Persistent Expression of Experimental Autoimmune Encephalomyelitis (EAE)-Specific Vβ8.2 TCR Spectratype in the Central Nervous System of Rats with Chronic Relapsing EAE¹

Multiple sclerosis (MS)³ is a chronic progressive or relapsing remitting form of human autoimmune disease characterized by the presence of demyelinating plaques in the central nervous system (CNS). Several pieces of evidence suggest that T cells reactive with neuroantigens such as myelin basic protein (MBP) and proteolipid apoprotein are responsible for the development of MS lesions. However, controversial results have been reported so far with regard to the nature of MS-associated T cells for the following reasons (1, 2, 3). First, in contrast to inbred experimental animals, MHC haplotypes of humans are different among individuals. Therefore, the TCR phenotype of MS-associated T cells could be different from one person to another even if an Ag recognized by these T cells is the same. Second, it is difficult to obtain brain tissue, including MS plaques, for examination at an appropriate time point. When brain tissue obtained at autopsy is used, bystander T cells, which infiltrate the CNS at a later stage of the disease, may interfere with the analysis of MS-associated T cells. It is also possible that T cells recognizing an neuroantigen at a certain period would be substituted with those recognizing a different Ag as a result of inter- or intramolecular epitope spreading (4, 5).

We have recently applied CDR3 spectratyping for the analysis of oligoclonality of TCR of T cells which are responsible for the development of experimental autoimmune encephalomyelitis (EAE), an animal model for MS (6). In rat acute EAE, several groups including us reported that T cells bearing Vβ8.2 TCR, which is frequently used by in vitro-established encephalitogenic T cell clones (7, 8), appear in the CNS parenchyma, especially at the early stage of the disease, and that a large number of these Vβ8.2 TCR clones possess a common motif in their CDR3 region (9, 10, 11, 12). Based on these findings, we recently screened 22 spectratypes (Vβ1–20) derived from T cells isolated from spinal cord lesions by CDR3 spectratyping and found that Vβ8.2 spectratype with the shortest CDR3 is clonally expanded throughout the course of acute EAE (6). Furthermore, the EAE-specific nucleotide sequence is highly preserved in the CDR3 region. These results indicate that CDR3 spectratyping and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones can be applicable for the detailed analysis of TCR of MS patients, the character of which is much more complicated than that of animal models as mentioned above.

In the present study, we performed CDR3 spectratyping analysis using spinal cord T cells and PBL isolated from rats with chronic relapsing EAE. Special attention was paid to the following points. First, is the EAE-specific spectratype pattern recognized in acute MBP-induced EAE also found in chronic relapsing EAE induced with spinal cord homogenate? Second, do changes in EAE-specific spectratype pattern occur in the course of chronic relapsing EAE? Consequently, it was found that EAE-specific spectratype pattern seen in acute EAE was also detectable in chronic relapsing EAE throughout the disease, suggesting that an encephalitogen in spinal cord homogenate is the same as that used for acute EAE induction. Thus, the determination of CDR3 size by spectratyping has been shown to be a powerful tool with which to analyze the T cell repertoire of T cell-mediated autoimmune diseases. Analysis of MS patients by CDR3 spectratyping may provide useful information about the nature of MS-associated T cells.

Lewis rats were purchased from Seiwa (Fukuoka, Japan) and used at 8–12 wk of age. Chronic relapsing EAE was induced by immunization of rats with 200 μl of guinea pig spinal cord homogenate in CFA (Mycobacterium tuberculosis H37Ra, 5 mg/ml) as described previously (13) with a few modifications. One gram of spinal cord tissue was homogenized in 1 ml of PBS, and the suspension was emulsified with an equal volume of CFA. Starting from the day of immunization, rats were given i.p. injections of cyclosporin A (Sandoz, Tokyo, Japan) at a dose of 4 mg/kg three times a week until day 21 postimmunization (PI). Acute EAE was induced by immunization with guinea pig MBP as described previously (14). Each rat was injected in the hind footpads on both sides with an emulsion containing 100 μg of guinea pig MBP in CFA. The clinical severity of EAE was divided into four stages (grade 1, floppy tail; grade 2, mild paraparesis; grade 3, severe paraparesis; grade 4, tetraparesis or moribund condition) (15). In this study, tissue sampling was performed at the early (day 10–12 PI), peak (day 13–15 PI), and recovery (day 21–23 PI) stages of acute EAE and at the first attack (day 14–17 PI), remission (day 20–23 PI), second attack (day 25–27 PI), and full recovery stage (day 34–37 PI) of chronic relapsing EAE. Under ether anesthesia, blood was aspirated via cardiac puncture, and the whole spinal cord was removed. PBL and spinal cord T cells were then isolated by proteolytic enzyme treatment and the density gradient method as described previously (16).

A single immunoperoxidase staining was performed using mAbs against TCR Vβ8.2 (R78), Vβ8.5 (B73), and Vβ10 (G101) (17) as described previously (14). Briefly, frozen sections of the spinal cord were air-dried and fixed in ether for 10 min. After incubation with normal horse serum, sections were allowed to react with mAb, biotinylated horse anti-mouse IgG (Vector, Burlingame, CA), and horseradish peroxidase (HRP)-labeled Vectstain Elite ABC Kit (Vector). HRP binding sites were detected in 0.005% diaminobenzidine and 0.01% hydrogen peroxide.

RNA was extracted from PBL and spinal cord T cells using RNAzol B (Biotecx Laboratories, 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 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 Vβ1–20 were the same as those used in the previous study (9). Two types of Cβ primers, Cβ outer (5′-TGTTTGTCTGCGATCTCTGC-3′) and Cβ inner (5′-TCTGCTTCTGATGGCTCA-3′), were used in this study. Primers for Vα which were the same as those used in the previous study (18) and two types of Cα, Cα outer (5′-AGGGTGATGTTATCAGACTGG-3′) and Cα inner (5′-GGATCTTTCAGCTGGTACACA-3′), were used for TCR α-chain analysis. Cα and Cβ primers were labeled with Cy-5 or rhodamine or remained unlabeled.

CDR3 spectratyping was performed as described previously (19) with a few modifications. Three individual rats were examined at each time point. cDNA was amplified with Vα- or Vβ-specific and rhodamine-labeled Vα or Cβ 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 30 min at 50°C. Then, the fluorescence-labeled DNA profile on the gel was directly recorded using FMBIO fluorescence image analyzer (Hitachi, Yokohama, Japan). Spectratypes revealed by this analysis usually consisted of five to seven bands. We designated each band as I, II, or III in order of molecular size; e.g., the band representing the smallest Vβ8.2 PCR products was Vβ8.2 band I.

cDNA in PCR products or isolated from bands (in most cases, band I) on the acrylamide gel was reamplified with Vβ and unlabeled Cβ 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 instructions. The plasmid DNA was then sequenced using Cy5-labeled Cβ inner primer and Autoread Sequencing Kit on a 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 Vβ segments and ending before the GXG box in the Jβ region as described previously (20).

In MBP-induced acute EAE, Vβ8.2⁺ T cells are the predominant population in spinal cord T cells throughout the course of the disease (9). We wished to know whether Vβ8.2 predominance is observed in chronic relapsing EAE induced by immunization with spinal cord homogenate. For this purpose, spinal cord sections taken from rats at various stages of EAE were immunohistochemically stained using currently available Vβ-specific mAbs, and the proportion of Vβ8.2⁺, Vβ8.5⁺, and Vβ10⁺ T cells was determined by counting stained cells. As shown in Fig. 1, Vβ8.2⁺ T cells were the largest population among three phenotypes at all the stages examined. It is of interest to note that although the number of Vβ8.5⁺ and Vβ10⁺ T cells roughly paralleled the clinical course, that of Vβ8.2⁺ T cells remained relatively unchanged during the remission, second attack and recovery stage after a steep decline after the first attack (Fig. 1).

For comparison, CDR3 spectratyping analysis was performed using T cells isolated from the spinal cord of a rat with MBP-induced acute EAE. A typical spectratype pattern is shown in Fig. 2. As reported previously (6) and confirmed here, oligoclonal expansion of Vβ8.2 and Vβ17 TCR with the shortest CDR3 (band I) (arrow and arrowhead in Fig. 2, respectively) was obvious at this stage. However, only Vβ8.2 showed persistent expansion throughout the course of acute EAE (data not shown). Using spinal cord T cells isolated from rats during chronic relapsing EAE, CDR3 spectratyping was performed and the spectratype pattern was compared with that obtained in acute EAE (Fig. 3, A–D). It is clearly demonstrated that Vβ8.2 band I was markedly expanded during the first attack (arrow in Fig. 3,A). In addition, there was oligoclonal expansion of Vβ12 band I (open arrow in Fig. 3,A). Expansion of Vβ8.2 band I persisted during the remission phase, while that of Vβ12 band I was not recognized at this stage (Fig. 3,B). In Fig. 3,B, there was oligoclonal expansion of Vβ19 band I (arrowhead), but this finding was observed only in one of three rats examined. The spectratype pattern at the second attack was similar to that at the first attack in which both Vβ8.2 and Vβ12 band I was clonaly expanded (arrow and open arrow in Fig. 3,C). Vβ8.2, but not Vβ12, band I expansion was still noticed at the full recovery stage (Fig. 3 D).

We also examined the TCR α-chain repertoire at the first and second attacks of the disease. As shown in Fig. 4, oligoclonal expansion of Vα6 and Vα23 was observed at the first attack (arrows). A similar finding was obtained at the second attack (data not shown).

We then extracted cDNA from Vβ8.2 and Vβ12 band I which showed oligoclonal expansion, amplified it by nested PCR, then cloned and determined the nucleotide sequences of the CDR3 region of each clone (Tables I and II). During the first attack, the most frequently found sequence in Vβ8.2 band I was DSSYEQYF (58.3%) (Table I) which is reported to be the representative sequence of in vitro-established MBP-specific encephalitogenic T cell clone (7). The percentage of this sequence in Vβ8.2 band I at the remission increased to 75% (Table I). Interestingly, the predominance of the DSSYEQYF sequence remained unchanged during the second attack and became more marked at the full recovery stage (Table I). We also sequenced the CDR3 region of clones derived from Vβ12 band I (Table II) during the first and second attacks. Quite unexpectedly, the predicted amino acid sequence of the CDR3 region of the most frequently found Vβ12 clones was identical to that of Vβ8.2 clones, i.e., DSSYEQYF. However, the predominant Vβ12 clone isolated at the first attack is not the same as that found at the second attack because the nucleotide sequence of these clones was different (Table II).

We also examined the spectratype pattern of TCR of PBL (Fig. 5). Since the CNS lacks the lymphatic system, precursor cells may enter the CNS via the blood stream. CDR3 size spectratyping was performed at the first attack, remission, second attack, and full recovery stages using PBL. Unlike spinal cord T cells, oligoclonal expansion was marked only in Vβ8.2 TCR (data not shown). As shown in Fig. 5, Vβ8.2 band I expansion was observed during the first and second attacks in PBL as seen in the spinal cord. However, this EAE-specific spectratype pattern was not visible in PBL at the remission and full recovery stage.

EAE has long been investigated as a model for MS. However, in contrast to MS which is a remitting and relapsing disease of chronic nature, the clinical course of some types of mouse and most rat EAE is acute and monophasic. To analyze the pathomechanisms of relapse of autoimmune diseases, we induced chronic relapsing EAE in Lewis rats by immunization with spinal cord homogenate and administration of a low dose of cyclosporin A. By this approach, we were able to examine the T cell repertoire of spinal cord T cells during the remitting and relapsing course and obtained several interesting findings.

We first determined the number of T cells bearing different Vβ phenotypes using currently available Vβ-specific mAbs (Fig. 1). As previously demonstrated by us (9) and others (10), Vβ8.2⁺ T cells infiltrate the CNS at the early stage of MBP-induced acute EAE and become a predominant population. If Vβ8.2 predominance is not observed in the spinal cord of rats with chronic relapsing EAE induced by immunization with spinal cord homogenate, then encephalitogenic Ags other than MBP would be involved. However, this was not the case. As clearly shown in this study, Vβ8.2⁺ T cells are the predominant population throughout the course of chronic relapsing EAE, suggesting that Ag involved in chronic EAE is similar to that in acute EAE.

CDR3 spectratyping analysis revealed that EAE-specific spectratype pattern, i.e., oligoclonal expansion of Vβ8.2 with the shortest CDR3 (band I), was found in the spinal cord throughout the course of chronic relapsing EAE and that it was essentially the same as that of acute monophasic EAE (6). Furthermore, determination of nucleotide and amino acid sequences showed that the CDR3 region of the majority of clones constituting Vβ8.2 band I was the same as that of MBP-specific encephalitogenic T cell clones (7, 8) and remained unchanged as the disease progressed. The most straightforward explanation for these findings is that in chronic relapsing EAE of this type, encephalitogenic Ag is not switched from one to another and is similar to that of acute EAE, possibly MBP. Another characteristic finding was oligoclonal expansion of Vβ12 band I of spinal cord T cells. Vβ12 band I expansion was observed in the spinal cord, and not in PBL, only at the active stage of the disease (during the first and second attacks). Furthermore, the most frequently found CDR3 amino acid sequence was identical to that of Vβ8.2. Although the exact function of T cells bearing this spectratype remained to be elucidated, we consider at the moment that they act as additional effector cells which are activated during active stages of chronic relapsing EAE. The possibility that they are regulatory T cells seems to be less likely because Vβ12 band I expansion was not observed at the recovery stage. Collectively, these findings suggest that upon immunization with CNS homogenate including heterogeneous Ags, T cells recognize the major component of encephalitogens, expand and develop EAE lesions.

We have also examined TCR α-chain by CDR3 spectratyping. Our previous studies regarding the Vα repertoire in acute EAE revealed that Vα1 and Vα2 expansion was observed at preclinical and early stages, whereas that of Vα23 was detected mainly at the clinical stages (G. Kim, K. Kohyama, N. Tamuma, and Y. Matsumoto, unpublished data). Thus, the use of Vα-chain by spinal cord T cells is not strict compared with that of Vβ-chain. In the present study, we found that Vα6 and Vα23 showed oligoclonal expansion at the first and second attacks of chronic relapsing EAE. Therefore, preferential usage of Vα23 by spinal cord T cells is the same finding in both acute and chronic relapsing EAE although Vα6 expansion was not observed in acute EAE. These Vαs are thought to pair with Vβ8.2 or Vβ12, which expanded oligoclonally in chronic relapsing EAE. However, it is also possible that multiple chains are expressed on a single cell as reported previously (21, 22). Very recently, Wang et al. (23) reported very interesting findings with regard to the role of TCR α- and β-chains in Ag specificity. Using mice carrying single Vα-chain (a TCR α-chain transgenic mouse in a TCR α-deficient background), they found that a specific amino acid residue of β-chain plays a critical role in determining the specificity of TCR-Ag interactions. The fact that different TCR Vβs possess highly preserved sequences in the CDR3 region suggest that polyclonally activated T cells in the CNS recognize the same or structurally similar Ags during the course of the disease.

Analysis of PBL by CDR3 spectratyping also revealed an interesting finding with regard to relapse of EAE. As clearly shown in Fig. 5, Vβ8.2 band I expansion in PBL was observed at the first and second attacks, but not at the remission and full recovery stage. In addition, the majority of TCR clones comprising this band possessed EAE-specific sequence in their CDR3 region (our unpublished data). These findings imply that encephalitogenic T cells are activated in the lymphoid organ and supplied via the blood stream to the CNS only during the clinical phase of the disease. Recently, it was demonstrated that myelin protein expression is increased in lymph node at a later stage of chronic relapsing EAE resulting in reactivation of encephalitogenic T cells (24). This event may correspond to reexpansion of Vβ8.2 band I in PBL.

In the present study, we analyzed a complete set of TCR β-chain family of spinal cord T cells and PBL of Lewis rats with chronic relapsing EAE by CDR3 spectratyping and then determined the nucleotide sequences of the spectratype-derived CDR3 of interest. We show here that EAE-specific spectratype pattern in the spinal cord remains unchanged throughout the course of the disease, thus strongly suggesting that encephalitogenic epitope recognized by T cells is not changed during the course. When considering TCR-based immunospecific therapy for MS patients, CDR3 spectratyping analysis is indispensable to obtain important information.

We thank Y. Suzuki and Y. Kawazoe for technical assistance.