The CNS T cell repertoire was analyzed by RT-PCR, spectratyping, and nucleotide sequencing of the amplified products at different times following adoptive transfer of a CD4+, Th1, VB2+ encephalitogenic SJL/J proteolipid protein peptide 139–151-specific T cell clone. The third complementarity-determining region of TCR B chains in the spinal cord was used as an indicator of T cell heterogeneity. Spectratypic analysis revealed that a single peak corresponding to the third complementarity-determining region of the initiating T cell clone predominated during the acute phase. During recovery and relapse the complexity of the spectratype increased. DNA sequence analysis revealed that the donor clone predominated at the acute phase. By the first relapse the donor clone, although represented most frequently, was a minority of the total. Spectratypic analysis of the same samples for several other VB families revealed their presence during acute disease or relapses but, with the exception of VB17, their absence during the recovery stage.

Experimental autoimmune encephalomyelitis (EAE)3 can be induced in genetically susceptible animals of a number of different species by immunization with neuroantigens such as myelin basic protein, proteolipid protein (PLP) or myelin oligodendrocyte protein. In the SJL/J mouse chronic, relapsing disease is mediated by CD4+, Th1, neuroantigen-specific T cell lines or clones and can be adoptively transferred to naive syngeneic animals following activation of the T cells by a period of in vitro culture (1, 2, 3).

As it has been hypothesized that determinant spreading contributes to relapsing disease, there is considerable interest in the T cell repertoire in the CNS throughout the course of EAE. Increasing heterogeneity of the CNS T cell population would be predicted as it is during the relapsing phases that determinant spreading has been detected.

There are multiple ways in which CNS T cell heterogeneity may arise during the course of EAE. Following active immunization using CFA both neuroantigen-specific T cells and mycobacteria-specific T cells are raised in response to immunization. There is consensus that activated T cells, regardless of specificity, are able to readily access the CNS (4, 5, 6, 7, 8, 9). Thus, both neuroantigen-specific and mycobacteria-specific T cells will be found in the CNS. A second source of heterogeneity is the neuroantigen used as immunogen. If a protein is used, there will be a response against multiple epitopes. This is also true, although to a lesser degree, when peptides are used for immunization. Also, the emulsion containing the Ag and mycobacteria persists at the site of injection for weeks or longer, providing a continuing source of stimulation of the immune system. Finally, inflammation at the site of the lesions may render the blood-brain barrier permeable to an influx of host cells, which increases T cell heterogeneity within the CNS.

The use of adjuvant may be avoided by adoptive transfer of encephalitogenic T cell lines to syngeneic recipients. With a few exceptions, the T cell lines used for adoptive transfer were heterogeneous. Expansion of minor populations of T cells in the line after transfer to the recipient may lead to increased donor T cell heterogeneity in the CNS. Because nonspecific T cells from the recipient cross into the CNS during the course of disease, another layer of complexity is added to the CNS T cell population. The use of a well-characterized encephalitogenic T cell clone to induce passive EAE would eliminate heterogeneity contributed by the donor T cell population and allow visualization of the host T cell CNS component.

Recently, we generated several encephalitogenic PLP peptide 139–151-specific T cell clones, and found that by using the CDR3 region of one of these T cell clones, 3-19, as an idiotypic marker, the clone could be tracked in the CNS during the acute and relapse phases of EAE (3, 10). It also allowed ready differentiation of the clone from host-derived T cells, as the marker was unique to the clone.

To assess the degree of heterogeneity within the CNS at different stages of disease following adoptive transfer of an encephalitogenic T cell clone, cDNA prepared from the spinal cord of recipients of T cell clone 3-19 at different stages of disease was amplified using primers for VB gene segments present in the SJL mouse and a CB primer labeled with fluorescein. The amplified products were analyzed by spectratyping (11, 12, 13, 14) to assess CDR3 size heterogeneity.

The spectratyping technique provides a minimal measure of TCR heterogeneity, as each band may be composed of multiple CDR3 regions of equal size but differing compositions. To assess heterogeneity at the single-cell level, it is necessary to clone and sequence the PCR products that form each band. As it would be a very large task to sequence every band in the spectratype for each VB family, we restricted this part of the study to the VB2 band that corresponded to the size of the CDR3 of clone 3-19 for two reasons. First, as the CDR3 sequence of clone 3-19 was known, we could readily differentiate the encephalitogenic CDR3 from other TCR CDR3 regions of this size, and second, it was likely that this band was representative of other CDR3 regions. The band was isolated from the CNS spectratype at different stages of disease and cloned, and the nucleotide sequence was determined. At the acute stage of disease 100% of the sequences obtained were identical with T cell clone 3-19. At remission very few CDR3 sequences were that of the clone, while at relapse, the number of 3-19 sequences obtained was increased relative to the number of heterogeneous sequences. Detailed findings of these studies are reported below.

Female SJL/J (H-2s) were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the Animal Resource Center of the Medical College of Wisconsin (Milwaukee, WI). Mice were used between 8 and 12 wk of age.

PLP peptide 139–151 was synthesized by the Nucleic Acid and Protein Core Facility of the Cancer Center of the Medical College of Wisconsin. The amino acid sequence of this peptide is H-S-L-G-K-W-L-G-H-P-D-K-F, where S is substituted for the C found at position 140 in the authentic PLP sequence.

Female SJL/J mice were immunized with PLP peptide 139–151 (100 μg/mouse) emulsified in IFA supplemented with Mycobacterium tuberculosis H37Ra (50 μg/mouse). A total of 0.1 ml of emulsion was injected into four sites on the flanks. At 24 and 72 h following the initial injections, 400 ng of Bordetella pertussis toxin (Sigma) was administered i.v. to mice injected with PLP peptide 139–151. Ten days following initial immunization the draining lymph nodes were removed, and a single-cell suspension was prepared and cultured without further separation at 3 × 106/ml, 2 ml/well, in 24-well tissue culture plates. The culture medium was RPMI 1640 supplemented with 10% FCS, 5 × 105 M 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 10 mM HEPES buffer, and 5 μg/ml peptide 139–151. After 4 days at 37 C blast cells were isolated on a Ficoll-Hypaque gradient and resuspended at 2 × 105/ml with irradiated (3000 rad) syngeneic spleen cells at 1 × 106/ml. Erythrocytes in spleen cell preparations were lysed with ammonium chloride. Cells were cultured in the absence of nominal Ag in tissue culture flasks for a 10-day rest period followed by a 4-day restimulation period during which the surviving cells were cultured at 1 × 105/ml with peptide 139–151 (5 μg/ml) and fresh irradiated syngeneic spleen cells at 2.5 × 106/ml in 24-well culture plates. Ag-specific T cells were cloned by limiting dilution in 96-well round-bottom culture plates from primary cultures of lymph node cells. Irradiated syngeneic spleen cells were added at 5 × 105/well along with Ag and 10% Con A supernatant from a rat spleen cell culture as a source of lymphokines. Positive clones or subclones were transferred at ∼2 wk to 24-well plates for expansion. They were maintained throughout by subculture with Ag, fresh irradiated spleen cells (5 × 106/ml), and 10% Con A supernatant. T cell clones were assessed for monoclonality by PCR analysis using a set of primers for each VB gene expressed in the SJL mouse and a B chain constant region primer. Only those clones that reacted with a single VB primer were used for further study. Clone 3-19 was found to be a VB2, CD4+, Th1 T cell and was highly encephalitogenic following adoptive transfer to syngeneic naive mice.

Activated 3-19 T cells (8 × 105 to 1 × 106) were injected i.v. into irradiated (500 rad) female SJL/J mice. Mice were observed daily for clinical signs of EAE throughout the experimental period. Clinical signs of EAE were scored on a scale of 1–4 as previously described (15).

Total RNA was isolated from spinal cords of recipients of T clone 3-19 and unmanipulated control mice (16). Quantification of RNA was performed by spectrophotometry at 260 nm. For preparation of cDNA 1 μg of total RNA was incubated with 10 μl of 5× buffer, 5 μl of poly(T) (20 μM) 2.5 μl of dNTPs (10 mM), and water to a final volume of 42 μl. The mixture was heated at 68°C for 2 min, then cooled in ice, and 0.5 μl of RNAguard (Pharmacia, Piscataway, NJ), 5 μl of DTT (0.1 M), and 2.5 μl Moloney murine leukemia virus reverse transcriptase were added. The mixture was incubated at 42°C for 45 min. PCR amplification of cDNA was performed as follows. One microliter of cDNA was incubated with 2.5 μl of 10× buffer, 0.5 μl of dNTPs (10 mM), 1 μl of 50 mM MgCl2, 1 μl of primers (20 μM), and water to a volume of 24 μl. The mixture was heated to 95°C, and 1 μl (1 U) Taq polymerase was added. The amplification program was conducted at 94°C for 1 min, the appropriate annealing temperature (55–61°C depending on the Tm of the primers) for 1 min, and 72°C for 1 min. At the end of the program there was a final extension at 72°C for 7 min.

The amplified products were analyzed by agarose gel electrophoresis. To verify that the products were authentic, they were transferred to a nylon membrane and hybridized with a 32P-labeled oligonucleotide with a sequence internal to that of the primers used for amplification. The oligonucleotides (5′-3′) used in this study are as follows: VB2, ATG AGC CAG GGC AGA ACC TTG TAC; VB3, GAA ATT CAG TCC TCT GAG GCA GGA; VB4, CTA AAG CCT GAT GAC TCG GCC ACA; VB6, GCC CAG AAG AAC GAG ATG GCC GTT; VB10, CTT CGA ATC AAG TCT GTA GAG CCG G; VB16, CTC TGA AAA TCC AAC CCA CAG CAC TGG; and VB17, GAA ATC CTA TCC TCT GAA GAA GAC. The B chain CDR3 probe for the peptide 139–151-specific 3-19 T cell clone was TGCAGTGCAAACAGG. The B chain constant region internal probe was GGCTCAAACAAGGAGACCTTGGGTGGA. The B chain constant region external primer was CCAAGCACACGAGGGTAGCCT. The CD3δ primers were GGA ACA CAG CGG GAT TCT GG (sense) and CAC CAG CCA TGG TGC CCG AG (antisense). The hypoxanthine phosphoribosyltransferase (HPRT) primers were GTT GGA TAC AGG CCA GAC TTT GTT G (sense) and GAT TCA ACT TGC GCT CAT CTT AGC C (antisense).

At various times after adoptive transfer of the 3-19 T cell clone, mice were sacrificed, and the spinal cord was removed. Total RNA was prepared by the procedure of Chomzynski and Sacchi (16) and was reverse transcribed to prepare cDNA. To assess total CNS T cells, cDNA prepared from mice at different stages of disease was amplified using CD3 and HPRT primers. The CD3 signal was normalized to the HPRT signal from the same sample at each time point. Oligonucleotide primers specific for VB2 and the external B chain constant region were used in PCR amplification of the cDNA obtained from the tissues. cDNA from clone 3-19 was used as a positive control. The integrity of the cDNA prepared was assessed by PCR amplification with oligonucleotide primers for (HPRT). Following amplification with the VB2 and the external B chain constant region primers, the amplification products were transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH) and probed with either a 32P-labeled internal B chain constant region probe or with the 32P-labeled 3-19 CDR3 probe. The blots were then analyzed by a phosphorimager and by autoradiography. cDNA prepared from normal unmanipulated mice served as the negative control.

cDNA prepared from the spinal cords of normal mice or recipients of T cell clone 3-19 was amplified using a VB2 primer and a CB primer labeled with fluorescein. The amplified products were then diluted 1/50, added to an equal volume of formamide, and heated at 98°C for 2 min. Seven microliters of the samples were applied to a prewarmed 5% acrylamide sequencing gel. The gels were run at 30 W for 3 h on a Pharmacia ALF automated DNA sequencing apparatus. Analysis of the separated products was performed using Fragment Manager software (Pharmacia). cDNA prepared from normal mouse spleen or thymus served as a positive control. In some instances 20 μl of the undiluted amplified products were separated at 60 W on a 5% denaturing polyacrylamide sequencing gel until the region corresponding to 120–160 bp was near the bottom of the gel. The fluorescein-labeled products were analyzed on a Storm II apparatus (Molecular Dynamics, Sunnyvale, CA), and the data were analyzed with ImageQuant software (Becton Dickinson, Mountain View, CA). Spectratypic analyses were performed on at least three mice at each stage of disease. Representative data are shown in the figures.

Amplified products from the band corresponding to the 3-19 T cell clone were isolated from the acrylamide gel, ligated into the pTAdv vector, and cloned using the AdvanTage PCR cloning kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The plasmid DNA was purified using Qiagen Spin Plasmid Miniprep using the manufacturer’s instructions (Qiagen, Valencia, CA), and sequenced using the Autoread Sequencing kit (Pharmacia) on the ALF DNA sequencer.

We recently reported that when EAE was induced by a single T cell clone, the clone could be detected in the spinal cord during disease phases, but not during remissions (10). As a TCR molecular idiotypic marker was used to identify the donor clone after transfer to the recipients, no information was available with respect to other T cells that might be present in the spinal cord at these times. Was the loss of the donor clone during remission mirrored by the total T cell population, or was it specific for the encephalitogenic clone? Secondly, did the T cell population in the spinal cord become more heterogeneous during the course of disease as would be predicted by the epitope spreading hypothesis? To answer these questions, cDNA was prepared from the spinal cord of mice at the acute, remission, and relapse stages of passive EAE. Total T cell cDNA was assessed by RT-PCR using primers for the CD3δ chain. TCR spectratyping of several cDNA VB families was performed to assess the overall heterogeneity of the CNS T cell population. Heterogeneity was assessed at the single-cell level by cloning and sequencing of the TCR CDR3 regions found in cDNA from mice at different stages of EAE.

The first question to be answered was whether there was a reduction in total T cell number in the spinal cord during remission. To address this question cDNA prepared from spinal cord at the acute phase, remission, and relapse was coamplified using oligonucleotide primers for CD3 and HPRT. The products were separated on an agarose gel and analyzed on an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA). The ratio of the density of the CD3 band at 325 bp to the density of the HPRT band (165 bp) at each time point was determined. cDNA from thymus served as a positive control, and cDNA from normal SJL spinal cord was the negative control. The conditions of the assay were such that the HPRT signal was not amplified to saturation. Tabulation of the ratios of CD3 signal to HPRT signal showed that the CD3 signal was diminished at the recovery stage with respect to the HPRT control, indicating a reduction in the total T cell population in the CNS (Table I). The experiment was repeated twice with similar results. Similar results were found when cDNA from several other mice at the recovery stage was amplified in this manner.

Table I.

Ratio of density of CD3 product to HPRT product after coamplification and separation on 1% agarose gel of cDNA derived from spinal cord of mice with EAE

Disease StageCD3/HPRT
Acute 0.28 
Remission 0.10 
Relapse 1 0.25 
Relapse 2 0.25 
Disease StageCD3/HPRT
Acute 0.28 
Remission 0.10 
Relapse 1 0.25 
Relapse 2 0.25 

The next question concerned the heterogeneity of the T cell population in the spinal cord at different disease phases. Spinal cord cDNA was analyzed by PCR-based spectratyping to assess the total TCR heterogeneity of several VB families. In the second set of experiments in which DNA sequence analysis was performed, the analysis was limited to the VB2+ T cell population, the TCR family of the donor T cell clone, to keep the complexity of the analysis to a manageable level.

Fig. 1 shows the results of a spectratypic analysis of spleen T cells from a normal SJL mouse performed by amplification across the CDR3 region of the B chain of the TCR using VB family-specific primers and a fluoresceinated CB primer. The amplified products were then separated on an automated DNA sequencing apparatus (A.L.F., Pharmacia) and analyzed using Fragment Manager software (Pharmacia). As the CDR3 regions of different TCR vary in size, a family of products was generated for each VB family. Each band represents CDR3 regions of the same size amplified from the cDNA of one or more T cell clones within the spleen. The bands differ by 3 bp as determined by using standards of known base pair number. Typically, the spectratypic analysis of normal spleen or thymus T cells results in a series of six to eight bands of varying intensity, with the most intense band located centrally, and bands of decreasing intensity on either side. The distribution of intensities normally approximates a Gaussian curve. Skewing from a normal distribution is presumptive evidence for expansion or reduction of a particular subset of T cells in the immune response.

FIGURE 1.

Spectratypic analysis of splenic T cells from a normal SJL/J mouse. cDNA from the spleen was amplified with various VB primers and a fluorescent CB primer. The amplified products were separated on an automated DNA sequencing apparatus (A.L.F., Pharmacia) and analyzed using Fragment Manager software (Pharmacia).

FIGURE 1.

Spectratypic analysis of splenic T cells from a normal SJL/J mouse. cDNA from the spleen was amplified with various VB primers and a fluorescent CB primer. The amplified products were separated on an automated DNA sequencing apparatus (A.L.F., Pharmacia) and analyzed using Fragment Manager software (Pharmacia).

Close modal

Spectratypic analyses of spinal cord cDNA samples from mice at different stages of EAE are shown in Fig. 2. Each panel represents a different mouse in which disease was initiated by adoptive transfer of encephalitogenic T cell clone, 3-19. Upon amplification with a VB2 and a CB primer, the product of clone 3-19 was 184 bp, as verified by reactivity with a 32P-labeled 3-19 clonotypic probe. The CDR3 nucleotide and protein sequence of clone 3-19 was known (3), making it possible to differentiate donor CDR3 sequences from recipient CDR3 sequences in the 184-bp band. All other size bands as well as bands generated by amplification with non-VB2 primers must be derived from recipient T cells.

FIGURE 2.

Spectratypic analysis of VB2+ T cells in the spinal cord of SJL/J mice at various stages of disease. Normal spinal cord cDNA amplified in the same manner yielded no detectable products.

FIGURE 2.

Spectratypic analysis of VB2+ T cells in the spinal cord of SJL/J mice at various stages of disease. Normal spinal cord cDNA amplified in the same manner yielded no detectable products.

Close modal

The TCR spectratype of several VB families amplified from spinal cord cDNA prepared from tissue harvested during acute disease are shown in Fig. 3,A. In each analysis the same quantity of material was used. Size heterogeneity in each family is indicated by multiple peaks. It is of interest that there was a single predominant peak with VB4, -6, and -17 at this stage of disease. As the spectratype of normal splenic T cells approximated a normal curve with respect to CDR3 size (Fig. 1), this implied that there was selectivity of host T cell entry or expansion in the CNS during this stage of disease. If migration were a random event, the pattern should approximate a normal distribution.

FIGURE 3.

Spectratypic analysis of spinal cord cDNA from SJL/J mice at various stages of EAE with different VB family primers.

FIGURE 3.

Spectratypic analysis of spinal cord cDNA from SJL/J mice at various stages of EAE with different VB family primers.

Close modal

The spectratypes of the same VB families amplified from cDNA prepared from animals in remission are shown in Fig. 3,B. It is apparent from this panel that there was an overall reduction in TCR expression during this phase of disease, in agreement with the data presented in Table I. Only VB17+ TCR appeared to be undiminished, and, in fact, this family is more heterogeneous than during the acute phase.

Fig. 3, C and D, shows the results of analyses of first and second relapses, respectively. It is noteworthy that VB3 TCR were not present in either mouse at this stage of disease. Each of the other VB families was more heterogeneous than at the acute phase. In each instance two or three bands became prominent, in sharp contrast to the predominance of a single band during the acute phase. As each band probably contained several different CDR3 regions, the heterogeneity of the CNS T cell population increased dramatically during the relapsing stage of disease. Again, the data indicated either selectivity of entry of certain VB families into the CNS, or selective expansion of certain families after entry. These findings are representative of several mice tested in this manner.

Several things are apparent from the spectratypic patterns shown in Fig. 2. First, during acute disease, the predominant band size (184 bp) corresponded to that of the donor T cell clone, 3-19. However, one or more bands of larger size appeared at this stage of disease, which indicated that a small number of host-derived VB2+ T cells was present at the initial stage of disease. Table II shows the percentage of the total area of each peak in the spectratype. During the acute phase the 184-bp peak was by far the major peak in each case. Interestingly, during remission, this peak was reduced from 83 to 26% and from 62 to 12% of the total in Fig. 3, A and B, respectively. Other bands at 187, 190, 193, and 196 bp increased from the low levels seen in the acute phase. At relapse the 184-bp band again increased relative to the larger bands in the spectratype from 26 to 35% in Fig. 3,A and from 12 to 32% in Fig. 3 B.

Table II.

Percentage of total area represented by each peak of the CNS VB2 TCR spectratype at different stages of EAE

Clinical StagePeak (bp)
184187190193196
Expt. A      
Acute 89a    
Remission 18 15 42  25 
Relapse 15 19 24 30 12 
      
Expt. B      
Acute 47 30 
Remission 14 13 27 24 23 
Relapse 26 17 21 25 10 
Clinical StagePeak (bp)
184187190193196
Expt. A      
Acute 89a    
Remission 18 15 42  25 
Relapse 15 19 24 30 12 
      
Expt. B      
Acute 47 30 
Remission 14 13 27 24 23 
Relapse 26 17 21 25 10 
a

Percentage of total area of all curves in spectratype.

The next question concerned the heterogeneity of TCR within each band. As it was impracticable to clone and sequence each band in the spectratype, the 184-bp band, which corresponded to the TCR clone used to induce disease, was chosen as a model for CDR3 heterogeneity. Using this band allowed differentiation of the clone CDR3 from recipient CDR3. cDNA was prepared from spinal cord RNA taken from several different mice at the acute, remission, and relapse phases of disease. The deduced amino acid sequences and frequency of the 184-bp DNA nucleotide sequences were assessed for two mice at each stage of EAE and are reported in Table III. Sequence 1 was that of the donor T cell clone, 3-19. Twenty-three of 23 sequences cloned from PCR-amplified cDNA taken at acute disease were that of the encephalitogenic T cell clone. None of the other sequences of this size were found at the acute stage of disease, although a small number of bands of larger size were noted. During remission the same sequence was present in 7 of 24 clones, while sequence 2 comprised 6 of 11 clones. At relapse, 10 of 21 clones were of the original sequence, 1 of 11 comprised sequence 2, and the remaining 9 sequences were unique, indicating that numerous host-derived T cells of new specificities were present in the spinal cord at this later time.

Table III.

TCR β-chain CDR3 protein sequences deduced from cloned spinal cord cDNA from SJL mice at different stages of EAE

SequenceFrequency During
AcuteRemissionRelapse
1. TCSANRYEQYFGPGTa 23/23b 7 /24 10 /21 
VB2 DB1 JB2S6    
2. TCSADKGLQYFGPGT  6 /11 1 /11 
VB2 DB2 JBS5    
3. TCSASPGQLYFGEGS  5 /24  
VB2 DB1 JB2S2    
4. TCSAGGNEQYFGPGT  1 /11  
VB2 DB1 JB2S6    
5. TCSAQAPEVFFGKGT  2 /13  
VB2 DB1 JB1S1    
6. TCSADLAEQFFGPGT  1 /13  
VB2 DB2 JB2S1    
7. TCSAGTDEQYFGPGT  1 /13  
VB2 DB2 JB2S6    
8. TCSADLAEQFFGPGT  1 /13  
VB2 DB1 JB2S1    
9. TCSAGTSEVFFGKGT   1 /11 
VB2 DB1 JB1S1    
10. TCSADQNTLYFGAGT   1 /11 
VB2 DB1 JB2S4    
11. TCSADRYEQYFGPGT   1 /11 
VB2 DB1 JB2S6    
12. TCSDWGDTQYFGPGT   1 /11 
VB2 DB2 JB2S5    
13. TCSRDQNTLYFGAGT   1 /11 
VB2 DB1 JB2S4    
14. TCSGTDQAQHFGEGT   1 /11 
VB2 DB1 JB1S5    
15. TCSADGYEQYFGPGT   2 /10 
VB2 DB2 JB2S6    
16. TCSAGDTEVFFGKGT   1 /10 
VB2 DB1 JB1S1    
17. TCSAADSDYTFGSGT   1 /10 
VB2 DB1 JB1S2    
SequenceFrequency During
AcuteRemissionRelapse
1. TCSANRYEQYFGPGTa 23/23b 7 /24 10 /21 
VB2 DB1 JB2S6    
2. TCSADKGLQYFGPGT  6 /11 1 /11 
VB2 DB2 JBS5    
3. TCSASPGQLYFGEGS  5 /24  
VB2 DB1 JB2S2    
4. TCSAGGNEQYFGPGT  1 /11  
VB2 DB1 JB2S6    
5. TCSAQAPEVFFGKGT  2 /13  
VB2 DB1 JB1S1    
6. TCSADLAEQFFGPGT  1 /13  
VB2 DB2 JB2S1    
7. TCSAGTDEQYFGPGT  1 /13  
VB2 DB2 JB2S6    
8. TCSADLAEQFFGPGT  1 /13  
VB2 DB1 JB2S1    
9. TCSAGTSEVFFGKGT   1 /11 
VB2 DB1 JB1S1    
10. TCSADQNTLYFGAGT   1 /11 
VB2 DB1 JB2S4    
11. TCSADRYEQYFGPGT   1 /11 
VB2 DB1 JB2S6    
12. TCSDWGDTQYFGPGT   1 /11 
VB2 DB2 JB2S5    
13. TCSRDQNTLYFGAGT   1 /11 
VB2 DB1 JB2S4    
14. TCSGTDQAQHFGEGT   1 /11 
VB2 DB1 JB1S5    
15. TCSADGYEQYFGPGT   2 /10 
VB2 DB2 JB2S6    
16. TCSAGDTEVFFGKGT   1 /10 
VB2 DB1 JB1S1    
17. TCSAADSDYTFGSGT   1 /10 
VB2 DB1 JB1S2    
a

Bold letters indicate amino acids in CDR3 region.

b

Number of times sequence found/total number of clones sequenced.

In general, the CDR3 amino acid sequences were unique to a given mouse. Sequence 2 was isolated from two different mice, as was sequence 3. However, each of the other CDR3 amino acid sequences were unique to the mouse from which it was isolated. There was no predominant usage of a particular JB in the clones, as many were represented in the isolates, nor was a particular DB used predominantly.

The issue of T cell heterogeneity within the spinal cord during murine EAE is important to understanding the mechanisms of disease and, in particular, in the design of therapies to alleviate disease. EAE in the SJL mouse model is a well-studied chronic relapsing disease; however, the mechanisms of recovery and relapse remain unresolved. The purpose of the present experiments was to assess the heterogeneity of the T cell population in the spinal cord during the course of passive EAE. Of particular interest was whether there was increasing T cell diversity throughout the course of disease. To examine this issue, EAE was induced by transfer of an encephalitogenic peptide-specific T cell clone. TCR heterogeneity in the CNS of the recipients was analyzed by spectratyping and, at the single-cell level, by DNA and protein sequence analysis of the TCR CDR3 regions of the B chain. As no adjuvants were used for induction of disease, cells entering the spinal cord should be a consequence of inflammation initiated by the encephalitogenic T cell clone.

Initially, the relative T cell content in the spinal cord at different disease states was determined by PCR analysis using CD3 primers for amplification. The CD3 signal was normalized to the HPRT signal and was found to be stronger during disease stages and reduced at remission. This finding is consistent with our recent report that an encephalitogenic T cell clone was readily detected in the spinal cord during the acute and relapse stages of disease, but was not found during remission (10). Others have reported a decrease in CNS T cell number following acute EAE (17, 18). The mechanism of T cell decline in the spinal cord may be apoptosis (19, 20, 21, 22).

TCR spectratyping has been used previously to analyze T cell populations in the spinal cord of rats (23). T cells were very heterogeneous during both the acute phase and the recovery stage. All VB families were present, and each family consisted of two to seven bands. Since EAE was induced by active immunization with myelin basic protein in complete adjuvant, the number of activated T cells able to enter the CNS would be expected to be quite large and would not necessarily represent only neuroantigen-specific T cells. As it has been reported that encephalitogenic VB8.2+ T cells in the rat contain short TCR B chain CDR3 regions (24), the smallest band of this family was selected for sequencing. Nucleotide sequence analysis revealed that a single short sequence predominated during the early and acute stages of disease (15 of 21), but that this sequence was less predominant at recovery. Unfortunately, neither cDNA prepared from the spinal cords of normal control rats nor that from rats injected with CFA alone was included in the study, so the contribution of T cells activated by mycobacteria in the adjuvant to overall T cell heterogeneity in the spinal cords could not be determined.

In the Lewis rat model of EAE it has been speculated that the encephalitogenic T cells were of early ontogenic origin, as the CDR3 regions of the TCR B chains were very short (24). Interestingly, the SJL T cell clone used in the present experiments also had a very short CDR3 region; however, in the mouse model, the CDR3 regions of the TCR B chains of encephalitogenic T cells are more diverse with respect to size and VB family (10).

It has been hypothesized that epitope spreading is a primary cause of relapsing disease (25, 26, 27, 28, 29, 30, 31, 32). In contrast, there are reports of failure to find convincing experimental evidence for epitope spreading (18, 33). It was hypothesized by the latter that that relapses are due to waxing and waning of the response against the immunodominant epitope, and that apparent epitope spreading is the result of the in vitro stimulation of neuroantigen-specific T cell found in the lymphoid organs. These differences have been ascribed to the different experimental techniques used for disease induction (34).

The use of active and passive EAE models in the investigation of epitope spreading has resulted in confusion, as the Ag depot that remains after active immunization provides a source of continued antigenic stimulation by the neuroantigen as well as mycobacteria over the course of months. In passive disease only the donor effector T cells serve to initiate disease; therefore, it was assumed that new specificities would have to come from sensitization of host T cells. A caveat is that many of the studies of epitope spread to this point have used heterogeneous neuroantigen-specific T cell lines to transfer disease. As these lines may contain minor populations of T cells of specificity other than for the immunodominant peptide, it is possible that increased diversity of T cell specificity resulted from expansion of these minor populations in vivo. In the present study EAE was induced by adoptive transfer of an encephalitogenic peptide-specific T cell clone to eliminate the potential contribution of responses to multiple epitopes associated with the encephalitogenic protein and mycobacteria to heterogeneity of the T cell repertoire in the spinal cord.

The results of the spectratypic analyses are relevant to the issue of epitope spread. The spectratype of normal spleen or other lymphoid tissues normally is seen as a series of several bands in a Gaussian-like distribution around a central prominent band. Although the reason for this distribution is not known, it is assumed that for each VB family a certain size CDR3 region is optimal for the binding pocket in the TCR, and that larger or smaller size CDR3 regions are less frequent. During an immune response, selective expansion of specific T cell clones with different size CDR3 regions may result in skewing of the spectratype. The findings with the VB2 family are of interest. During the acute phase of disease the donor clone was predominant in the spectratype. Later during remission and relapse, multiple bands were found, but they were not typical of the normal distribution. This implies selective expansion of host T cell clones. If there were simply a nonspecific influx of peripheral T cells into the spinal cord due to inflammation, a normal distribution would be predicted.

When other VB families were examined in this manner, the findings were similar. Interestingly, VB3 and VB10 spectratypes were not seen in all mice, again implying a specific response rather than a nonspecific accumulation of T cells in the spinal cord. These findings are in accord with a short previous report of spectratypic analyses of spinal cord T cells in SJL and B10.RIII mice (11). Based on the evidence provided by the spinal cord spectratypes, the possibility of epitope spread as being responsible for the expansion of specific T cell clones is likely. It is unfortunate that due to technical limitations serial samplings of individual mice cannot be analyzed in this manner, as this would be more informative.

As each band may consist of multiple CDR3 regions of identical size, the 184-bp band that corresponded to the VB2 CDR3 of the T cell clone used as a model for study of intraband heterogeneity as the nucleotide sequence of the CDR3 region of the clone was known and could be differentiated from host CDR3 regions. One hundred percent of the VB2 TCR B chain CDR3 region sequences obtained at the acute stage were from the transferred T cell clone. As disease progressed through recovery and relapse, increased heterogeneity was observed in this band, although the original sequence was still the majority of those cloned. Spectratypic analysis also showed increasing heterogeneity and skewing in terms of CDR3 size as disease progressed.

In summary, spinal cord T cells in mice in which EAE was induced by a single encephalitogenic T cell clone become increasingly heterogeneous as disease progresses due to an influx of recipient T cells. The data indicate that increased complexity is not due to a simple accumulation of host T cells, but is due to expansion of specific T cells clones that may be the result of sensitization of recipient T cells by additional epitopes during the inflammatory process.

1

This work was supported by Grant AI30605 from the National Institutes of Health.

3

Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; CDR3, third complementarity-determining region; HPRT, hypoxanthine ribophosphoryl transferase; CB, constant region of TCR B-chain.

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