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Tracking of V8.2-Positive Encephalitogenic T Cells by Complementarity-Determining Region 3 Spectratyping and Subsequent Southern Blot Hybridization in Lewis Rats after Neuroantigen Sensitization¹

Hiroshi Sakuma^*,, Kuniko Kohyama^*, Youngheun Jee^2,* and Yoh Matsumoto^3,*

^* Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan; and Department of Pediatrics and Developmental Biology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR - and -chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use V8.2 TCR and clonal expansion of the V8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of V8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that V8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of V8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organ-specific autoimmune diseases are mediated by T cells reactive with Ags in the target organ. In experimental autoimmune encephalomyelitis (EAE)⁴ induced in Lewis rats by immunization with myelin basic protein (MBP), encephalitogenic T cells mainly use the V8.2 of the TCR and respond to the immunodominant epitope residing in the 68–88 sequence of MBP molecules (1). Identification of pathogenic T cells and analysis of their kinetics during the course of EAE would provide useful information to elucidate the pathogenesis of EAE. However, it has been difficult to identify encephalitogenic T cells in situ before and after the onset of clinical EAE because the frequency of the T cells is very low throughout the course of the disease (2). FACS analysis revealed that the percentage of V8.2-positive T cells in the lymphoid organ during EAE is not significantly different from that in normal animals (3). Fluegel et al. (4) identified the in situ localization and cytokine profile of encephalitogenic T cells by the use of GFP gene-transduced encephalitogenic T cells after adoptive transfer into naive animals. Although this is an excellent method for detecting pathogenic T cells during the effector phase, T cells activated in the induction phase cannot be detected. The MHC class II tetramer presenting encephalitogenic peptides is potentially a powerful tool for this purpose, but preparation of the tetramer for individual diseases is very difficult.

We demonstrated in previous studies that expansion of the shortest V8.2 spectratype persists in the spinal cord lesion throughout the course of EAE and that the complementarity-determining region 3 (CDR3) sequence of the majority of TCR clones is the same as that of MBP-reactive encephalitogenic T cell clones (5, 6). These findings raise the possibility that clonal expansion of encephalitogenic T cells in the lymphoid organ of unmanipulated animals (not TCR transgenic mice) is detectable by CDR3 spectratyping even at the very early stage of the disease. In the present study we examined the presence or the absence of V8.2 spectratype expansion in regional and distant lymph nodes (LNs), spleen, thymus, and spinal cord of normal, preclinical, clinical, and recovered rats. Furthermore, Southern blot analysis with a probe that binds specifically to the encephalitogenic T cell-specific CDR3 sequence (7) demonstrated V8.2 TCR expansion more sensitively than CDR3 spectratyping. Interestingly, the analysis revealed that clonal expansion of encephalitogenic T cells bearing V8.2 in the entire body starts as early as day 3 of sensitization, suggesting that pathogenic T cells are activated and disseminated much earlier than previously assumed. Thus, sensitive detection of pathogenic TCR in organ-specific autoimmune diseases provides useful information to elucidate the pathomechanisms of the disease and to develop specific immunotherapy.

	Materials and Methods

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Rats and EAE induction

Lewis rats were purchased from Japan SLC (Shizuoka, Japan). Active EAE was induced as described previously (8). Each rat was injected in the hind footpads on both sides with an emulsion containing 100 µg of guinea pig myelin basic protein (MBP) in CFA (M. tuberculosis H37Ra, 5 mg/ml). The clinical stage of EAE was divided into four groups (grade 1, floppy tail; grade 2, mild paraparesis; grade 3, severe paraparesis; grade 4, tetraparesis or moribund condition) (9). At different time points, rats were killed under ether anesthesia, and the popliteal, inguinal, and mesenteric LNs; spleen; PBL; thymus; and spinal cord were removed and stored at –80°C until use.

cDNA synthesis and PCR amplification

RNA was extracted from frozen tissues using RNAzol B (Biotecx Laboratories, Houston, TX). cDNA was then synthesized by RT using the SuperScript Preamplification System (Invitrogen Life Technologies, Gaithersburg, MD) and was amplified in a thermal cycler (PerkinElmer, Norwalk, CT) using primer pairs for TCR. Cycling conditions for 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 V1–20 were the same as those used in the previous study (3). Two types of C primers, C outer (5'-TGTTTGTCTGCGATCTCTGC-3') and C inner (5'-TCTGCTTCTGATGGCTCA-3'), were used in this study. They were labeled with Cy-5 or rhodamine or remained unlabeled.

CDR3 spectratyping and Southern blot analysis with CDR3 probes

CDR3 spectratyping was performed as described previously (5). Briefly, PCR products were added to an equal volume of formamide/dye loading buffer and heated at 94°C for 2 min. The amplified PCR products were electrophoresed on polyacrylamide sequencing gels, and the fluorescence-labeled DNA profile on the gels was directly recorded using an FMBIO fluorescence image analyzer (Hitachi, Yokohama, Japan). The presence or the absence of contaminations of the reagents used in PCR was examined every 10 PCR analyses by performing PCR without the templates. When present, all reagents used and all results obtained during the period were discarded.

Southern blot analysis with encephalitogenic TCR-specific probes (designated as the DSS probe) was performed as described previously (7). Briefly, oligonucleotide probes corresponding to the CDR3 sequences were synthesized (Life Technology, Tokyo, Japan) and subsequently labeled with digoxigenin-dUTP using a DIG Oligonucleotide Tailing Kit (Roche, Indianapolis, IN). Electrophoresed spectratypes were blotted from the gel to the positively charged nylon membrane (Biodyne; Pall, New York, NY) by capillary transfer. DNA was then fixed by exposure to UV and prehybridized at 37°C in 5x SSC, 1% blocking solution, 0.1% N-lauroylsarkosine, and 0.02% SDS with 0.1 pmol/ml poly(A) to prevent nonspecific hybridization signals. Hybridization was performed in a buffer containing 5x SSC, 1% blocking solution, 0.1% N-lauroylsarkosine, 0.02% SDS, and 0.5 pmol/ml digoxigenin-labeled CDR3 spectratype-derived sequence probe at 55°C for 24 h. The temperature for hybridization for each probe was determined separately. The detection of DNA hybrid products was performed using the Digoxigenin Luminescent Detection Kit (CSPD; Roche) according to the manufacturer’s instructions. The membrane was then exposed to x-ray film for 20 min at room temperature.

Sequencing of PCR products

cDNA in PCR products or isolated from bands on the acrylamide gel was reamplified for five cycles with V and unlabeled C inner primers. Then PCR products were ligated into the pT-Adv vector and cloned using the AdvanTAge PCR Cloning Kit (BD Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The plasmid DNA was sequenced using Cy5-labeled C inner primer and the Autoread Sequencing Kit on an ALFexpress DNA sequencer (Pharmacia Biotech, Tokyo, Japan). CDR3 length is defined as the region starting from an amino acid residue after the CASS sequence of most V segments and ending before the GXG box in the J region as described previously (10).

Immunoprecipitation and immunoblotting

Spinal cord tissue was homogenized in lysis buffer (40 mM Tris-HCl, 120 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, pH 7.5) with homogenizer (226A2; As One, Osaka, Japan) in a microtube. The homogenate was centrifuged at 12,000 rpm for 30 min, and the supernatant was harvested. For immunoprecipitation assay, the supernatants containing 1,000 µg of protein were saved and precleared by incubation with 15 µl of protein G-Sepharose (Amersham Biosciences, Tokyo, Japan) at 4°C for 1 h. Clarified lysates were mixed with 5 µg of R73 (anti-TCR mAb) and gently rocked at 4°C overnight. The immune complexes were captured by additional incubation with 20 µl of protein G-Sepharose for 2 h. The TCR immune complexes bound to Sepharose beads were collected by centrifugation at 3,000 rpm for 2 min and washed three times with ice-cold lysis buffer. Conjugates were extracted by boiling in 20 µl of Laemmli sample buffer for 5 min, and supernatants were collected by centrifugation. The proteins were run on 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Tokyo, Japan). After blocking with 5% nonfat milk, the blots were incubated with biotinylated R78 (anti-V8.2 mAb; 1/1,000) at 4°C overnight, followed by incubation with ABC-HRP (Vector Laboratories, Burlingame, CA) for 1 h. The blots were developed by ECL reagents (Immunostar Kit; Wako Biochemicals, Tokyo, Japan) according to the manufacturer’s instructions.

Proliferative responses of lymphocytes against MBP and MBP peptides

Proliferative responses of LN and spleen cells were assayed in microtiter wells by uptake of [³H]thymidine. After being washed with PBS, LN cells (2 x 10⁵ cells/well) or spleen cells (3 x 10⁵ cells/well) were cultured with the indicated concentrations of MBP for 3 days with the last 18 h in the presence of 0.5 µCi [³H]thymidine (Amersham Biosciences, Tokyo, Japan). The cells were harvested on glass-fiber filters, and label uptake was determined using standard liquid scintillation techniques.

Lymphadenectomy and thymectomy

Lymphadenectomy of the draining, i.e., popliteal, LN was performed on days 1, 3, and 6 postimmunization (PI; n = 2 at each time point). In this experiment rats were immunized in the right foot pad only with MBP (75 µg/rat) emulsified with CFA, and the LN on the same side was removed at the indicated time point. At operation, not only the LN, but also the surrounding adipose tissue, were removed as much as possible. Thymectomy was performed by the usual method as described previously (11).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
V8.2 spectratype expansion and clonal expansion of encephalitogenic TCR were found in rats with clinical EAE

We have recently established a method to detect activated pathogenic TCR in situ in lymphoid and target organs during the course of organ-specific autoimmune diseases such as EAE (5, 6) and experimental autoimmune carditis (12). The representative results obtained from normal and EAE rats are shown in Fig. 1. Under normal conditions, the spectratype profile of the lymphoid organ shows a Gaussian distribution without any spectratype expansion (Fig. 1A). Sequencing of the PCR product extracted from one spectratype revealed that TCR clones possessed completely heterogeneous CDR3 sequences with the same size, whereas the whole V8.2 PCR product contained TCR clones with different sequences and sizes (Fig. 1A, box). In MBP-induced EAE in Lewis rats, V8.2 spectratype expansion was observed in both lymphoid and target organs, as shown in Fig. 1B and reported previously (5, 6). Sequencing of TCR clones derived from the expanded spectratype revealed clonal expansion of TCR clones with the EAE-specific CDR3 sequence, CASSDSSYEQYFGPG (Fig. 1B, box) (1).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. CDR3 spectratyping and the CDR3 sequence of TCR clones derived from the V8.2 spectratype of PBL of normal (A) and EAE (B) rats. CDR3 spectratyping was performed using PCR products amplified with TCR V1–20-specific primers. cDNA was extracted from bands representing the shortest spectratype of V8.2. Each sample was reamplified by nested PCR, cloned, and sequenced, as shown in the boxes. The representative results of three or more independent examinations are shown.

V8.2 spectratype profile in various lymphoid organs and spinal cord in normal and EAE rats

We then examined the V8.2 spectratype profile of various lymphoid organs and the spinal cord of normal rats, control rats immunized with keyhole limpet hemocyanin (KLH)/CFA and rats immunized with MBP/CFA (Fig. 2 and Table I). In normal rats, the V8.2 spectratype in all organs examined showed a normal pattern without any spectratype expansion (Fig. 2A). Southern blot hybridization with a probe that binds to the EAE-specific CDR3 sequence, CASSDSSYEQYFGPG (designated in this study as the DSS probe), gave no signal (Fig. 2B). In control rats that had been immunized with KLH/CFA and examined on day 14 PI, there was no V8.2 spectratype expansion and no signals in Southern blotting (Fig. 2, M and N), indicating that KLH does not induce the expansion of V8.2, at least during the examination period. We next examined the presence or the absence of EAE-specific V8.2 spectratype expansion and positive signals with the DSS probe in Southern blotting in rats immunized with MBP. In a previous study (7) we demonstrated, using several TCR clones with the known CDR3 sequence, that the DSS probe hybridizes only with the encephalitogenic CDR3 sequence, DSSYEQYFGPG, and detects clonal expansion more sensitively than CDR3 spectratyping. Thus, the positive signal in the V8.2 PCR product obtained by Southern blotting indicates clonal expansion of encephalitogenic T cells in Lewis rats immunized with MBP. The results are summarized in Table I, and the representative profiles are shown in Fig. 2. Between days 1 and 6 PI, there was no V8.2 spectratype expansion in the LNs, spleens, thymi, and PBL examined (Fig. 2, C, E, and G, and Table I). Interestingly, Southern blot analysis revealed that weak, but definite, signals were present in the popliteal and/or inguinal LNs on day 1 (samples referred to as day 1 included those processed between 24 and 30 h after immunization; Fig. 2D) and day 3 (Fig. 2F) PI. We repeated the examination using the same samples taken on days 1 and 3 and obtained the same results. The signal was also detected in the spinal cord on day 6 PI (Fig. 2H).

View larger version (74K): [in this window] [in a new window]   FIGURE 2. CDR3 spectratyping (A, C, E, G, I, and K) and subsequent Southern blot analysis (B, D, F, H, J, and L) of normal (A and B), MBP-immunized (C–J), and KLH-immunized (K and L) rats. Pop, popliteal LN; Ing, inguinal LN; Mes, mesenteric LN; Spl, spleen; Thy, thymus; SC, spinal cord. The sample number of each rat is shown in parentheses. The representative results of the examinations listed in Table I are shown. n.t., Not tested.

View this table: [in this window] [in a new window]   Table 1. Summary of CDR3 spectratyping analysis with a V8.2 primer pair and subsequent DNA hybridization with a DSS probea

Washout of circulating lymphocytes by PBS perfusion modulates the spectratype profile

A certain number of naive CD4 T cells stimulated with Ag presented by activated dendritic cells leave the LN via efferent lymphatic or blood vessels (13). Therefore, the majority of Ag-stimulated T cells seem to reside within the vessels during the early induction phase. By perfusion with PBS, we tried to determine whether encephalitogenic V8.2-positive T cells were within the vessels or if they had already migrated into the perivascular space. As clearly shown in Fig. 3, B, D, and F, positive signals found in nonperfused rats on days 1 and 3 PI disappeared after PBS perfusion. On day 6 PI, the signals found in the spinal cord and PBL disappeared, whereas those in the popliteal and inguinal LNs persisted after perfusion (Fig. 3H).

View larger version (100K): [in this window] [in a new window]   FIGURE 3. CDR3 spectratyping analysis of V8.2 TCR in various organs of normal and MBP-immunized rats after PBS perfusion. Positive signals found in nonperfused rats on days 1 and 3 PI disappear after PBS perfusion. On day 6 PI, the signals found in the spinal cord and PBL disappear, whereas those in the popliteal and inguinal LNs persist after perfusion (arrow).

To identify the presence of V8.2-positive cells at the protein level, Western blot analysis for the V8.2 protein was performed after immunoprecipitation with anti-TCR mAb (Fig. 4). After PBS perfusion, spinal cord tissues were taken from rats 1, 3, and 6 days after MBP/CFA immunization. The V8.2 signal was detected on day 1 PI and increased in intensity on days 3 and 6 (Fig. 4). These findings suggest that V8.2-positive cells in the subarachnoid space or trapped in blood vessels may be the source of these signals. We also tried to detect V8.2-positive cells with immunohistochemical staining using frozen sections, but did not detect positive cells, probably because of the paucity of cells (data not shown).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Immunoprecipitation and Western blot analysis of the TCR protein in the spinal cord (SC) after PBS perfusion. Spinal cord tissues were taken days 1, 3, and 6 PI and processed for immunoprecipitation with anti-TCR mAb. The proteins were run on 12% SDS-polyacrylamide gel and transferred and stained with biotinylated R78 (anti-V8.2 mAb). Lanes 1 and 2, Day 1 SC; lanes 3 and 4, day 3 SC; lanes 5 and 6, day 6 SC; lanes 7 and 8, day 1 spleen.

We measured the proliferative responses to MBP of lymphocytes isolated from popliteal and inguinal LNs and spleens on days 1, 3, 6, and 9 PI. As shown in Fig. 5, popliteal LN and spleen cells showed no response to MBP on day 1 PI (Fig. 5A). However, there was a vigorous MBP response of the popliteal LN cells on days 3 and 6 PI (diamonds in Fig. 5, B and C), whereas inguinal LN and spleen cells did not respond to MBP at these time points (squares and triangles). On day 9 PI, inguinal LN cells responded to MBP most strongly (triangles in Fig. 5D), and popliteal LN cells became less responsive (diamonds). Spleen cells reacted to MBP slightly at this stage. These findings correspond well with clonal expansion of MBP-specific T cells revealed by spectratyping and Southern blot analyses (summarized in Table I).

View larger version (32K): [in this window] [in a new window]   FIGURE 5. Proliferative responses of LN and spleen T cells from perfused rats after MBP immunization. Lymphocytes from the popliteal and inguinal LNs and spleens of rats that had been immunized 1 (A), 3 (B), 6 (C), and 9 (D) days earlier and the single-cell suspensions were cultured in the presence or the absence of MBP (10–100 µg/ml) for 3 days. Each symbol represents the mean values of triplicate assays, and SEs were within 10% of the mean values. At each time point, two rats were examined, and essentially the same results were obtained.

All the above studies strongly suggest that encephalitogenic T cells disseminate very rapidly immediately after sensitization and reside in the lymphoid and target organs. To determine whether these disseminated encephalitogenic T cells were sufficient for the development of clinical EAE, we performed lymphadenectomy of the draining popliteal LN on days 1, 3, and 6 after immunization with MBP. Special care was taken to remove not only the LN but also the surrounding adipose tissue as much as possible to block potential collateral circulation. Fig. 6 shows very clearly that removal of the draining LN as early as day 1 (Fig. 6A) as well as on days 3 (Fig. 6B) and 6 (Fig. 6C) did not prevent the development of EAE. The severity of EAE was almost the same as that of unmanipulated EAE (Fig. 6D). Before designing this type of experiment, we noticed that amputation of the immunized limb was more suitable for the above purpose because the collateral circulation could be completely blocked by this method. However, we decided not to perform amputations for ethical reasons. Removal of the thymus before Ag sensitization did not alter the clinical course of EAE (Fig. 6E).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Clinical course of EAE in rats that had received lymphadenectomy or thymectomy. Rats were immunized with MBP in the left hind foot pads, and the draining (popliteal) LNs on the same side along with the surrounding adipose tissue were removed on days 1 (A), 3 (B), and 6 (C) PI. The clinical courses of individual rats are shown in A–C. D, Mean clinical course of five untreated animals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There is very little information about the localization and kinetics of Ag-specific T cells in the induction phase of immune reactions, mainly because the frequency of such T cells is too low to detect. It was reported that by limiting dilution analysis, the frequency of MBP-reactive T cells is estimated at between 1 in 380 (17) and 1 in 12,000 (2) even at the peak of EAE. By injection of naive T cells taken from OVA TCR transgenic mice into wild-type mice and subsequent Ag challenge, a rapid increase in Ag-specific T cells was observed in a variety of organs within 10 days (18).

Identification of autoreactive T cells with autoantigen-presenting MHC class II tetramers is an ideal method theoretically. Several groups have shown using H2^g7/mimetope tetramers that diabetogenic T cells are positively selected in the thymus and accumulate in pancreatic LN and pancreatic islets of NOD mice (19, 20). In EAE, the optimal conditions for identifying encephalitogenic T cells with MHC/neuroantigen tetramers have been reported (21). However, there are several difficulties for general application of this method to autoimmune disease analysis. First, it is technically difficult to prepare suitable MHC tetramers. Second, and more importantly, the detection of autoreactive T cells is not always easy, because the frequency and affinity to MHC/Ag of autoreactive T cells are much lower than those to foreign Ag-reactive T cells. These are the reasons why MHC class II tetramers do not become popular compared with class I tetramers.

Molecular tracking of autoreactive T cells by TCR analysis has been attempted by several groups and has been shown to be a powerful tool to dissect the pathomechanism of autoimmune diseases (22, 23, 24, 25, 26). Muraro et al. (25) performed quantitative PCR analysis using primer pairs that amplified the junctional region of TCR - and -chains of Ag-specific T cells and showed that the frequency of candidate pathogenic T cells reflected well the clinical status of neurological immune-mediated disorders. It is also possible to detect a single autoreactive T cells in the target tissue by combining laser microdissection, single-cell PCR, and CDR3 spectratyping (26).

In the present study we identified clonal expansion of encephalitogenic T cells in Lewis rats that had been immunized with MBP by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the CDR3 sequence of the major encephalitogenic TCR clone. We took advantage of the fact that encephalitogenic T cells of Lewis rats with MBP-induced EAE mainly use V8.2 TCR with the DSSYEQYFGPG sequence in the CDR3 region (1, 7). This V8.2 predominance remains unchanged in the lymphoid and target organs throughout the course of EAE (5, 27). Consequently, we found in this study with ordinary CDR3 spectratyping analysis and subsequent DNA hybridization that encephalitogenic T cells were first activated and increased in the regional (popliteal) LN within the first 72 h of MBP immunization. Then activated encephalitogenic T cells disseminated and reached the spinal cord by day 6, 3–4 days before the onset of the disease. Consistent with these finding, the V8.2 protein in the spinal cord detected by immunoprecipitation increased on days 3 and 6 PI. At the peak of EAE, V8.2 spectratype expansion was marked in the spinal cord, and that in lymphoid organs became less obvious. The proliferative responses of T cells in LNs and spleens reflected the expansion of the V8.2 spectratype. The present study has demonstrated for the first time that encephalitogenic T cells, once activated, disseminate into the whole body much earlier than previously assumed. Perfusion of immunized rats provided useful information with regard to the location of T cells. Because washout of blood and lymphatic vessels on day 3 PI removed the positive signals in the LNs, activated encephalitogenic T cells reside within the vessels. In contrast, perfusion on day 6 did not eliminate the signals in the same site, suggesting that some encephalitogenic T cells infiltrate the tissue parenchyma. Taken together, these findings suggest that immunotherapy targeting encephalitogenic T cells in the vessels could be possible when therapy is initiated at the early stage or during remission of the autoimmune disease. It should be noted that the thymus always showed normal spectratype patterns and gave no signal in the Southern blot analysis. This finding indicates that the size of the V8.2-positive encephalitogenic T cell clone was too small to detect by our methods and further suggests that encephalitogenic T cells that had emigrated from the thymus to the peripheral lymphoid organs are sufficient for the induction of EAE. As clearly shown in Fig. 5E, rats that had received thymectomy developed full-blown EAE in the absence of the thymus.

In summary, we have been able to demonstrate early expansion and dissemination of encephalitogenic T cells in unmanipulated animals by using CDR3 spectratyping and Southern blot analysis. Introduction of new technology into TCR analysis enabled us to elucidate the dynamic expansion of pathogenic T cells in autoimmune diseases.

	Acknowledgments

 
We thank Y. Kawazoe for technical assistance. H.S. is grateful to Prof. S. Mizutani (Department of Pediatrics and Developmental Biology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan) for his continuous support.

	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 in part by grants-in-aid from the Ministry of Education, Japan.

² Current address: Department of Veterinary Medicine, Cheju National University, Jeju City 690-756, Korea.

³ Address correspondence and reprint requests to Dr. Yoh Matsumoto, Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai, 2-6 Fuchu, Tokyo 183-8526, Japan. E-mail address: matyoh{at}tmin.ac.jp

⁴ Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; CDR3, complementarity-determining region 3; KLH, keyhole limpet hemocyanin; LN, lymph node; PI, postimmunization.

Received for publication April 1, 2004. Accepted for publication August 2, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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