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The Journal of Immunology, 1998, 160: 509-513.
Copyright © 1998 by The American Association of Immunologists

CDR3 Size Spectratyping and Sequencing of Spectratype-Derived TCR of Spinal Cord T Cells in Autoimmune Encephalomyelitis1

Giok Kim, Naoyuki Tanuma, Takashi Kojima, Kuniko Kohyama, Yoko Suzuki, Yoko Kawazoe and Yoh Matsumoto2

Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To characterize the nature of autoimmune disease-inducing T cells in the target organ, oligoclonal expansion of spinal cord T cells of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined by complementarity-determining region 3 (CDR3) size spectratyping. It is known that TCR of in vitro-established myelin basic protein-specific T cell clones and lines have a short CDR3 and that the amino acid sequence in this region is highly preserved. On the basis of these findings, we analyzed 22 spectratypes of the TCR ß-chain (Vß1–20). Among them, only Vß8.2 and Vß17 showed oligoclonal expansion of TCR with a short CDR3 at the early stage of EAE. More interestingly, the spectratype profile of Vß8.2 seen at the early stage was preserved throughout the course of EAE, whereas that of Vß17 became more diverse at the peak stage of the disease. Analysis of nucleotide and predicted amino acid sequences of Vß8.2 CDR3 derived from the spectratypes revealed that the clones with CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population not only at the early stage but also at the peak and recovery stages (71, 71, and 60%, respectively). These findings imply that although the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, disease-associated TCR spectratype(s) are preserved throughout the course of the disease. Thus, CDR3 size spectratyping is a powerful tool for the screening of disease-inducing T cells in an autoimmune disease of unknown pathomechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE)3 is inducible in susceptible strains of rats and mice by immunization with neuroantigens such as myelin basic protein (MBP) or by adoptive transfer of MBP-specific T cells into naive animals. Recent analysis indicates that EAE-inducing T cells bear CD4 molecules and use a limited number of {alpha}- and ß-chains of the TCR (1). Furthermore, the complementarity-determining region 3 (CDR3) of TCR of long term-cultured encephalitogenic T cell clones is rather short, and some amino acid residues are conservatively preserved (2, 3). In the rat, encephalitogenic T cell clones established from guinea pig MBP-immunized animals mainly use Vß8.2 (4). Moreover, we (5) and others (6) have demonstrated by immunohistochemical and flow cytometric studies that Vß8.2+ T cells infiltrate the central nervous system (CNS) at the early stage of EAE and become a predominant population throughout the course of EAE. However, more detailed analysis by these methods is difficult because a complete set of mAb specific for rat TCR ß-chain family is not available at present. Conventional Vß analysis using PCR products of TCR also has limitations. As clearly demonstrated by Karin et al. (7), diverse Vß gene transcripts were detected in the CNS with full blown EAE even though Vß8.2+ T cells are still a predominant population.

The determination of CDR3 size by spectratyping is a powerful tool with which to analyze the T cell repertoire under normal and pathologic conditions (8). First, this method provides a profile of the T cell repertoire without any need for in vitro T cell expansion. Second, oligoclonal expansion of T cells that bear a particular TCR phenotype in mixtures of lymphocytes is readily detectable as a dense band on a gel. Thus, we applied this method to analysis of the repertoire of spinal cord T cells to determine whether T cells in the target organ show characteristics similar to long term-cultured encephalitogenic T cells. For this purpose, cDNA prepared from spinal cord tissue with inflammatory lesions at various stages of actively induced EAE was amplified and analyzed by CDR3 size spectratyping. Furthermore, cDNA of TCR showing oligoclonal expansion was extracted from bands on a gel, cloned, and sequenced. It was revealed that oligoclonal expansion of Vß8.2 with a short CDR3 occurred throughout the course of EAE and that the majority of Vß8.2 spectratype-derived cDNA clones had the same nucleotide sequence as that of in vitro-established encephalitogenic T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rats

Lewis rats were purchased from Seiwa (Fukuoka, Japan) and used at 8 to 12 wk of age.

EAE induction

Active EAE was induced in Lewis rats as described previously (9). Each rat was injected in the hind footpads on both sides with an emulsion containing 100 µg of guinea pig MBP in CFA (M. tuberculosis H37Ra, 5 mg/ml). The clinical stage of EAE was divided into four (grade 1, floppy tail; grade 2, mild paraparesis; grade 3, severe paraparesis; grade 4, tetraparesis or moribund condition) (10). At different times, rats were killed under ether anesthesia, and several segments of the lumbar spinal cord were snap-frozen in OCT compound. Twenty sections 20 µm thick were cut in a cryostat and stored at -80°C until use. In this study, the early, peak, and recovery stages of EAE generally refer to days 10 to 11 (grade 1), days 13 to 14 (grade 3 or 4), and day 18 to 20 (grade 0), respectively.

cDNA synthesis and PCR amplification

RNA was extracted from frozen sections using RNAzol B (Biotecx Lab, Houston, TX). cDNA was then synthesized by reverse transcription with SuperScript Preamplification System (Life Technologies, Gaithersburg, MD) and amplified in a thermal cycler (Perkin Elmer, Norwalk, CT) using Amplitaq Gold (Perkin Elmer) and primer pairs for TCR. Cycling conditions for PCR and nested PCR were as follows: 95°C for 10 min for denaturation and hot start; 55°C for 1 min for annealing; and 72°C for 1 min for extension followed by 40 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Primers for Vß1–20 were the same as those used in the previous study (5). Two types of Cß primers, Cß outer (5'-TGTTTGTCTGCGATCTCTGC-3') and Cß inner (5'-TCTGCTTCTGATGGCTCA-3'), were used in this study. They were labeled with Cy-5 or rhodamine or remained unlabeled.

CDR3 size spectratyping

CDR3 size spectratyping was performed as described previously (11) with a few modifications. cDNA was amplified with Vß-specific and rhodamine-labeled Cß outer primers, and undiluted or diluted PCR products were added to an equal volume of formamide/dye loading buffer and heated at 94°C for 2 min. Two microliters of the samples were applied to a 6% acrylamide sequencing gel. Gels were run at 30 W for 3 h and 30 min at 50°C. Then, the fluorescence-labeled DNA profile on the gel was directly recorded using an FMBIO fluorescence image analyzer (Hitachi, Yokohama, Japan). Spectratypes revealed by this analysis usually consisted of 5 to 7 bands. We designated each band as band I, II, or III in order of molecular size; e.g., the band representing the smallest Vß8.2 PCR products was Vß8.2 band I.

Sequencing of PCR products

cDNA in PCR products or isolated from bands (in most cases, band I) on the acrylamide gel was reamplified with Vß and unlabeled Cß inner primers. Then, PCR products were ligated into pT-Adv vector and cloned using the AdvanTAge PCR Cloning Kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instruction. The plasmid DNA was then sequenced using Cy5-labeled Cß inner primer and an Autoread Sequencing Kit on an ALFexpress DNA sequencer (Pharmacia Biotech, Tokyo, Japan). CDR3 length is defined as a region starting from an amino acid residue after the CASS sequence of most Vß segments and ending before the GXG box in the Jß region as described previously (12). Thus, Vß-CASSDSSYEQYFGPG-Jß would count as eight amino acids.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We performed CDR3 size spectratyping of TCR at the early, peak, and recovery stages of EAE. At least three rats were individually examined at each time point, and essentially the same results were obtained. Representative results are shown in Figures 1Go, 2 and 3. It is known that encephalitogenic, but not nonencephalitogenic, T cells have a short CDR3 in their TCR (2, 3). Therefore, we first checked the profile of Vß1–20 spectratypes at the early stage of EAE (Fig. 1Go). Although dense bands indicating oligoclonal expansion of TCR were recognized in Vß2, Vß4, Vß8.2, Vß9, Vß14, Vß15, Vß17, and Vß19, only Vß8.2 and Vß17 showed expansion of TCR with the shortest CDR3 (indicated by an arrow and an arrowhead, respectively). At the peak stage of EAE, the oligoclonal expansion seen at the early stage became less pronounced, and each Vß generally had five to seven spectratypes with one or two dense bands in their middle portion (Fig. 2Go). Vß8.2 was the only exception. Spectratypes consisting of Vß8.2 were rather rare compared with other Vßs, and the band representing the shortest CDR3 was very dense (arrow in Fig. 2Go). With regard to Vß17, the spectratype profile seen at the early stage did not persist and at the peak stage exhibited a pattern typical of other Vßs except Vß8.2 (in Fig. 2Go, arrowhead). We wished to establish whether there are clones that expand at the recovery stage; such clones would suggest the presence of regulatory T cells in the target organ. However, no newly expanded clones (except Vß8.2) were detected at this stage, and the spectratype pattern was essentially the same as that at the peak stage (Fig. 3Go). In Figure 4Go, spectratypes of Vß8.2 and Vß17 at the early, peak, and recovery stages of EAE are depicted together. The spectratype pattern of Vß8.2 remained unchanged throughout the course of EAE, and band I always showed oligoclonal expansion. In sharp contrast, Vß17 spectratypes showed oligoclonal expansion only at the early stage and diversified later on (Fig. 4Go).



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  		FIGURE 1. CDR3 size spectratyping of spinal cord T cells at the early stage of EAE. Total RNA was isolated from spinal cord tissue, reverse-transcribed into cDNA, and amplified with Vß1–20-specific and Cß-specific primers. PCR products were run on a denaturing polyacrylamide gel and analyzed with a fluorescence image analyzer operated on a Macintosh computer. Spectratypes representing oligoclonal expansion of Vß8.2 and Vß17 cDNA with the shortest CDR3 are indicated by an arrow and an arrowhead, respectively.

 


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  		FIGURE 2. CDR3 size spectratyping of spinal cord T cells at the peak stage of EAE.

 


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  		FIGURE 3. CDR3 size spectratyping of spinal cord T cells at the recovery stage of EAE.

 


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  		FIGURE 4. Profile of Vß8.2 and Vß17 spectratypes at the early, peak, and recovery stages of EAE. Spectratypes shown in Figures 1Go, 2Go and 3 were recorded as a PICT image using Canvas software on a Macintosh computer and Vß8.2 and Vß17 spectratypes were cut and are depicted together.

 
On the basis of the findings obtained by CDR3 size spectratyping, we decided to determine the nucleotide sequence of the CDR3 of Vß8.2 and Vß17 spectratypes. For this purpose, cDNA was extracted from Bands I of Vß8.2 and Vß17 on a polyacrylamide gel and reamplified by nested PCR. The cDNA was then cloned, and the nucleotide and amino acid sequences of the CDR3 of each clone were determined. As shown in Table IGoA, a large number of clones possessed the DSSYEQYF sequence which was frequently seen in in vitro-established encephalitogenic T cell clones (2, 3) at the early stage of EAE (83% of sample 1518 and 63% of sample 1780, average 71%). The average percentage of clones that possessed this sequence was much higher than that of randomly sequenced spinal cord-derived clones. In the latter case, clones that had the DSSYEQYF sequence accounted for 34% (13). More interestingly, all the clones without DSSYEQYE possessed Asp-Ser or (X)-Ser at positions 1 and 2 which are also reported as common motifs of encephalitogenic T cells (2). The T cell repertoire of Vß8.2 band I-derived clones at the peak stage of EAE was essentially the same as that at the early stage (Table IGoB). The predominance of the DSSYEQYF sequence in spectratype-derived cDNA clones persisted at the recovery stage, although it became less obvious (Table IGoC). TCR that did not have the common motif (DVWETTQYF) was the second largest population. With regard to Vß17, the predominance of a certain sequence was not observed (Table IIGo). However, all the sequenced clones possessed His and Gly at positions 1 and 2.


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  		Table I. Amino acid and nucleotide sequences of the CDR3 of Vß8.2 TCR extracted from spectratype band I of spinal cord T cells at various stages of EAEa

 

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  		Table II. Amino acid and nucleotide sequences of the CDR3 of Vß17 TCR extracted from spectratype band I of spinal cord T cells at various stages of EAE

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent studies have demonstrated that the TCR of encephalitogenic T cells in the rat has several unique characteristics. First, using in vitro-established T cell clones, it was demonstrated that encephalitogenic T cells mainly use Vß8.2 (4). Later, the predominance of Vß8.2+ T cells was confirmed in situ in the CNS of Lewis rats with EAE by immunohistochemistry (5, 14). However, Sun et al. claimed that TCR ß-chain usage of encephalitogenic T cells is not restricted to Vß8.2 (15, 16). Indeed, transfer of MBP-reactive Vß10+ T cells induced EAE indistinguishable from that induced by Vß8.2+ T cells (17). Recently, it was clearly demonstrated that preferential usage of Vß8.2 by encephalitogenic T cells is shaped in the intact Lewis thymus (18). Therefore, the non-Vß8.2 T cells involved in the induction of EAE seem to make up only minor populations. Second, the TCR of encephalitogenic T cells has an unique CDR3. Gold et al. (2) and Zhang et al. (3) first described that the TCR of encephalitogenic Vß8.2+ T cells has a short CDR3, suggesting that these T cells are ontogenically old and generated before terminal deoxynucleotidyl transferase appears, i.e., before day 4 after birth (19, 20). Furthermore, positions 1 and 2 were highly preserved and were Asp-Ser or (X)-Ser in most cases (3, 17). We (13) and others (21) reported that these common motifs were also frequently found in the CDR3 of TCR isolated from the spinal cord, but not from the lymph node.

Although these studies, i.e., random sequencing of CDR3 using T cell clones and lines or T cells isolated from the target organ, provided useful information, they have limitations. In the present study, we used CDR3 size spectratyping to detect oligoclonal expansion of certain Vß transcripts. As shown in Figures 1Go, 2Go and 3, screening of a complete set of Vß family transcripts could be easily achieved using uncultured samples, thereby avoiding the bias that can be produced from culture. CDR3 size spectratyping performed at the early, peak, and recovery stages of EAE revealed several intriguing findings. First, oligoclonal expansion of Vß8.2 with a short CDR3 persisted throughout the course of EAE. This finding is, at least in part, attributable to the fact that encephalitogenic T cells always recognize the immunodominant epitope of MBP (68–88 sequence) in MBP-induced EAE in the rat and that there is no inter- or intramolecular determinant spread (22). Even under such conditions, it was difficult by routine RT-PCR analysis to find Vß8.2 dominance especially at the peak or later stage of the disease (7). Second, oligoclonal expansion similar to Vß8.2 was seen in Vß17 transcripts at the early stage. Since T cells reacting with MBP89–101, an encephalitogenic peptide sequence in both mice and rats (23, 22), bear Vß17 in SJL/J mice (24), it is possible that Vß17+ T cells in Lewis rats are also encephalitogenic. Last, no Vß other than Vß8.2 showed oligoclonal expansion at the recovery stage. This finding suggests that regulatory T cells that expand at the later stage are not involved in the recovery from EAE. Alternatively, regulatory T cells may exist in the expanded Vß8.2 population. In addition, it is possible that regulatory T cells may rely more on restricted V{alpha} expression.

Determination of nucleotide sequences of Vß8.2 spectratype-derived TCR confirmed the findings reported by Buenafe et al. (21) that the Asp-Ser CDR motif is frequently present in spinal cord-derived cDNA clones after random sequencing. Moreover, we demonstrated that the majority of Vß8.2 clones extracted from an expanded spectratype (60–70% depending on the stage of EAE) have the DSSYEQYF sequence in the CDR3, and the rest except one found at the recovery stage (DVWETQYF) have either Asp-Ser or (X)-Ser motif (Table IGo). Percentages of these sequences are much higher that those obtained by random sequencing because most TCR clones with a longer CDR3 (bands II and III according to the nomenclature in this study) do not have the CDR3 common motif (our unpublished observation). The CDR3 profile of Vß8.2 at the recovery stage was slightly different from those seen at the early and peak stages. The frequency of the DSSYEQYF sequence decreased slightly (60%), and TCR clone without Asp-Ser or (X)-Ser motif constituted the second largest population. As mentioned in the previous paragraph, T cells that bear this sequence might have a regulatory function.

In the present study, we analyzed a complete set of TCR ß-chain family of spinal cord T cells of Lewis rats with active EAE by CDR3 size spectratyping and then determined the nucleotide sequence of the spectratype-derived CDR3 of interest. Since the TCR of encephalitogenic T cells has been extensively investigated and well characterized mainly using long term-cultured T cell clones, we were able to detect one definite and several possible TCR spectratypes in the target organ associated with EAE development according to the criteria of TCR of encephalitogenic T cells. Conversely, it is highly possible to identify autoimmune disease-associated TCR spectratype(s) based on characteristics of spectratypes. We are currently investigating rat experimental autoimmune carditis (EAC) by the strategy used in this study. EAC is inducible by immunization with cardiac myosin (25) and transferable to naive animals by sensitized T cells (26), but the exact nature of carditis-inducing T cells is unknown. We screened Vß1–20 spectratypes of heart-infiltrating T cells and found several candidate spectratypes that expanded clonally and thus may be associated with the development of EAC (manuscript in preparation). Likewise, CDR3 size spectratyping is a powerful tool for the screening of autoimmune-associated TCR, and the strategy, including the methods shown in this study, can be applied for all T cell-mediated autoimmune diseases.


   Footnotes
 
1 This study was supported in part by grants-in-aid from the Ministry of Education, Japan, Toyama Chemical Co., and Naito Foundation. Back

2 Address correspondence and reprint requests to Dr. Yoh Matsumoto, Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6, Fuchu, Tokyo 183, Japan. Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; CDR3, complementarity-determining region 3; CNS, central nervous system; EAC, experimental autoimmune carditis. Back

Received for publication June 26, 1997. Accepted for publication September 15, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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