1 Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan 2 Laboratory of Developmental Immunology, Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511, Japan 3 NeuroResource, Institute of Neurology, London WC1N 1PJ, UK 4 Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan
Correspondence to: T. Yamamura; E-mail: yamamura{at}ncnp.go.jp
Transmitting editor: L. Steinman
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Abstract |
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Keywords: chronic inflammatory demyelinating polyneuropathy, invariant V7.2J
33 T cell, multiple sclerosis, NKT cell, single-strand conformation polymorphism
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Introduction |
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Several lines of evidence indicate that the T cell repertoire in humans contains invariant T cells distinct from CD1d-restricted NKT cells (2,57). It has recently been proposed that T cells expressing an invariant AV19AJ33 TCR (the canonical hV7.2J
33 or mV
19J
33 TCR rearrangement) represent a second invariant T cell subset that would develop in the absence of CD1d (811). Independent studies by Lantz et al. (8,11) and Shimamura et al. (9,10) have shown that the V
19J
33 T cells do not require CD1d for their development and expansion in vivo. They are present in TAP-1 knockout mice (8), but are absent in ß2-microglobulin-deficient mice (8,9), suggesting that they probably recognize a non-peptide antigen associated with a non-classical MHC class Ib molecule other than CD1d. Although surface phenotypes of the invariant T cells remain to be fully characterized, the V
19J
33 T cells were enriched in the NK1.1+CD3+ population isolated from CD1d-deficient mice (9), allowing us to refer to the invariant T cells as a second type of NKT cells. Very recently, Lantz et al. (11) have reported that the invariant T cells are enriched in the gut lamina propria, and that their selection and/or expansion require B cells and commensal flora. The distribution of the cell population would indicate that the novel invariant T cells are possibly involved in the host response at the site of pathogen entry. Finally, MR1, a non-classical class I-related molecule (12), has been identified as a restriction element involved in the selection of the V
19J
33 T cells.
It is of note that the novel MR1-restricted NKT cells share several characteristics with CD1d-resticted NKT cells. For example, both of the invariant populations can be detected in unrelated individuals with different ethnic backgrounds and a majority of the cells resides in the CD4CD8 T cell population (7,8). As noted, the TCR chain represents the canonical V
J
rearrangement, whereas the ß chain sequence is restricted by the use of particular Vß segments (hVß11 for CD1d-restricted NKT cells and hVß2 and 13 for MR1-restricted T cells). Both of the invariant populations have a natural memory phenotype (CD44high). Furthermore, the invariant TCR sequences as well as their restriction elements (CD1d and MR1) are highly conserved across the species. These characteristics are consistent with the idea that the invariant lymphocyte populations might exert an immediate response against phylogenetically conserved antigens at the frontier between innate and adaptive immunity (1). In accordance with this idea, CD1d-restricted NKT cells produce large amounts of IL-4 and IFN-
within hours of TCR engagement (3,1315). Through the explosive release of cytokines and chemokines, they are capable of initiating a cascade of immunological events involved in regulation of autoimmunity and vital defense against microbial agents or tumor cells (1). In contrast, very little is known about the function of the MR1-resricted T cells. Although accumulating data indicate that they may promptly respond to antigen by producing IL-4 (Shimamura et al., unpublished observations), their ability to produce cytokines and chemokines needs to be systematically analyzed in the coming years.
A numerical reduction or functional alterations in CD1d-rectricted NKT cells bearing the V24J
Q invariant chain have been documented in various human autoimmune diseases (3,4). Using three different approaches, the RT-PCR single-strand conformation polymorphism (SSCP) clonotype method (16), anti-V
24 and anti-Vß11 antibodies, and glycolipid-loaded CD1d tetramers (17), we have recently revealed that the V
24J
Q NKT cells are greatly reduced in number in the peripheral blood of multiple sclerosis (MS), a putative autoimmune disease mediated by Th1 autoimmune T cells (18,19). We also examined the distribution of the V
24J
Q NKT cells in the central nervous system (CNS) lesions from patients with MS as well as in the peripheral nerve biopsy samples derived from chronic inflammatory demyelinating polyneuropathy (CIDP) patients (16). Although expression of non-invariant V
24 rearrangement was ubiquitous in the CNS samples of MS, the V
24J
Q clonotype specific for the NKT cells appeared to be missing in most of the CNS lesions. In contrast, the NKT cell clonotype could be readily detected in a large majority of the biopsy samples of CIDP, which is a chronic demyelinating disease of the peripheral nervous system (PNS) with a presumed autoimmune origin (20).
Using the same samples previously analyzed for the V24J
Q NKT cells (16), we conducted experiments to address the following questions. (i) Are the invariant V
7.2J
33 T cells reduced in the peripheral blood of MS? (ii) Are they involved in the inflammatory lesions of MS and CIDP or could they be missing from the lesions? (iii) Are they present in the cerebrospinal fluid (CSF) derived from MS? Here, we report that unlike the V
24J
Q NKT cells, the V
7.2J
33 T cells are not reduced in the peripheral blood of MS. More strikingly, they were detected in some of the pathological samples obtained from MS and in the majority of nerve biopsy samples from CIDP, and could be detected in the CSF samples from MS. Comparison with other T cell populations indicated a selective accumulation of the V
7.2J
33 T cells in the inflammatory lesions. We propose that the invariant V
7.2J
33 T cells do not only play a role in protection against pathogen entry in the gut (11), but also in the regulation of autoimmune tissue inflammation.
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Methods |
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Peripheral blood mononuclear cells (PBMC), CSF, CNS and PNS samples
Heparinized blood (20 ml) was taken and PBMC were isolated by Ficoll density gradient centrifugation. CSF samples were obtained within 1 week after the onset of exacerbation. The sural nerve biopsy samples were taken for other diagnostic purposes with standard procedure (23). Samples were snap-frozen and were stored at 70°C along with frozen brain samples until analysis had been performed. The histopathological characterization of the MS plaques was performed as described previously (24).
Isolation of mRNA and synthesis of cDNA
mRNAs were isolated from 107 PBMC and from CSF sediment using the QuickPrep Micro mRNA purification kit (Amersham-Pharmacia Biotech, Uppsala, Sweden). The air-dried pellet was resuspended in 20 µl of RNase-Free Water and used for cDNA synthesis by the First-Strand cDNA synthesis kit (Amersham-Pharmacia Biotech, Uppsala, Sweden) using oligo-dT as primer. The mRNAs previously isolated from autopsy CNS samples and sural nerve biopsy samples (16) were also converted to cDNA by the same approach.
SSCP analysis
We conducted RT-PCR SSCP following amplification with V and C
primers as described previously (16,25). Primers and probes were designed based on the previously published sequences (2,3,5,8,26). To detect the V
7.2J
33 invariant chain, 1 µl of the diluted cDNA was used for each PCR reaction with V
7.2-specific sense primer (GTCGGTCTAAAGGGTA CAGT) and anti-sense C
-specific primer (CAGCTGAGAG ACTCTAAAT). cDNAs obtained from PBMC samples were amplified by PCR for 39 cycles, while cDNAs from autopsy/biopsy samples and CSF were amplified for 40 cycles. Quantities of 0.2 µg of sense and 0.2 µg of anti-sense primers (30 pmol) were added to 50-µl reactions containing 5 µl of 10 x ExTaqBuffer, dNTPs and 2.5 U of ExTaq DNA polymerase (Takara, Tokyo, Japan). Amplified DNAs were diluted (1:3) and heat-denatured. Aliquots of 4 µl of the diluted samples were electrophoresed in non-denaturing 4% polyacrylamide gel. DNAs were transferred to Immobilon-S (Millipore Intertech, Bedford, MA) and hybridized with biotinylated C
-specific (AAATATCCAGAACCCTGACCCTGCCGTGTACC), J
33-specific (TATCAGTTAATCTGGGGCGCTGGGACCAA GCT) or invariant V
7.2J
33-specific internal probe (TGTGC TGTGAGAGATAGCAACTATCAGTTAATCTG). To detect the V
24J
Q invariant chain, cDNAs were PCR amplified with V
24-specific sense primer (ACACAAAGTCGAACGGAAG) and C
-specific anti-sense primer (GATTTAGAGTCTCTCA GCTG), and then hybridized with a probe specific for the invariant V
24J
Q sequence (TGTGTGGTGAGCGACAGAG GCTCAACCCTG) as previously described (16).
SSCP clonotypes were visualized by incubation with streptavidin, biotinylated alkaline phosphatase and a chemiluminescent substrate system (Phototope; New England Biolabs, Bedford, MA). cDNAs for human IL-4 and IFN- were amplified by RT-PCR as described previously (16).
Real-time V7.2 clonotypic RT-PCR
Quantitative RT-PCR (LightCycler; Roche Molecular Bio chemicals, Mannheim, Germany) was performed with the V7.2 sense primer and with an anti-sense primer matching the CDR3
region of the V
7.2J
33 T cells (TGATAGTTG CTATCTCTCAC). An aliquot of 1 µl of the cDNA was amplified by PCR for 40 cycles using quantification with a commercial kit (LightCycler DNA Master SYBR Green I; Roche Molecular Biochemicals). Quantities of 0.2 µg of sense and 0.2 µg of anti-sense primers (30 pmol) were added to 50-µl reactions containing 5 µl of 10 x ExTaqBuffer, dNTPs and 2.5 U of ExTaq DNA polymerase (Takara). All PCR reactions were controlled by ß-actin expression (sense primer: AGAGA TGGCCACGGCTGCTT; anti-sense primer: ATTTGCGGTGG ACGATGGAG) (27). Based on the standard values of control samples, the relative expression for each sample was determined with the LightCycler software.
TCR DNA sequencing
In brief, small areas of the SSCP gel corresponding to the clonotypes of interest were cut out and DNAs were eluted as described previously (25). A second PCR was performed with the corresponding V-specific and C
-specific primers using the eluted DNAs as template. The PCR products were cloned into pCR 2.1-TOPO Vector using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and recombinants were sequenced using an ABI 377 DNA sequencer (Applied Biosystems, Foster City, CA).
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Results |
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Invariant V7.2J
33 T cells do not expand in response to
-galactosylceramide (
-GalCer)
V24V
11 NKT cells are known to proliferate in response to
-GalCer, which is a prototype ligand for the NKT cells (4,17). Additionally, we examined if the invariant V
7.2J
33 T cells respond to
-GalCer. In brief, PBMC from healthy individuals were stimulated with
-GalCer as previously described (17) and the
-GalCer-stimulated PBMC cultures were harvested at different time points for SSCP analysis. We observed that the invariant V
7.2J
33 clonotype would gradually diminish, while the V
24V
11 clonotype remarkably expanded shortly after stimulation with
-GalCer (data not shown). This result confirms that
-GalCer is not a stimulatory ligand for the invariant V
7.2J
33 T cells.
Invariant V7.2J
33 T cells are not reduced in the peripheral blood of MS
In previous studies, we have demonstrated that the V24J
Q NKT cells are remarkably reduced in the peripheral blood of MS, particularly in the remission phase (16,17). In fact, SSCP analysis for the V
24J
Q clonotype detected the NKT cell clonotype in all the HS (18 of 18, 100%) (16), but the clonotype was not found in any of the MS patients in remission (Table 1). Given the notable similarities between CD1d-restricted NKT cells and MR1-resricted T cells, we speculated that the invariant V
7.2J
33 clonotype might also be reduced in the peripheral blood of MS. However, the invariant clonotype could be readily detected by the SSCP method in 13 of 15 peripheral blood samples from MS patients in remission (Fig. 2 and Table 1). To further evaluate the frequency of the V
7.2J
33 T cells in PBMC of MS, we applied a real-time RT-PCR with V
7.2- and V
7.2J
33 clonotype-specific primers for quantitative analysis. Using this assay, we measured relative expression of the invariant V
7.2J
33 mRNA in the PBMC derived from five MS in remission and from five HS. As shown in Fig. 3, there was no significant difference between HS and the patients with MS. Taken together, we conclude that MR1-restricted T cells are conserved in number in the patients with MS.
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Accumulation of the invariant V7.2J
33 T cells in the autopsy CNS lesions of MS
Next we asked if the V7.2J
33 T cells may infiltrate into the autopsy CNS lesions of MS. Using the SSCP clonotype method, we analyzed 14 CNS lesions obtained from five autopsied cases with MS, including seven acute plaques, six subacute plaques and one chronic plaque. V
7.2+ TCR could be amplified by RT-PCR from seven out of the 14 lesions, although they were detected only in one of the six control CNS samples (Table 1). We found that all the V
7.2+ plaques (four acute plaques, two subacute plaques and a single chronic plaque) expressed the message for the invariant V
7.2J
33 sequence (Fig. 5, upper panel and Table 1). In contrast, as reported previously (16), whereas V
24+ TCR could be amplified from eight of the 14 MS autopsy lesions, only a single subacute plaque expressed the V
24J
Q clonotype, i. e. the invasion of V
24J
Q NKT cells was mainly restricted to the CIDP lesions, whereas the V
7.2J
33 T cells were also found in some CNS lesions from MS. To verify the postulate that the V
7.2J
33 T cells are involved in the pathology of MS, we also analyzed CSF samples obtained at an acute stage of MS. We were able to detect the V
7.2J
33 invariant sequence in 73% of the samples examined (Fig. 5, lower panel and Table 1), supporting that the invariant V
7.2J
33 T cells are a component of the CNS infiltrates in MS.
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Discussion |
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Here, we documented that the autopsy CNS lesions from MS as well as the biopsy PNS lesions from CIDP are infiltrated with the invariant V7.2J
33 T cells. We also showed that the invariant V
7.2J
33 T cells are present in a large majority of the CSF samples obtained at relapse phases of MS. In contrast, V
19+ TCR could not be detected in any of the affected tissues from MS or CIDP after PCR amplification, although they can be detected in all the PBMC samples. Although the function of the invariant V
7.2J
33 T cells remains elusive, the present results demonstrated for the first time to our knowledge that the novel invariant T cells (811) are present in autoimmune inflammation affecting the nervous system.
In a very recent report (11), Lantz et al. showed that the MR1-restricted invariant T cells are preferentially located in the gut mucosa and therefore proposed to name the population as mucosal-associated invariant T cells (MAIT). In support of the special role of this cell population in the mucosa, the number of the cells in the gut mucosa was greatly reduced in germ-free mice. This observation indicated the role of commensal flora for selection of the invariant T cells. Although an interpretation for this could be that the MR1-resricted T cells would recognize the exogenous antigen bound to MR1, analysis of the TT hybridomas showed that they could recognize MR1 directly in the absence of bound ligand (11). If this is indeed the case, the MAIT cells could be autoreactive to MR1. To accommodate the autoreactivity with the requirement for commensal flora, it could be speculated that MR1 expression may require some ligand derived from or induced by commensal flora. An alternative possibility is that microbial products may facilitate translocation of MR1 to the cell membrane.
Provided that the invariant V7.2J
33 T cells are generated or expanded in the mucosa, how would they accumulate into the inflammatory lesions of MS and CIDP? Although this remains a conundrum, we would speculate that inflammation-associated signals such as chemokines play a role in the initial step. In fact, CD1d-restricted NKT cells would behave like inflammatory cells and rapidly accumulate into certain granuloma lesions (30), and they could be detected in non-autoimmune inflammatory lesions (31,32). Given a number of similarities between V
7.2J
33 and V
24J
Q NKT cells, we would postulate that the V
7.2J
33 T cells also might be preferentially recruited to the inflammatory sites. To support this idea, non-autoimmune inflammatory lesions are reported to express the invariant V
7.2J
33 TCR (32).
The second critical step may be the interaction of the invariant T cells with B cells expressing MR1 in the site of lesions, given that the vast majority of the inflammatory lesions are infiltrated with B cells. We could expect the V7.2J
33 T cells to regulate local immune responses by producing cytokines. If IL-4 is the major cytokine produced by the novel invariant T cells, their interaction with B cells may lead to augmentation of antibody production. Even though the encephalitogenic Th1 autoreactive T cells are down-regulated by IL-4, the direct interaction between B cells and the V
7.2J
33 T cells may substantially augment the tissue damage or alter the type of lesions. However, if suppressive cytokines such as transforming growth factor-ß are the major products of the V
7.2J
33 T cells in vivo, they may down-regulate the B cells as well as inflammatory cells in the vicinity. Although we can only speculate about how they would deal with autoimmunity, it is possible that they would play an active role in the regulation of autoimmune inflammation. To verify this hypothesis, we need to systematically analyze the functions of the V
7.2J
33 T cells with regard to ligand recognition and cytokine production. It is also important to know if the presence or absence of the invariant T cells may correlate with the type of pathology. The present data indicate that it is indeed a rewarding attempt.
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Acknowledgements |
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Abbreviations |
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CIDPchronic inflammatory demyelinating polyneuropathy
CNScentral nervous system
CSFcerebrospinal fluid
HShealthy subject
MAITmucosal-associated invariant T cell
MSmultiple sclerosis
ONDother neurological disease
PBMCperipheral blood mononuclear cell
PNSperipheral nervous system
SSCPsingle-strand conformation polymorphism
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References |
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