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*
Neuroimmunology Unit, Center of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; and
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular mechanisms behind the skewed usage of particular TCRBV products are largely unknown. Components of the trimolecular complex, the TCR, the MHC molecule, and the antigenic peptide, are obvious candidates for such influences and, consequently, the subjects of interest in this study (17, 18). The importance of MHC molecules is also nourished by other observations. Although susceptibility/resistance for both MS and EAE is polygenically controlled (19, 20), the strongest association described is with alleles within the MHC. For instance, the HLA-Dw2 haplotype is a decisive susceptibility gene region in MS (21). Circumstantial evidence strongly suggests that the impact of allele-specific MHC molecules on induction and maintenance of autoimmune diseases is related to their ability to selectively present antigenic peptides to T cells, both in the thymus and in the periphery (22).
The aims of the present study of LEW rat EAE were twofold: firstly, to evaluate the dependence of TCRBV8S2 usage by encephalitogenic T cells on MHC allele-specific products in conjunction with autologous and heterologous MBP63-88 peptides, and, secondly, to quantify the preferential recruitment of TCRBV8S2+ T cells. Inbred MHC congenic LEW rats (LEW (RT1l), LEW.1AV1 (RT1av1), and LEW.1W (RT1u)) were selected for studies on the basis of previously demonstrated MHC restriction patterns (23, 24). EAE was induced in all LEW rat strains immunized with the 63 to 88 peptide of the GP MBP (MBPGP63-88) and, in addition, in LEW rats immunized with the corresponding autologous rat MBP peptide (MBPRAT63-88). Quantification of activated TCRBV8S2+ T cells from blood, draining lymph node (LN), and CNS was examined, as measured by FACS analyses. Only LEW rats immunized with the heterologous MBPGP63-88 sequence displayed T cell responses characterized by preferential TCRBV8S2 expression. It was herein demonstrated that the differential usage of TCRBV8S2 in the congenic LEW rat strains is determined by the relationship between allele-specific MHC class II products and the actual sequence of the encephalitogenic MBP peptide.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male rats (Table I
), 8 to 12 wk of age, were used in all
experiments. LEW (RT1), LEW.1AV1 (RT1av1), and LEW.1W
(RT1u) rats were originally obtained from the
Zentralinstitut für Versuchstierzucht, Hannover, Germany (25).
Subsequently, they were locally bred in filter boxes and routinely
tested for specific pathogens. Breeding pairs were checked for
homozygosity by examination of a microsatellite marker located within
the RT1 region.
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MBPGP63-88 (AARTTHYGSLPQKSQRSQDENPVVHF), MBPRAT63-88 (HTRTTHYGSLPQKSQRTQDENPVVHF), and MBPRAT/GP89-101 (VHFFKNIVTPRTP) sequences were synthesized by F-moc/HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate) strategy (Dr. A. Engström, Department of Medical and Physiologic Chemistry, University of Uppsala, Sweden). Peptides were purified by reversed-phase chromatography and, subsequently, analyzed by plasma desorption mass spectroscopy. The degree of purity of the used peptides was >99%. The N-terminally biotinylated peptide MBPGP72-85 (LPQKSQRSQDENPV) was a generous gift from Dr. G. Jung, Institute of Organic Chemistry, University of Tübingen, Germany.
mAbs and reagents
All mAbs used for FACS analysis were purchased from PharMingen
(San Diego, CA): FITC-labeled anti-rat TCR(A/B) (clone R73, mouse
IgG1,
), phycoerythrin (PE)-labeled anti-rat TCRBV8S2 (clone R78,
mouse IgG1,), PE-labeled anti-rat TCRBV10 (clone G101, mouse
IgG2a,), FITC-labeled anti-rat CD25 (IL-2R 
-chain, clone
WT.1, mouse IgG2a,), FITC-labeled mouse IgG1, (clone 107, 3), and
PE-labeled mouse IgG2a, (clone G155-178). The anti-rat TCRBV8S2
mAb (clone R78, mouse IgG1,) was also used for in vivo-depletion
studies. The hybridoma MRC-OX-6 (European Collection of Cell Cultures,
Salisbury, U.K.), secreting a mouse anti-rat RT1.B-specific Ab
(IgG1), was cultured in DMEM (Life Technologies, Paisley, Scotland)
supplemented with 10% FCS (Life Technologies), 1%
penicillin/streptomycin (Life Technologies), and 1% glutamine (Life
Technologies). The OX-6 Abs were purified on a protein G column
(Pharmacia, Uppsala, Sweden). Con A was purchased from Sigma (St.
Louis, MO).
Induction and evaluation of EAE
For induction of EAE, rats were injected intradermally at the base of the tail with a total volume of 200 µl of inoculum containing 200 µg of either MBPGP63-88 or MBPRAT63-88 in saline mixed (1:1) with CFA, which consisted of IFA (Sigma) and 1 mg of heat-inactivated Mycobacterium tuberculosis (strain H37 RA; Difco Laboratories, Detroit, MI). Immunizations were performed under inhalation anesthesia with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL).
Animals were clinically scored and weighed on a daily basis up to 25 days post-immunization (p.i.). Symptoms were scored as follows: grade 1, tail weakness or tail paralysis; grade 2, hind leg paraparesis; grade 3, hind leg paralysis; grade 4, complete paralysis (tetraplegy), moribund state, or death.
Fractionation and cultivation of mononuclear cells (MNCs) from LN, spleen, and blood
Under deep anesthesia, draining inguinal LN were dissected out and put in DMEM. MNCs were isolated by careful disruption of the LN; washed twice in DMEM; resuspended in complete medium (CM) containing DMEM supplemented with 1% rat serum, 1% penicillin/streptomycin, 1% glutamine, and 50 µM 2-ME (Life Technologies); and flushed through a 70-µm plastic strainer (Falcon; Becton Dickinson, Mountain View, CA). MNC from spleen were prepared in the same way as for LN with the difference that RBC were lysed with lysing buffer, consisting of 0.15 M NH4Cl, 1 mM KHCO3 and 0.1 mM Na2 EDTA adjusted to pH 7.4.
MNC were cultured at a concentration of 2 x 106 cells/ml in 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) with 100 µl of cell suspension per well at 37°C in a humidified atmosphere containing 5% CO2. For each rat/Ag combination, MNC were cultured for 72 h, washed twice in DMEM, adjusted to a concentration of 20 x 106 cells/ml, and immunostained for FACS analysis.
Blood was drawn by heart puncture under deep anesthesia and collected in heparinized blood containers (Becton Dickinson). Blood MNC were then isolated on Lymphoprep density gradients (Nygaard, Oslo, Norway), washed twice in DMEM, and resuspended in CM at a concentration of 20 x 106 cells/ml for FACS analysis.
Recovery of MNC from CNS
Deeply anesthetized animals were perfused with 75 ml of PBS. CNS was carefully dissected out and transferred to a 50-ml centrifugation tube (Falcon) containing 35 ml of DMEM, gently agitated to release cells into the medium, and floated through a 70-µm plastic strainer (Falcon). Contaminating RBC were removed through Lymphoprep density gradient centrifugation, and MNC were collected from the interphase, washed twice in DMEM, and resuspended in CM for immunostaining.
Proliferation assay
All proliferative experiments were performed in triplicates in 96-well round-bottom microtiter plates. A total of 2 x 105 MNC/well were cultivated in CM with or without the relevant Ags for 60 h and, subsequently, pulsed with 0.5 mCi of [3H]TdR (Amersham, Buckinghamshire, U.K.) per well for an additional 12 h. DNA was collected on glass fiber filters (Skatron, Sterling, VA) and [3H]TdR incorporation was measured in a beta counter (Beckman, Palo Alto, CA).
Enumeration of cells secreting Ag-specific IFN-
To enumerate T cells secreting IFN- after Ag exposure, the
ELISPOT method was used (23, 24). Nitrocellulose-bottomed 96-well
plates (MAHA; Millipore, Molsheim, France) were coated with the mAb DB1
(a generous gift of Dr. Peter van der Meide, TNO Primate Centre,
Rijswijk, the Netherlands), which reacts with rat IFN-. Following
washing with PBS, the plates were blocked with DMEM containing 5% FCS
(Life Technologies). A total of 4 x 105 cells
per well in 200 µl of CM were added to the plates and incubated for
48 h at 37°C in a humidified atmosphere containing 5%
CO2. For each Ag, triplicate determinations were performed.
Afterward cells were discarded and plates were washed four times with
PBS. Secreted and bound IFN- was visualized with biotinylated DB12
(also a generous gift of Dr. Peter van der Meide), which has another
binding site on IFN- than DB1, avidin-biotin peroxidase (Vector
Laboratories, Burlingame, CA) and, subsequently, by staining with
carbazole (Sigma).
Immunostaining and flow cytometry
For flow cytometric analysis, cells isolated from blood, LN, and CNS as well as cultured cells were immunostained in 96-well microtiter plates. Predetermined optimal concentrations of mAbs were added to each cell suspension (50 µl). Plates were then incubated in the dark on ice, washed twice in PBS, and resuspended in 200 µl of PBS containing 1% paraformaldehyde. Finally, samples were analyzed by use of a Becton Dickinson FACSort flow cytometer.
To obtain large enough numbers of relevant TCRBV+ T cells for reliable analysis of CD25 co-expression, specific acquisition gates were used. This procedure allowed saving of events signifying lymphocytes expressing either TCRBV8S2 or TCRBV10 products, as defined by light scatter properties in combination with positive signals visualized in the second fluorescence channel (FL2). By this approach, 2000 to 5000 cells per sample of each specific TCRBV+ T cell subset could be analyzed for CD25 co-expression in the first fluorescence channel (FL1). In general, CNS samples contained large amounts of cellular debris that frequently co-localized with double-stained T lymphocytes visualized in the regular flow cytometry plots. To sort out irrelevant events (debris), the third fluorescence channel (FL3) was utilized (26). In the FL1 vs FL3 plot, TCR(A/B)+ T cells could be gated and these events were then further analyzed for expression of specific TCRBV products in an FL1 vs FL2 plot.
Frequencies of the relevant TCRBV+ T cell subsets are represented as percentages of the total number of TCR(A/B)+ T cells. In most T cell samples, the relevant TCRBVs and CD25 were co-expressed at a level equal to that of the negative controls. Hence, to define CD25+ cells, the fluorescence intensity-level marker was set directly above the negative population. The same marker position was consistently used for both TCRBV+ T cell subsets.
Depletion of TCRBV8S2+ T cells
For depletion studies, a total volume of 200 µl containing 150 µg of the R78 mAb in PBS was injected i.p. in each rat on day 8 p.i. The degree of depletion of TCRBV8S2+ T cells was tested on day 9 p.i. by FACS analysis of blood cells from each treated rat.
Purification of RT1.Bl molecules
RT1.Bl molecules were purified from LEW rat LN, thymic, and splenic tissues by affinity chromatography using the OX-6 mAb (anti-RT1.B) coupled to CNBr-activated Sepharose-4B (Pharmacia) (27). Purity of the eluted proteins was assessed by SDS-PAGE and subsequent silver staining.
Peptide binding assay
For the peptide binding affinity assay, biotinylated MBPGP72-85 was used as the reference peptide. Briefly, RT1.Bl molecules (200 nM) were incubated with the reference peptide (500 nM) in the presence of various concentrations of each of the unlabeled relevant peptides, MBPGP63-88, MBPRAT63-88, and MBPGP/RAT89-101. The binding buffer consisted of 2 mM EDTA, 25 mM Na2CO3, 50 mM Tris/HCL, 0.01% azide, 0.1 mM PMSF, and 0.1% Nonidet P-40 (Boehringer, Mannheim, Germany), titrated to pH 5 with a citrate solution. After 72 h of incubation at room temperature, the peptide-MHC complexes were quantified by ELISA (28). The signal intensity of the enzymatic reaction was directly proportional to the concentration of the newly formed peptide-MHC complexes under the conditions used. The relative binding affinity of the relevant peptide is expressed as the peptide concentration needed for 50% inhibition of the binding of the reference peptide (IC50). IC50 values were determined in three separate experiments.
Statistical analysis
Student’s t test was used for normally distributed variables. When the data did not fulfill the criteria of being normally distributed, nonparametric statistics (Mann-Whitney U test) was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT1 congenic LEW rats immunized with highly purified MBP peptides,
MBPGP63-88 or MBPRAT63-88, were monitored
for EAE disease courses over a period of 25 days (Fig. 1). Following immunization with
MBPGP63-88, LEW and LEW.1AV1 rats displayed initial overt
clinical signs on average at day 10 p.i. (Fig. 1A). Both rat strains generated a self-limiting
monophasic disease profile, reaching a mean maximum disease score of
grade 3. The semiresistant LEW.1W rat strain also developed a
monophasic disease profile, but with a later onset (day 14 p.i.)
and a lower mean maximum disease score (grade 1.6). Moreover, LEW rats
immunized with MBPRAT63-88 elicited a clinical course
similar to that obtained from immunization with MBPGP63-88
(Fig. 1B). None of the CFA-injected controls
exhibited any signs of EAE.
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Next, we determined the relationship between the specific set of expressed MHC allelic products and the relative number of activated TCRBV8S2+ T cells in MBP peptide-induced EAE. RT1 congenic LEW, LEW.1AV1, and LEW.1W rats were immunized with MBPGP63-88 and, in addition, LEW rats with MBPRAT63-88. Because of the known bias toward preferential TCRBV8S2 usage in LEW rat EAE, the activation state within the respective TCRBV8S2+ T cell subset was analyzed for CD25 co-expression on day 12 p.i. Since TCRBV10+ T cells have never been reported to be expanded or retracted during anti-MBP responses in the rat, each tested cell population was double stained in parallel with anti-TCRBV10 and anti-CD25 mAbs as control.
Within both the PBL- and the LN-derived T cell pool from
MBPGP63-88-primed LEW rats, the number of
TCRBV8S2+ T cells co-expressing CD25 was significantly
increased (p < 0.0003), whereas the frequency
of TCRBV10+/CD25+ T cells was unaffected (Fig. 2). Such a selective, increased
expression of CD25 in the TCRBV8S2+ T cell subset was not
observed in LEW rats immunized with MBPRAT63-88. Likewise,
MBPGP63-88-primed LEW.1AV1 and LEW.1W rats, CFA-injected,
and naive controls exhibited unchanged frequencies of both the
TCRBV8S2+/CD25+ and the
TCRBV10+/CD25+ T cell subset. Due to the
delayed onset of clinical signs in the LEW.1W strain (Fig. 1
A), we assessed CD25 expression on
TCRBV8S2+ T cells also on day 16 p.i. No preferential
activation was observed (data not shown).
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We also examined whether the specific activation of
TCRBV8S2+ T cells in LEW rats immunized with
MBPGP63-88 caused measurable expansions. On day 12
p.i., a significant, albeit modest expansion of TCRBV8S2+ T
cells was demonstrated in PBL from the LEW rat immunized with
MBPGP63-88 (p < 0.05) (Fig. 3, A and B),
but not in the draining LN-derived T cell population (Fig. 3B). Importantly, neither LEW rats immunized with the
autologous MBPRAT63-88 peptide nor LEW.1AV1 and LEW.1W rats
immunized with MBPGP63-88 exhibited preferential expansions
of TCRBV8S2+ T cells. The frequencies of
TCRBV10+ T cells in immunized animals were
indistinguishable from those observed in CFA-injected and naive
controls. As expected, LEW.1W rats immunized with
MBPGP63-88, analyzed on day 16 p.i., did not display
expansions within either of the TCRBV8S2+ and
TCRBV10+ T cell subsets (data not shown).
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In view of the over-representation of activated
TCRBV8S2+ T cells in blood from
MBPGP63-88-primed LEW rats, we investigated the degree of
passage through the blood-brain barrier into the CNS compartment. The
relative presence of TCRBV8S2+ T cells was thus determined
at the site at which the autoantigenic MBP target molecule is exposed.
LEW, LEW.1AV1, and LEW.1W rats were immunized with
MBPGP63-88 or MBPRAT63-88, and CNS-infiltrating
cells were recovered on day 12 p.i. Almost 30% of T lymphocytes
from the CNS of LEW rats immunized with
MBPGP63-88 expressed TCRBV8S2
(p < 0.0003) (Fig. 4). This relative frequency of
CNS-associated TCRBV8S2+ T cells was markedly higher than
that recorded in LEW rat T cell populations derived from blood
(p < 0.0003) and LN (p
< 0.0003). Importantly, no increased frequency of
TCRBV8S2+ T lymphocytes was recorded in LEW rats immunized
with the autologous MBPRAT63-88 peptide, or
LEW.1AV1 and LEW.1W rats immunized with MBPGP63-88. MNC
recovered from LEW.1W rats on day 16 p.i. showed normal
frequencies of TCRBV8S2+ and TCRBV10+ T cells
(data not shown).
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Since LEW rats had shown marked differences after immunization
with either MBPGP63-88 or MBPRAT63-88 in
the preferential activation and recruitment of TCRBV8S2+ T
cells in vivo, we also tested for cross-reactivity with both peptides
in vitro. Cells from draining LN of LEW rats immunized either with
MBPGP63-88 or MBPRAT63-88 were isolated on day
12 p.i. and cultured with the MBP63-88 peptides, Con A, or medium
alone. Irrespective of choice of immunogen, over 80% of Con
A-stimulated TCRBV8S2+ and TCRBV10+ T cells
co-expressed CD25 (data not shown). After immunization with
MBPGP63-88 and in vitro restimulation with the same
peptide, the number of TCRBV8S2+ T cells co-expressing CD25
was selectively increased as opposed to that of the
TCRBV10+/CD25+ T cell subset
(p < 0.01). Interestingly, this was also true,
but to a lower extent, after culture with MBPRAT63-88
(p < 0.05) (Fig. 5A). Thus,
TCRBV8S2+ T cells preferentially expanded due to the
heterologous MBPGP63-88 peptide, which could recognize and
maintain an increased CD25 expression in response to the syngeneic
MBPRAT63-88 peptide. No significant changes in expression
of CD25 on TCRBV8S2+ cells were recorded after secondary
stimulation in vitro with MBPRAT63-88 and
MBPGP63-88 of cells from MBPRAT63-88-primed LEW
rats (Fig. 5B).
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A). T
cells from MBPRAT63-88-primed LEW rats proliferated roughly
at similar levels after culture with MBPGP63-88 and
MBPRAT63-88 (Fig. 6B). Even more
conspicuous and discriminating MBP peptide responses were recorded by
using production of the proinflammatory cytokine IFN- as the
outread. Lymphoid cells from MBPGP63-88-immunized LEW rats
showed high numbers of IFN--secreting cells after restimulation in
vitro with MBPGP63-88. Such cells were detected, although
at lower numbers, also after culture with MBPRAT63-88 (Fig. 6C). MBPRAT63-88-immunized LEW rats had
high numbers of IFN--secreting cells already at low peptide
concentrations after culture with MBPGP63-88, while
MBPRAT63-88 stimulated stronger at higher peptide
concentrations (Fig. 6D).
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To assess the clinical importance of TCRBV8S2+ T
cells, MBPGP63-88-primed LEW and LEW.1AV1 rats
and, in addition, MBPRAT63-88-primed LEW rats were injected
i.p. with anti-TCRBV8S2 mAb on day 8 p.i. Depletion was determined
by FACS analysis of T cells from blood on day 9 p.i. In all cases, the
reductions in numbers of TCRBV8S2+ T cells exceeded 50%
and the TCR densities on the remaining TCRBV8S2+ T cells
were markedly down-regulated (data not shown). As expected, the
TCRBV10+ T cell subset was unaffected (data not shown).
Following injections with the anti-TCRBV8S2 mAb, only LEW rats
pre-immunized with MBPGP63-88 exhibited mitigation of the
EAE disease course, as distinguished from both LEW rats pre-immunized
with MBPRAT63-88 (p <
0.05) and LEW.1AV1 rats pre-immunized with MBPGP63-88
(p < 0.05) (Fig. 7).
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The RT1.Bl molecule is the restriction element
for most encephalitogenic T cells in MBP63-88-induced EAE in LEW rats
(29). To test whether the affinities of MBPGP63-88 vs
MBPRAT63-88 for the groove of the RT1.Bl
molecule may differ, which would at least partly offer an explanation
for the variations in recruitment patterns of TCRBV8S2-expressing T
cells, we performed competitive binding studies including the two
MBP63-88 peptides and OX-6 affinity-purified RT1.Bl
molecules (Fig. 8). RT1.Bl
molecules were incubated with a biotinylated reference peptide
(MBPGP72-85; 500 nM) and serial dilutions of the respective
relevant peptide (5 nM-250 µM). Practically equally strong inhibition
of binding was observed for both MBP63-88 peptides (IC50:
MBPGP63-88, 2.5 µM; MBPRAT63-88, 0.7 µM).
In comparison, the MBPRAT/GP89-101 peptide, which has been
reported to associate with the RT1.Dl allelic product (30),
showed weak competitive capacity and only at high molar concentrations
(IC50>250 µM).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our flow cytometric approach confers improved precision and sensitivity on quantitative analyses of activation and expansion levels of T cell subsets expressing particular TCRBVs. It also has the advantage that the results would not be biased by strong selective pressures that may affect data obtained from long-term growth of MBP-specific T cell lines and clones, hitherto used for characterizations of TCR repertoires (11). In draining LN and blood from LEW rats immunized with MBPGP63-88, we detected increased numbers of TCRBV8S2+/CD25+ T cells. Additionally, in the blood, but not in the LN, significant expansions of TCRBV8S2+ T cells were detected and, interestingly, roughly 30% of the CNS-derived T lymphocytes expressed the TCRBV8S2 product. These observations indicate that Ag-specific activation of TCRBV8S2+ T cells in the draining LN leads to a ready release of these lymphocytes into the bloodstream. On passing through CNS vessels, and because they are in an activated state expressing appropriate sets of adhesion molecules, the TCRBV8S2+ T cells fulfill the requirements for being able to cross the blood-brain barrier and thus accumulate at the site at which MBP is exposed (1). In contrast to meningeal and cerebrospinal fluid-derived T cells, a considerable quantity of T cells in parenchymal CNS lesions appears to undergo Ag-specific apoptosis (33, 34), which may explain why only a minority of infiltrating cells recovered from the CNS parenchyma is Ag-specific (35). The fact that depletion of TCRBV8S2+ T cells led to amelioration of EAE (Ref. 36, and the present study) and that our CNS data are based on cells recovered from surface areas of the CNS and the meninges, indicate that a substantial fraction of CNS-associated T cells might be encephalitogenic and Ag specific.
The MBPGP63-88 and MBPRAT63-88 peptides differ in three amino acid positions. Positions 63 and 64 contain alanine residues in the MBPGP63-88 sequence, whereas MBPRAT63-88 has histidine at position 63 and threonine at position 64. Both positions are located outside the minimal core region of the sequence important for induction of EAE (29). Inside the core region, at position 79, threonine (T79) in MBPRAT63-88 is replaced by serine (S79) in MBPGP63-88. Consequently, this single amino acid replacement most likely constitutes the basis of the differential MBP63-88-specific TCRBV repertoires. In support of this inference, it has previously been reported that a variant of the moth cytochrome c-derived 88-103 peptide, in which glutamate is substituted for threonine in position 102, converted a T cell response characterized by TCRBV3 overusage into a T cell response encompassing principally normally distributed TCRBV products (18).
Potentially, a reciprocal amino acid exchange in a given peptide sequence can affect the distribution of the epitope-specific TCR repertoire by contributing to differences either in binding affinities of the two peptides to the restricting MHC class II molecule or in selective involvement in recognition of the respective relevant amino acid, exposed as a TCR contact residue. The ligand-binding motif of the RT1.Bl molecule has recently been defined by combining the pool sequencing approach with sequencing of individual ligands (37). Interestingly, the side chains of the two alternative core amino acids of the MBP63-88 sequence, T79 and S79, both appear to qualify as anchor residues for pocket 6 of the allele-specific RT1.Bl groove. However, another recently published study, in which a panel of alanine-substituted MBPGP72-85 analogues was tested for each member’s relative binding strength to the RT1.Bl motif, indicated that T79 and S79 would rather act solely as TCR contact residues (38). Nevertheless, despite the fact that the single T79/S79 substitution has to be considered conservative and that competitive binding studies of MBPGP63-88 and MBPRAT63-88 excluded differences in their affinities for the RT1.Bl molecule, the two alternative peptides have distinct capacities for evoking differential TCR repertoire patterns. It can thus be deduced that an utmost minimum structural difference in the MBP63-88 core sequence may lead to radical conformational changes in the TCR-identifiable part of the peptide/RT1.Bl complex. On the assumption that T79 and S79 interact with pocket 6 of the RT1.Bl groove, their differential contribution to the three-dimensional structure of the peptide/RT1.Bl complexes can be accounted for by distinct specific effects on the orientation of the identical flanking core TCR contact amino acids of the MBP63-88 peptide. Alternatively, though less likely, T79 and/or S79 may not only be involved in an anchoring function but also be partly exposed outwards, allowing them to act as direct TCR contact units. However, ultimate elucidation of the exact role of each amino acid core residue of MBPGP63-88 and MBPRAT63-88, respectively, in the TCR/peptide/RT1.B trimolecular complex awaits its resolution, possibly by virtue of employing x-ray crystallography.
The peripheral TCR repertoire is established by the effects of phenomena such as intra- and extrathymic selection events (39), multiple allelism of TCR-coding gene segments (40), and endogenous expression of superantigens (41). In addition, incessant challenges by exogenous immunogens in the periphery impose continuous modifications on the composition of the pre-immune TCR repertoire (42). By the use of inbred MHC congenic LEW rats, kept under identical physical conditions, we can preclude germline-encoded TCR polymorphism, occurrence of endogenous superantigens, and unintentional environmental influences from acting as factors contributing to the over-representation of the TCRBV8S2+ T cell subset in the MBPGP63-88-primed LEW rat.
The capacity for eliciting preferential peripheral activation of MBPGP63-88-specific TCRBV8S2+ T cells may depend on central tolerance induction. Since only LEW rats with intact thymi exhibit preferential TCRBV8S2 usage (39), it can be presumed that the autoreactive MBPRAT63-88-specific TCR repertoire is shaped to a high degree by self-peptides presented by RT1.Bl molecules on thymus-associated APCs during the course of T cell maturation (43). The tolerogenic peptides should originate from the self-MBP itself (44) and/or other self-molecules, such as RT1l Ags, sharing homology sequences with self-MBP (45). According to the affinity/avidity selection model of thymocyte development, it can be argued that a substantial portion of the TCRBV8S2+ pre-T cells escape negative selection by binding at low avidity to the self-peptide/RT1.Bl complex (46, 47). These pre-T cells will eventually reach a full immunocompetent state, thus constituting part of the naive peripheral T cell pool, which can become specifically activated through high-avidity binding to the nonself-MBPGP63-88/self-RT1.Bl complex. After immunogen challenge, the immune system operates by favoring T cells with high avidity for Ag (48). As a consequence, preferential recruitment of MBPGP63-88-specific T cells expressing TCRBV8S2 may prevail.
Differences in disconcordance rates for MS within monozygotic (70%) and dizygotic (95%) twins, respectively, argue for involvement of both genetic predispositions and environmental factors in contributing to the probability of developing MS (49). Various infectious agents have been hypothesized to be a crucial etiologic factor. A plausible mechanism of induction of MS may thus be based on a multistep molecular mimicry hypothesis, in which microorganisms trigger autoimmune reactions by providing immunogenic nonself-peptides, cross-reactive with self (50, 51). Since MBPGP63-88 can be considered the equivalent of any foreign peptide sharing homology sequences with a self-molecule, the LEW rat EAE may be a valid model for studying the T cell-related immunopathogenicity of MS. Admittedly, the situation in humans may be more complex in view of its chronicity and B cell-mediated pathogenesis (52). Our LEW EAE data suggest that thymic selection over the RT1l-encoded allele-specific molecules in conjunction with the collection of available self-peptides shapes a peripheral pre-immune TCR repertoire encompassing low-avidity anti-self-TCRBV8S2+ T cells. Following systemic challenge, these T cells will become prevalently activated and expanded through selective high-avidity recognition of the MBPGP63-88/RT1l class II complex. This series of events may result in autoaggressive cross-reactivity with the MBPRAT63-88/RT1l class II complex, thus being instrumental for induction of EAE. Indeed, we observed that MBPGP63-88-primed TCRBV8S2+ T cells responded to MBPRAT63-88 in vitro, and there were immune cross-reactivities both with proliferation and proinflammatory cytokine production. A similar multistep scenario may underlie the skewed TCRBV repertoire described in certain HLA-Dw2+ MS patients (7, 8, 9). By cross-reactivity with immunogenic peptides derived from common bacteria and viruses, low-avidity, TCR repertoire-restricted self-MBP-reactive T cells may become activated and expanded (50, 51). Thus, as in the LEW rat EAE model, nonself, self-mimicking exogenous Ag-derived sequences would participate in peptide/MHC complexes recognized at high avidity by autoreactive encephalitogenic T cells expressing particular TCRBVs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert Weissert, Neuroimmunology Unit, Center of Molecular Medicine L8:04, Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; MS, multiple sclerosis; MBP, myelin basic protein; LEW, Lewis; GP, guinea pig; LN, lymph node; MNC, mononuclear cell; p.i., post-immunization; IC50, 50% inhibitory concentration; PE, phycoerythrin; CM, complete medium; FL, fluorescence channel; T79, threonine at position 79; S79, serine at position 79; ELISPOT, enzyme-linked immunospot.
Received for publication June 4, 1997. Accepted for publication October 1, 1997.
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and Vß chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different. J. Exp. Med. 169:27.ß+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis. Eur. J. Immunol. 26:1328.[Medline]
CDR3-specific motif associated with Lewis rat autoimmune encephalomyelitis and basic protein-specific T cell clones. J. Immunol. 158:5472.[Abstract]
. Pedersen, E. J. Roosenboom, W. Hensen, W. van Eden, S. Buus. 1994. Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules. Int. Immunol. 6:751.This article has been cited by other articles:
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  N. Fissolo, S. Haag, K. L. de Graaf, O. Drews, S. Stevanovic, H. G. Rammensee, and R. Weissert Naturally Presented Peptides on Major Histocompatibility Complex I and II Molecules Eluted from Central Nervous System of Multiple Sclerosis Patients Mol. Cell. Proteomics, September 1, 2009; 8(9): 2090 - 2101. [Abstract] [Full Text] [PDF]
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 D. Zhou, R. Srivastava, S. Nessler, V. Grummel, N. Sommer, W. Bruck, H.-P. Hartung, C. Stadelmann, and B. Hemmer Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis PNAS, December 12, 2006; 103(50): 19057 - 19062. [Abstract] [Full Text] [PDF]
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 R. Weissert, K. L. de Graaf, M. K. Storch, S. Barth, C. Linington, H. Lassmann, and T. Olsson MHC Class II-Regulated Central Nervous System Autoaggression and T Cell Responses in Peripheral Lymphoid Tissues Are Dissociated in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., June 15, 2001; 166(12): 7588 - 7599. [Abstract] [Full Text] [PDF]
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 H. Hammarberg, O. Lidman, C. Lundberg, S. Y. Eltayeb, A. W. Gielen, S. Muhallab, A. Svenningsson, H. Linda, P. H. van der Meide, S. Cullheim, et al. Neuroprotection by Encephalomyelitis: Rescue of Mechanically Injured Neurons and Neurotrophin Production by CNS-Infiltrating T and Natural Killer Cells J. Neurosci., July 15, 2000; 20(14): 5283 - 5291. [Abstract] [Full Text] [PDF]
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A. Lobell, R. Weissert, S. Eltayeb, C. Svanholm, T. Olsson, and H. Wigzell Presence of CpG DNA and the Local Cytokine Milieu Determine the Efficacy of Suppressive DNA Vaccination in Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 1999; 163(9): 4754 - 4762. [Abstract] [Full Text] [PDF]
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R. B. Smeltz, M. H. M. Wauben, N. A. Wolf, and R. H. Swanborg Critical Requirement for Aspartic Acid at Position 82 of Myelin Basic Protein 73-86 for Recruitment of V{beta}8.2+ T Cells and Encephalitogenicity in the Lewis Rat J. Immunol., January 15, 1999; 162(2): 829 - 836. [Abstract] [Full Text] [PDF]
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R. Weissert, A. Lobell, K. L. de Graaf, S. Y. Eltayeb, R. Andersson, T. Olsson, and H. Wigzell Protective DNA vaccination against organ-specific autoimmunity is highly specific and discriminates between single amino acid substitutions in the peptide autoantigen PNAS, February 15, 2000; 97(4): 1689 - 1694. [Abstract] [Full Text] [PDF]
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