma}- and IL-4-producing cells were enumerated in splenocytes from naive mice by cellular ELISPOT assay as described earlier (35). In brief, splenocytes (5 x 106 cells/ml) were cultured for 48 h in 24-well plates either with medium alone or with B5 (20 µg/ml). Millititer HA nitrocellulose plates (Millipore) were coated overnight at 4°C with anti-IFN-g or anti-IL-4 Abs. After blocking the coated plates, Ag-stimulated cells were added at graded concentrations for 24 h at 37°C. The wells were then incubated with biotin-conjugated anti-IFN-{gamma} or anti-IL-4 mAbs followed by incubation with avidin peroxidase (Vector Laboratories). Spots were developed by the addition of 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich) and counted using a computerized image analysis system (Light-tools Research) and the image analyzer program, NIH Image 1.61.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TCR peptide B5-reactive regulatory T cells show restricted TCR V-gene usage

Using flow cytometric analysis we have previously demonstrated that TCR-peptide B5-reactive CD4+ Treg in B10.PL mice were oligoclonal and predominantly Vβ14+ and Vβ3+ (9). Depletion of the Vβ14+ T cell population could prevent spontaneous recovery, and also results in a chronic and more severe form of EAE in B10.PL mice. In the present study, we further examined the TCR repertoire of B5-reactive regulatory CD4+ T cells by cloning and sequencing their TCR {alpha}- and β-chain genes.

The CD4+ Treg cell clone MB5.1, reactive to the B5 peptide derived from the framework region 3 of the Vβ8.2 chain, has been shown to demonstrate regulatory function. Thus adoptive transfer of this T cell clone protects B10.PL mice from Ac1–9-induced EAE (9). Additionally, TCR peptide B5 was used as an immunogen to generate specific CD4+ T cell clones and hybridomas from B10.PL mice. Alternatively, CD4+ T cell clones were also obtained from mice recovering from MBPAc1–9-induced EAE. The CD4+ T cell clones and hybridomas (n = 11 shown in Table I) were examined for their TCR V{alpha} and Vβ usage, and their reactivity to TCR peptide B5 was measured by proliferation and cytokine release assays. All T cell hybridomas and clones were CD4+ and CD8–. Vβ TCR mRNA from these T cells was reverse transcribed, amplified, cloned, and sequenced as described in Materials and Methods. Six of the B5 peptide-reactive CD4+ T cell hybridomas and the functional T cell clone displayed the TCR Vβ14+ phenotype (Table I). Two hybridomas used TCRVβ3+, one used TCR Vβ4+ and one used TCR Vβ8.3+. Sequence analysis of the β-chain of these hybridomas showed that all these hybridomas had a somewhat similar CDR3 composition, most having both a serine and glycine in the NDN region with additional polar or hydrophobic residues. Three of the six Vβ14+ T cell hybridomas and the functional Vβ14+ Treg clone used the Jβ1.2 gene segment. In addition, the Vβ CDR3 region of the regulatory T cells possessed a conserved negatively charged amino acid (Glu or Asp) as well as Tyr and/or Leu either provided by the Jβ gene or by the N additions. TCR V{alpha}-chain analysis using flow cytometric or sequence analysis of the {alpha}-chain of the hybridomas revealed that there was much heterogeneity in the V{alpha} TCR usage among these TCR peptide B5 reactive CD4+ Treg cells (see Table II and III ). However, three of the six Vβ14+ hybridomas as well as the functional clone (MB5.2) used a TCR V{alpha}4.3 gene segment (Table II and III).


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  		Table I. The aa sequences of the junctional region of the TCR Vβ -chains of B5-reactive CD4+ Treg cellsa

 

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  		Table II. The aa sequences of the junctional region of the TCR V{alpha} -chains expressed by CD4+ Treg cellsa

 

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  		Table III. A summary of the TCR V{alpha} chain gene expression by CD4+ Treg cellsa

 
Immunoscope analysis of the Vβ14+ hybridomas and the MB5.2 T cell clone revealed that 3 of the 6 hybridomas and the clone displayed Vβ14-Jβ1.2 gene usage with a 7 aa CDR3 length (Fig. 1). The remaining Vβ14+ T cell hybridomas demonstrated either Jβ2.3 (8 aa CDR3), Jβ2.5 (9 aa CDR3), or Jβ2.6 (6 aa CDR3) gene segment usage.


Figure 1
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  		FIGURE 1. Immunoscope analysis of cloned B5-reactive Vβ14+CD4+ Treg cells. The Vβ14+ Treg clones and hybridomas were analyzed by CDR3 spectratyping or immunoscope analysis following RT-PCR amplification using the Vβ14-Cβ primer pair. The amplified products were further subjected to a run-off PCR using a panel of fluorescent Jβ primers, and the products were analyzed via automated CDR3 length analysis (ABI PRISM 310 genetic analyzer). Because Vβ14+ T cells were clonal, they showed only a single amplification peak in a particular Vβ-Jβ set as shown in the figure and accordingly no detectable amplification was seen with any of the other Vβ-Jβ combinations.

 
In summary, phenotypic characterization of the TCR peptide B5 reactive regulatory CD4+ T cells revealed a rather limited V gene usage: the majority of these T cells expressed TCR Vβ14 and the Jβ1.2 gene segment with a 7 aa CDR3 length. Furthermore, most of the Vβ14+CD4+ Treg cells used the TCR V{alpha}4 gene segment.

Vβ14-Jβ1.2 specific Treg expansions during the spontaneous recovery from EAE

Because there was dominant usage of the Vβ14-Jβ1.2 gene segment among the regulatory T cell hybridomas and clones, we analyzed Vβ14 TCR usage in individual mice spontaneously recovering from Ac1–9-induced EAE. Immunoscope analysis is designed to dissect the T cell repertoire to reveal clonal expansions from any bulk in situ population (e.g., from spleen, lymph nodes, or CNS). This RT-PCR based technique initially uses TCR Vβ-Cβ combinations to amplify a single TCR gene. These amplified Vβ-Cβ products are further used in combination with each of the Jβ primers that are fluoresceinated in a PCR run-off reaction. The run-off reactions are separated on an automated sequencer ABI Prism 310 Genetic Analyzer, modified to analyze the CDR3 length of the TCRs. In a nonimmunized mouse, a TCR "spectratype" for a particular Vβ-Jβ family will display a Gaussian distribution of ~8–10 peaks, with each peak representing a Vβ-Jβ CDR3 length within the Vβ-Cβ combination. When a particular CDR3 specific clonal expansion occurs, a single peak will rise above the other neighboring peaks. This technique subdivides the mouse Vβ T cell repertoire into ~2800 compartments and overcomes the requirement for T cell cloning to detect T cell expansions in each animal. It is especially potent in seeking known expansions, such as Vβ14-Jβ1.2 among individual animals.

EAE was induced in B10.PL mice with Ac1–9 CFA/PTx, and spleens were harvested from mice either at the onset of EAE (day 15) or during recovery from disease (day 27). Spleen cells were either unstimulated or stimulated in vitro with TCR peptide B5 (40 µg/ml) for three days. Total mRNA was extracted from the cells and amplified by PCR with the Vβ14-Cβ primer set. Each Vβ14-Cβ PCR product was further subjected to a run-off reaction with a panel of fluorescent Jβ primers and the samples were separated on a genetic analyzer as described in the Materials and Methods section. In vitro stimulation of spleen cells with the specific Ag allows the development of observable peaks in the CDR3 profile. As depicted in Fig. 2a, spleen cells from mice spontaneously recovering from EAE showed a high proliferative responsiveness to TCR peptide B5, but not to a control peptide, B1. Spleen cells from normal mice did not respond to either of the TCR peptides. These data are consistent with previously reported studies (11).


Figure 2
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  		FIGURE 2. Clonal expansion of a public Vβ14+ regulatory CD4+ T cell during recovery from EAE. a, The proliferative responses of spleen cells from either nonimmunized mice or from mice recovering from EAE (day 27) were assessed in response to an in vitro challenge with graded concentrations of TCR peptide B5 (filled histogram) or to control TCR peptide B1 (open histogram). Thymidine incorporation, at an optimal concentration of 40 µg/ml TCR peptide is shown. Representative data from two independent experiments are shown. b, Spleen cells from B10.PL mice spontaneously recovering from EAE were harvested and stimulated in vitro with 40 µg/ml TCR peptide B5 (left panel) and analyzed for various Vβ14-Jβ specific TCR clonal expansions using immunoscope, as described in the Materials and Methods section. A representative immunoscope profile of a Vβ14-Jβ1.2 TCR expansion with a dominant 7 aa CDR3 length is shown. Profiles of Vβ14-Jβ1.2 TCRs from unstimulated (center panel) or CFA-immunized B10.PL spleens (right panel) are also presented. c, RIS values of Vβ14-Jβ1.2 expansions were observed in spleens of individual B10.PL mice (n = 6), spontaneously recovering from EAE.

 
Clonal expansion of T cells with the Vβ14-Jβ1.2 gene segment (RIS > 3 or more) was observed in all mice at various stages of EAE. Furthermore, it was observed that all the Vβ14-Jβ1.2 TCR expansions had a 7 aa CDR3 length. In the mice recovering from EAE (7 in the group) there also was a dominant expansion of T cells using the Vβ14-Jβ1.2 gene segment with a RIS value >3 (Fig. 2, b and c). However, unlike the mice at the onset of EAE, the mice recovering from EAE had Vβ14-Jβ1.2 TCR expansions with both 7 aa. and/or 9 aa CDR3 lengths. Four of the mice had the Vβ14-Jβ1.2 TCR expansions with a 9 aa CDR3 length. One mouse had both the 7 and 9 aa CDR3 length expansions in the Vβ14-Jβ1.2 profile (Table IV).


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  		Table IV. Immunoscope analysis for Vβ14-Jβ gene segment usage in splenocytes from B10.PL mice either at the onset or recovery from MBPAc1–9-induced EAEa

 
Interestingly, dominant expansion of T cells with the Vβ14-Jβ1.5 gene segment was also observed. Contrary to the Vβ14-Jβ1.2 expansions detected in each mouse at the onset of EAE or the recovery phase, the Vβ14-Jβ1.5 expansion was observed in the majority but not all B10.PL mice (10/14). Furthermore, the Vβ14-Jβ1.5 expansions were of various CDR3 lengths (7–10 aa). Besides these Vβ-Jβ families, there were a few other Vβ14-Jβ expansions. The T cell clone MB 5.2 which used the Vβ14-Jβ1.2 gene segment, as determined by sequencing, also showed a single expansion, detected by spectratyping, within the Vβ14-Jβ1.2 gene segment (RIS > 12; 7 aa CDR3 length) (Fig. 1).

These results suggest that TCR peptide B5-reactive T cells dominantly express the Vβ14-Jβ1.2 gene segment with a 7 or 9 aa CDR3 length, with preference given to the former length. They are generated spontaneously during the onset of EAE and persist during recovery. It is not yet clear whether these and other T cell expansions, e.g., within the Vβ14-Jβ1.5 family, represent functional regulatory T cells. Because expansion of other irrelevant TCR peptide-reactive T cells do not modulate the course of EAE, it is unlikely that such T cells would be able to regulate nonspecifically by competing with MBP-reactive pathogenic Vβ8.2+ T cells for space or IL-2.

Vβ14-Jβ1.2 specific T cell clones are primed after immunization with TCR peptide B5

Immunization with TCR peptide B5 prevents mice from developing MBPAc1–9-induced EAE, presumably owing to activation/expansion of Vβ14-Jβ1.2+ T cells. We therefore examined whether direct immunization with the B5 peptide could expand Vβ14-Jβ1.2+ T cells. Table V shows that Vβ14-Jβ1.2 T cells with 7 aa CDR3 expansions were readily detected in both B10.PL and PL/J mice (both H-2u strains). Furthermore, it was observed that Vβ14-Jβ1.2 T cell expansions also occurred in mice that were protected from Ac1–9 induced EAE by prior immunization with TCR peptide B5. This suggests that the regulatory T cells that were spontaneously generated during the recovery from EAE were also intrinsically primed by TCR peptide B5, and that these Vβ14-Jβ1.2 T cells mediate protection from EAE.


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  		Table V. Immunoscope analysis of the Vβ14-Jβ expansion profile in immunized micea

 
Vβ14-Jβ1.2+ Treg clones are part of the naive repertoire of mice

Do the Vβ14-Jβ1.2 T cell expansions seen in the B10.PL mouse represent an expansion of a naive population of T cells or have these cells been primed previously? To address this issue, B5-reactive T cell lines were generated from the spleens of naive mice. TCR peptide B5-reactive T cells could be easily generated from B10.PL spleen cells derived from nonimmunized mice by repeated in vitro stimulation with the TCR peptide B5. Fig. 3a displays flow cytometric analysis of these T cell lines showing that a majority of the T cells used the Vβ14 TCR (line no. 1, 64% Vβ14+ T cells; line no. 2, 51% Vβ14+ T cells). Immunoscope analysis showed that both these TCR peptide B5-reactive lines developed by in vitro stimulation of naive spleen cells, displayed the dominant Vβ14-Jβ1.2 expansion, and that both of these Vβ14-Jβ1.2 gene segment expansions had CDR3 length of 7 aa (Fig. 3b). One of the T cell lines also showed a T cell expansion of the Vβ14-Jβ1.5 gene segment in addition to the dominant Vβ14-Jβ1.2 TCR expansion.


Figure 3
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  		FIGURE 3. Predominance of the TCR Vβ14 gene usage in B5-reactive CD4+ Treg cell lines. T cell lines were generated from spleens of two unimmunized or naive B10.PL mice by in vitro stimulation with TCR-peptide B5 (40 µg/ml) and IL-2, as described in Materials and Methods. After culturing for 4 wks, T cell lines were immunophenotyped for their TCR Vβ usage with a panel of Abs reactive to different TCR Vβ gene segments. Staining with anti-TCR Vβ14 mAb is shown (a). Immunoscope analysis also revealed a dominant Vβ14-Jβ1.2 gene segment usage (b) with a 7 aa CDR3 length in each of two lines. The RIS values were 4.5 and 5.1, respectively for the first and second T cell lines.

 
Vβ14 TCRs of both B5-reactive CD4+ T cell lines were amplified by RT-PCR and cloned into the pCR2.1 vector and sequenced using an automated sequencer (ABI Prism 310 Genetic Analyzer). Thirty clones were randomly picked from each of the T cell lines and subjected to sequence analysis. Clones containing the inset were picked at random and 8 or 9 of 30 had the Vβ14 TCR sequence. It was observed that most of the T cells used the Vβ14-Jβ1.2 gene segment in both T cell lines generated from naive mice (7/8 in the first and 7/9 in the second T cell line). The composition within the CDR3 region of the various Vβ14-Jβ1.2 clones from these naive mouse lines was very similar to the cloned B5-reactive T cell clones and hybridomas generated from mice immunized with MBP or the TCR-peptide B5. In fact, one of these T cell clones had an identical CDR3 sequence to that of the functional T cell clone MB 5.2, generated from a TCR-peptide B5-immunized mouse (9). Furthermore, there were at least four Vβ14-Jβ1.2 sequences that were repeated in each of these independently derived T cell lines (Table VI).


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  		Table VI. B5-reactive CD4+ Treg cells from naïve mice predominantly use the Vβ14-Jβ1.2 gene segmentsa

 
These observations confirm that CD4+Vβ14-Jβ1.2 T cells, reactive to the TCR peptide B5 are present as a part of the naive repertoire of the B10.PL mice. These regulatory CD4+ T cells become activated and expand during the course of MBPAc1–9-induced EAE and in collaboration with CD8+ Treg mediate the spontaneous recovery from and resistance for re-induction of disease. These regulatory CD4+ T cells can also be expanded following immunization with TCR peptide B5 resulting in protection from disease.

Frequency of B5-reactive CD4+ Treg in naive mice correlates with the severity and duration of EAE

Because B5-reactive CD4+ Treg clones have been shown to mediate spontaneous recovery from MBP-induced EAE in H-2u mice and are also present in the naive repertoire, we have examined whether a difference exists in the frequency of these Treg precursors in the two H-2u strains and F1 strain, and whether their number in naive animals has a relation to the severity or duration of EAE. We have used two different approaches, namely classical limiting dilution and Elispot analysis to examine the frequency of B5-reactive CD4+ Treg cells in spleens derived from naive B10.PL or PL/J and (B10.PL x PL/J)F1 mice. As shown in Table VII, the frequency of B5-reactive CD4+ Treg in PL/J mice was 8–10-fold higher in comparison to that in B10.PL mice. Interestingly, the frequency of CD4+ Treg in F1 mice was similar to that in B10.PL animals suggesting dominance of the B10 background. Next, we compared the disease course following immunization of age- and sex-matched B10.PL, PL/J, or F1 mice with MBPAc1–9/CFA/PTx. As shown in Fig. 4, PL/J mice contracted disease of shorter duration (7 days) and lesser severity than in B10.PL or F1 mice. These data suggest that a higher level of B5-reactive CD4+ Treg cells in a naive repertoire correlates positively with a rapid spontaneous recovery from a less severe EAE. In parallel, a comparison of the frequency of the pathogenic MBPAc1–9-reactive T cells in these H-2u mouse strains did not reveal any significant difference (data not shown).


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  		Table VII. Frequency of TCR peptide B5-reactive regulatory CD4 T cells in naive H-2u mice

 

Figure 4
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  		FIGURE 4. A comparison of the clinical course of EAE between the B10.PL, PL/J and (B10.PL x PL/J)F1 mice. 6 wk old female mice (6–8 mice in each group) were immunized with 150 µg of MBPAc1–9/CFA/PT for the induction of EAE as described in Materials and Methods, and monitored daily for clinical symptoms.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The appearance of clinical autoimmunity not only requires activation of the self-reactive immune repertoire but also a dysregulation of peripheral tolerance mechanisms, which include a negative feedback mechanism involving TCR-peptide-reactive CD4+ and CD8+ Treg populations. Characterization of the TCR repertoire of Treg is important in following their development as well as for understanding their physiological function. In this report, we show that the TCR peptide-reactive CD4+ Treg population involved in the control of myelin basic protein-reactive pathogenic T cells is oligoclonal, using only a few dominant TCR V-genes. This was addressed using two approaches: 1) analyzing TCRs at the level of individual sequences from cloned CD4+ Treg both from naive mice and from mice recovering from MBP-induced EAE as well as from mice immunized with the relevant TCR-peptide; 2) CDR3 spectratyping or immunoscope analysis conducted directly ex vivo on peripheral lymphoid tissues from naive and immunized animals. Furthermore, determination of the frequency of Treg populations by limiting dilution in the naive repertoire allowed us to correlate disease severity and recovery with the extent of regulatory activity in two H-2u strains and their F1 progeny.

Analysis of the TCR repertoire of CD4+ Treg showed that the Treg TCR repertoire is oligoclonal and predominantly uses the TCR Vβ14 and Vβ3 gene segments. The oligoclonality in TCR usage by CD4+ Treg is quite reminiscent of the predominant usage of the TCR Vβ 8.2-Jβ2.7 gene segment by pathogenic T cells in both B10.PL and PL/J mice (31, 32, 36). It is also evident that pathogenic as well as Treg populations recognize self-peptides: pathogenic T cells recognize a N-terminal determinant of MBP, Ac1–9, whereas CD4+ Treg recognize a peptide derived from the conserved framework region 3 of the TCRVβ8.2 chain. It is likely that the self-peptide complexes displayed in the periphery restrict the TCR repertoire of CD4+ Treg populations. The antigenic specificity of the CD4+ Treg for the B5 region of TCRVβ8.2 is also consistent with the less diverse repertoire of these CD4+ Treg, in contrast to the much more diverse CD25+CD4+ Treg repertoire (37) that presumably has multiple Ag specificities. In contrast to the CD25+CD4+ Treg, our sequence data indicate no overlap in the TCR repertoires of the pathogenic and Treg populations. The other essential regulatory arm in the B10.PL system, residing within the CD8+ subset (4, 38), is an upstream determinant of the same Vβ8.2 peptide chain (aa 42–50) that binds to the Qa-1a molecule (39, 40). The CD8+ Treg population is CD8{alpha}{alpha}+, Qa-1-restricted and is able to induce apoptosis in activated Vβ8.2+ but not in naive or resting Vβ8.2+ or in Vβ8.2– cells (39). The TCR repertoire of CD8{alpha}{alpha}+ Treg population is also restricted, resulting in limited TCR diversity within each of the three relevant T cell populations in this system (41).

A number of observations suggest that the Treg populations described here are not part of the FoxP3+CD25+CD4+ Treg subset. We have recently conducted gene microarray studies and found that Treg in our system do not constitutively express high levels of CD25 or FoxP3 molecules. The limited expression of these molecules has further been confirmed by PCR and at the protein level using flow cytometry with appropriate mAbs (T. Smith, unpublished data). Furthermore, depletion of CD25+CD4+ T cells using anti-CD25 mAb does not influence the ability of B5-reactive CD4+ Treg to prevent EAE (L. Madakamutil, unpublished data). Interestingly, those CD4+ Treg are CD69+, indicating that they are of the memory phenotype even in naive animals (38). Experiments are in progress to further characterize cell surface molecules expressed by the CD4+ Treg.

TCR-peptide-reactive CD4+ Treg bind to the B5 peptide/I-Au complex with high avidity, as previously discussed (4, 42). A comparison of relative TCR avidity based upon the level of TCR peptide required for the stimulation of individual CD4+ Treg suggests that Vβ14+ Treg are of higher avidity, followed by the Vβ3+ CD4+ Treg population (L. Madakamutil, unpublished data). It is noteworthy that treatment of B10.PL mice with anti-TCR Vβ14 mAb results in incomplete recovery and chronic disease, whereas mice treated with anti-Vβ3 alone do not show a significant difference in the natural course of EAE (13). These data further suggest the critical role Vβ14+CD4+ Treg play in this model. We did not find any significant correlation with the usage of TCR V{alpha} gene segments of CD4+ Treg. It may be fruitful to examine whether the display of self-TCR ligand either in the thymus or in the periphery, influences the frequency of the TCR-peptide-reactive CD4+ Treg populations, and we will examinine these questions in a congenic mouse backcrossed onto the B10.PL background lacking the TCR Vβ 8 gene segment.

Our data also demonstrate that although the dominant or the public Vβ14-Jβ 1.2+ Treg are present in the naive repertoire, their frequency is too low to be detected by proliferative recall assays. The CD4+ Treg expand during the course of disease in concert with pathogenic Vβ8.2+ T cells which expand following immunization with MBP. This could readily occur if professional APCs such as dendritic cells are able to capture dying Vβ8.2+ T cells and cross-present TCR fragments to CD4+ Treg and CD8+ Treg, thereby leading to their expansion. Indeed, recently we have found evidence for cross priming of CD4+ Treg by dendritic cells using both in vitro and in vivo experiments (Trevor Smith and Vipin Kumar, unpublished data). Finally, the experiments presented here suggest that the efficacy of negative feedback regulation in controlling the expansion of a pathogenic T cell population bearing a particular TCR-Vβ-chain depends upon the frequency of TCR-Vβ-reactive Treg in the naive repertoire. It is not clear why the frequency of CD4+ Treg is so different in mice expressing the same H-2u haplotype but different background genes. It may relate to the number of Vβ8.2+ T cells or dendritic cells as well as to their ability to cross-present TCR peptides. In any case, comparison between these two strains reveals the importance of the CD4+ Treg population in the physiology of the immune response to MBP.

Another interesting point is the apparent bias toward the B10.PL rather than the PL/J response in the (B10.PL x PL/J)F1 strain. This is similar to the B10.PL bias in the pattern of responsiveness to the self Ag MBP or the foreign Ag hen egg lysozyme in (SJL x B10.PL)F1 mice (43, 44, 45). In earlier cases, bias in responsiveness had been suggested to correlate with lower expression or affinity of I-As molecules for the Ag. However, in (B10.PL x PL/J)F1 mice it appears that the background genes in B10 mice influence the frequency of the CD4+ Treg population and accordingly, susceptibility to disease and its severity.

The limited and dominant public clone used by the TCR peptide-specific CD4+ Treg allows us to track these cells in vivo under different experimental conditions. For example, we have examined whether these CD4+ Treg infiltrate into the CNS by monitoring the expansion of the dominant Vβ14-Jβ1.2 clonotype using spectratyping analysis. This is an important issue, as it enables the examination of regulation directly in the target tissue. In preliminary analyses we were not able to detect a clonal expansion of the Vβ14-Jβ1.2 gene segment with 7 aa. CDR3 length in CNS tissue at different time points during the course of EAE, suggesting that CD4+ Treg mediated regulation is orchestrated in the peripheral lymphoid organs such as the spleen or lymph nodes and not in the CNS.

How is the CD4+ Treg population able to regulate the anti-MBP pathogenic T cell response? As mentioned earlier, cytokines secreted by the CD4+ Treg are crucial in the induction of CD8+ Treg which are the ultimate effectors of regulation. CD4+ Treg clones secrete IL-2 and IFN-{gamma}, but undetectable levels of IL-4, IL-5, IL-10, and TGF-β. Earlier, we examined whether the cytokine secretion profile of CD4+ Treg influenced the cytokine profile of the MBP-reactive pathogenic T cells and their ability to mediate disease (46). To accomplish this we used nasal priming with the B5 peptide in the presence of IL-4 or IL-12 cytokines, deviating the CD4+ Treg population in a Th1 or Th2 direction, and determined the effect on the subsequent induction of EAE (46). The priming of type 1 CD4+ Treg results in deviation of the MBP-reactive effector T cell population in a type 2 direction and protection from disease. In contrast, induction of type 2 CD4+ Treg results in earlier onset and exacerbation of EAE with poor recovery and an increased frequency of Th1 effectors. This was further confirmed in experiments using vaccination with plasmid DNA or adenovirus vectors expressing Vβ8.2 along with IL-4 or IL-12 vectors (26, 29). We have now further examined the role of IFN-{gamma} secreted by CD4+ Treg in regulation using IFN-{gamma}–/– mice and found that CD4+ Treg generated from these mice failed to provide protection in either IFN-{gamma}+/+ or IFN-{gamma}–/– recipients (B. Pedersen and V. Kumar, unpublished data). These data clearly suggest that the secretion of IFN-{gamma} by CD4+ Treg is required for their regulatory function. We are currently examining whether IFN-{gamma} influences the activation/differentiation of CD8+ Treg directly or indirectly by modulating immunoproteasomes and thereby processing/presentation of TCR by dendritic cells.

The CD8+ Treg are specific for a different determinant near the CDR2 region from the Vβ8.2 chain are restricted by a nonclassical class 1b molecule Qa-1, and kill the target Vβ8.2+ pathogenic CD4 T cells (38, 39, 40, 41). Thus, MBP-reactive Vβ8.2+ Th1 cells are apoptotically depleted following the induction of regulation (30). This leads to a slow expansion of non-Vβ8.2+ and Th2 cells resulting in deviation of the anti-MBP response globally in a Th2 direction (28, 46). Accordingly, the effectiveness of CD4+/CD8+ Treg mediated regulation is dependent upon 2 distinct processes: one is the removal of highly pathogenic T cells; the other is the deviation of response toward Th2 leading to the engagement of regulatory cytokines, for example, IL-4, IL-10, or TGF-β (8, 47). Thus, expansion of pathogenic MBP-reactive CD4 Th1 cells is effectively neutralized by a specific negative feedback regulatory mechanism involving recognition of TCR-peptide/MHC complexes by CD4+ and CD8+ Treg populations. Furthermore, Th2-like cells are then able to suppress T cells expressing different TCRs or those reactive with other MBP determinants or other myelin proteins through a bystander mechanism (47). In summary, immune tolerance to self Ags is maintained at least in part by a negative feedback regulatory system based upon the recognition of conserved motifs within the TCR of the dominant pathogenic CD4+ T cell and involves both effector (disease-inducing) and regulatory (CD4+ and CD8+) lymphocytes bearing oligoclonal and nonoverlapping TCR repertoires.


   Acknowledgments
 
We thank Drs. Randle Ware and Trevor Smith for critical reading of the manuscript and other members of the laboratory for help.


   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 Grants from the National Institutes of Health, the National Multiple Sclerosis Society, and the Multiple Sclerosis National Research Institute (to V.K.). Back

2 Address correspondence and reprint requests to Dr. Vipin Kumar, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. E-mail address: vkumar{at}tpims.org Back

3 Abbreviations used in this paper: MBP, myelin basic protein; EAE, experimental autoimmune encephalomyelitis; PTx, Pertussis toxin; RIS, relative index of stimulation. Back

Received for publication July 13, 2007. Accepted for publication February 1, 2008.


   References
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 Materials and Methods
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 Discussion
 Disclosures
 References
 

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