close

Вход

Забыли?

вход по аккаунту

?

Common T-cell receptor V usage in oligoclonal T lymphocytes derived from cerebrospinal fluid and blood of patients with multiple sclerosis.

код для вставкиСкачать
Common T-cell Receptor Vp Usage in
Oligoclonal T Lymphocytes Derived from
Cerebrospinal Fluid and Blood of Patients
with Multiple Sclerosis
Soon Jin Lee, PhD, Kai W. Wucherpfennig, MD, PhD, Staley A. Brod, MD, Deborah Benjamin, BS,
Howard L. Weiner, MD, and David A. Hafler, MD
T-cell populations were investigated in the blood and cerebrospinal fluid of patients with multiple sclerosis and other
neurological diseases. Individual T cells were directly cloned from the cerebrospinal fluid and blood before in viuo
expansion, and their clonotypes were compared by Southern blot analysis of the rearrangement patterns of their T-cell
receptor p chain and y chain genes. This allowed the determination of whether two T cell clones shared the same Tcell receptor and thus arose from identical, clonally expanded (oligoclonal) progenitor T cells. As an extension of
previous studies, oligoclonal T-cell clones were identified in both cerebrospinal fluid and blood populations in 5 of 9
patients with inflammatory demyelinating disease among a total of 486 blood and cerebrospinal fluid T-cell clones. In
contrast, no clonally expanded T-cell populations were found among a total of 424 clones derived from either blood of
4 normal control subjects or blood and cerebrospinal fluid of 8 patients with other neurological diseases. Analysis of Tcell receptor V, genes among 4 oligoclonal T-cell populations derived from 3 patients with multiple sclerosis demonstrated common usage of the Vp12 gene segment. These data suggest that oligoclonal T cells share similar specificities
and that clonal expansion may have resulted from specific stimulation by an antigen. Moreover, identical clones
between blood and cerebrospinal fluid were observed in 3 of 9 patients with demyelinating disease, thus providing
further evidence of an equilibrium between peripheral and central nervous system immune compartments.
Lee SJ, Wucherpfennig KW, Brod SA, Benjamin D, Weiner HL, H d e r DA. Common T-cell receptor V,
usage in oligoclonal T lymphocytes derived from cerebrospinal fluid and blood of patients
with multiple sclerosis. Ann Neurol 1991;29:33-40
Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS)
{ 1, 23. Pathological studies have indicated that the majority of infiltrating cells at sites of active demyelination are activated T cells and macrophages { 3 ] . A
major hypothesis regarding the pathogenesis of multiple sclerosis is that T cells reactive with myelin protein
determinants migrate into the CNS and, either directly
or by recruitment of effector cells, mediate damage to
white matter tracts [4, 51. Thus, characterization of
immune T cells in the CNS and blood of patients with
multiple sclerosis is likely to be important in attempting to understand the pathogenesis of the disease.
It is now known that each T cell expresses a single
receptor that consists of either an a-p or y-6 heterodimer that recognizes antigen in association with major
histocompatibility gene complex proteins. The highly
diverse T-cell receptor (TCR) repertoire is generated
during T-cell ontogeny in the thymus by rearrangement of TCR genes, which consist of variable
(V), diversity (D), junctional
and constant (C) gene
regions {b}.Whether two T cells are the same and thus
derived from a common progenitor cell can be determined by analysis of these TCR gene rearrangements.
That is, Southern blot analysis of DNA from T cell
clones digested with different restriction enzymes and
probed with different TCR genes can identify two
clones that have a high probability of sharing the same
TCR gene rearrangements and thus can be assumed to
arise from the identical progenitor T cell [7].
If T-cell populations in the blood and cerebrospinal
fluid (CSF) of patients with multiple sclerosis are derived from a large number of progenitor cells, examination of TCR genes of many T-cell clones would
reveal different gene rearrangements. Alternatively,
there may be more restricted expansion of T cells in
From the Center for Neurologic Disease, Division of Neurology,
Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA.
Address correspondence to Dr HaAer, Center for Neurologic Disease, Brigham and Women’s Hospital, 75 Francis Street, Boston,
MA 02115.
Received Oct 19, 1989, and in revised form Apr
Accepted for publicatiun Jd 9, 1990.
u),
9 and Jul9, 1990.
Copyright 0 1991 by the American Neurological Association
33
Table 1, Oligoclonal T Lymphocytes fvom Patients with Multiple ScEerosis
T-cell Clonalitya
Patient
Disease
Multiple sclerosis
Gr
...
Ka
...
Mu
...
Po
...
Ca
...
Re
...
Da
...
Sh
...
Auf
...
Other neurological diseases
Ki
Myasthenia
sz
SSPE
Av
Lyme
Fe
Stroke
McK
NPH
Ga
GBS
Wi
SSPE
Ve
HZV
Normal controls
Pw
...
He
...
Ka
...
Ha
...
Age (yr)
Sex
34
F
F
M
F
M
39
38
51
22
55
26
40
5
30
20
60
65
65
52
16
63
42
28
30
36
M
E
F
F
F
F
M
F
F
F
F
M
Duration
of Disease
Oligoclonal
Bands
10 yr
8 Yr
8 mo
8 yr
5 Yr
10 yr
6 Yr
15 y t
4 mo
3 Yr
8 Yr
3 mo
1 mo
6 mo
4 mo
3 Yr
9 mo
Blood
Blood'
CSFb
& CSP
Totald
Yes
No
1 Band
No
No
No
Yes
No
Yes
2126
016
014 1
2/10
0133
18128
2
0
0
20154'
5/33'
ND
Yes
NO
No
0152
0119
0135
Oil 1
0114
NO
0116
0119
019
017
5127
0137
0130
0156
0140
0136
0118
2127
ND
019
018
012
012 5
No
Yes
No
ND
0126
0114
0133
0120
ND
F
...
...
I;
M
...
...
0166"
0128
...
...
012 8
M
...
...
0113
0178'
3
3160
0
4
0
0
0
0189
4/56
0155
0127
2/34
ND
0
0
0
0
0152
0128
014 3
ND
0
0113
0139
0126
0168'
ND
0120'
"Total number of oligoclonal T cells: barno% blood or CSF, 'berween blood and CSF, 'total among blood and CSF. See Results section for
description of clone patterns.
cPrcviously described clones [S}.
fPatietit with single episode of perivascular lymphocytic infiltrates and demyelination.
Myasthenia = myasthenia gravis; SSPE
subacute sclerosing panencephalitis; Lyme = Lyme meningoencephalitis; h'PH
hydrocephalus; GBS = Guillain Bar& syndrome; ND = not done; HZV = herpes zoster virus meningoencephalitis.
the immune system of patients with multiple sclerosis
resulting in either oligoclonal or possibly monoclonal
T-cell populations. Southern blot analysis of a large
number of T-cell clones derived from patients with
multiple sclerosis would then show common TCR
gene rearrangements. We investigated this in a previous study and found oligoclonal T-cell populations in
the CSF and blood of some but not all patients with
multiple sclerosis ES}. The observation that the T-cell
repertoire may be altered in autoimmune disease with
expansion of particular T-cell populations has subsequently been reported in other autoimmune diseases
r9, 101.
To determine whether oligoclonal T cells derived
from different patients share similar specificities of
their T-cell antigen receptor, Vp gene usage was determined by the polymerase chain reaction (PCR) by using a panel of TCR Vp primers specific for published
Vp families. Identification of a common TCR Vp gene
usage would suggest that the clonal expansion of these
cells is due to specific stimulation through the T-cell
34 Annals of Neurology Vol 29 No 1 January 1991
=
normal pressure
antigen receptor and that these cells may be important
in the iniciation or progression of the inflammatory
response leading to demyelination in the CNS.
Materials and Methods
Subjects
Patients with multiple sclerosis had clinically definite disease
by using standard criteria [ l l )with confirmation on magnetic
resonance imaging (MRI) scanning. The characteristics of the
patients are presented in Table 1. Patients with other neurological disease are also shown in Table 1 and include patients with myasthenia gravis, subacute sclerosing panencephalitis (SSPE) 123, Lyme meningoencephalitis (with pleocytosis
of 45 lymph~cyteslmm~
in CSF); Guillain-Barri. syndrome,
acute thromboembolic vascular disease of the brain (CVA),
normal pressure hydrocephalus, and varicella-zoster meningoencephalitis. Patients Gr, Ka, and Mu with multiple sclerosis and control subjects Wi, Ve, and Pw were previously
reported [8].Chronic progressive multiple sclerosis was
defined as a decline in the Kurtzke disability status scale in
the 9 months before venous and lumbar punctures [12]. The
2 subjects with relapsing-remitting W R ) multiple sclerosis
had acute clinical relapses within 2 weeks of venous and
lumbar punctures. Patient Au had a 4-month progressive
course of gait difficulty and behavioral changes with multiple
white matter lesions on MRI. CSF examination in this patient showed oligoclonal banding with normal immunoglobulin index, 19 lymph~cytesimm~,
and normal protein and
glucose. On brain biopsy, there was demyelination with
perivascular infiltrates of lymphocytes and monocytes. Peripheral blood and CSF was obtained from all patients after
informed consent.
Cell Preparation
Peripheral blood mononuclear cells (PBMC) were isolated
from heparinized venous blood by means of a Ficoll-Hypaque density gradient (Pharmacia Fine Chemicals, Piscataway, NJ), washed twice with Hanks balanced salt solution
(GIBCO, Grand Island, NY), counted, and resuspended in
standard media consisting of lo%, pooled human serum
(GIBCO, Pel Freeze, Arkansas) in RPMI, 2% glutamine
(GIBCO), and 1% penidhistreptomycin (GIBCO). CSF
was obtained by lumbar puncture within 2 hours of blood
drawing, and cells were prepared as previously described [S}.
T-cell Cloning
PBMC were directly cloned at less than one cell per well
with lo5 autologous irradiated (5,000 rads) PBMC and Phytohemagglutin P (PHA.P) (1.0 Fglml) (Wellcome, Dartford,
UK) in 96 well V-bottom plates. Forty-eight hours later, 0.1
ml of media containing either 10% interleukin (1L)-2 (delectinated T-cell growth factor that is column purified, from
ABI, Columbia, MD) and, in some patients, an additional 50
Uirnl of recombinant interleukin 4 (rIL-4) (Genzyme, Cambridge, MA) were added to each well. rIL-4 was used in
more recent clones to increase growth rate although cloning
efficiency was not changed f12). Cultures were fed with IL-2
and rIL-4 every 3 to 5 days until approximately day 12, when
all the wells are passed to 96 well U-bottom plates. Growthpositive wells were scored macroscopically by an inverted
microscope and transferred into V-bottom plates with 5,000
cloned T cells per well with lo5 allogeneic irradiated (5,000
rads) mononuclear cells (from leukopacs) and PHA.P (1.0
pg/ml) with IL-2 plus rIL-4. When there were approximately
20 to 100 x lo3 T cells per V-bottom well (usually 3-5
days), clones were transferred to flasks at a cell concentration
of 0.5 x lo6 cellslml. T-cell clones were restimulated every
10 to 14 days, as previously described, in V-bottom plates
with 5,000 cloned T cells per well, 10' allogeneic irradiated
mononuclear cells, PHA.P, IL-2, and rIL-4.
Southern Blot Analysis
Genomic DNA extractions were performed according to
standard methodology El3). DNA was digested with the
restriction enzyme EcoRI or HindIII, size-fractioned by electrophoresis through an 0.8% agarose gel, and transferred
onto nitrocellulose by the method of Southern blot analysis.
Filters were hybridized to nick-translated 32P-labeledprobes
of the TCR p chain gene and y chain gene and were washed
at 60°C in 0.1% sodium dodecylsulfate, 0.015 M sodium
chloride, and 0.0015 M sodium citrate before autoradiography. DNA probes were restriction enzyme DNA frag-
ments of previously cloned germline T-cell antigen receptor
p chain genes. J p t is a 2.6-kilobase (kb) HindII-Nd fragment containing the J p l gene segment cluster; Jp2 are
contiguous 1.9-kb PvtlII-PtwII and 1.5-kb PvtlII-EcoRV
fragments containing the 3' half of the Jp2 gene segment
cluster. The two probes do not cross-hybridize. Jv is a
0.8-kb HindIIT-EroRI fragment containing Jvl.It hybridizes
to both the Jvl and Jy2 gene segments CS}.
PCR Analysis
PCR amplification of genomic D N A (Patients [with multiple
sclerosis), Po and Ka) and cDNA (Patient {with multiple
sclerosis], Re) was performed as previously described 1141.
Briefly, RNA was extracted from T-cell clones by extraction
with guanidium-isothiocyanateiphenolchloroform and isopropanol precipitation. Single-stranded complementary
DNAs (cDNAs) were synthesized from 1 pg RNA by using
oligo-deoxythymidine (Sigma Chemical, St Louis, MO) and
Avian Myeloblastosis virus-reverse transcriptasc (Bethesda
Research Laboratories, Inc, Gaithersburg, MD). PCR amplification was done with a panel of 15 oligonucleotides corresponding to the CDR2 region of the TCR p chain (Vpl-16)
and a Cp primer (cDNA). For amplification of genomic
DNA, Vp primers were used in combination with both a J p l
and a Jp2 primer, which were located at the 3' end of the J p l
and Jp2 gene cluster, respectively. Amplifications were done
for thirty cycles (94°C for 1 minute, 55°C for 2 minutes,
72°C for 3 minutes) with 1 pg of each primer in 50-1-1.1reactions. Amplified products were separated in 1% agarose gels,
transferred to nitrocellulose, and hybridized with an internal
oligonucleotide probe. Probes were end-labeled with Y-'~Padenosine triphosphate and T4 polynucleotide kiriase (BRL)
to a specific activity of lo8 cpmipg and hybridized. Blots
were washed at a final stringency of 6 x SSCi70"C and
autoradiographed for 2 to 18 hours.
Sequences for J primers are as follows:J p l primer, 5' CCC
CCG AGT CAA GAG TGG AGC CCC CAT ACC 3'; Jp2
primer, 5' CCG AGG GGC TGG AAG GTG GGG AGA
CGC CCG 3'; J p l probe, 5' CCT GGT CCC ATT CCC
AAA GTG GAG GGG TGA 3'; and Jp2 probe, 5' TGA
CCG TGA GCC TGG TGC CCG GCC CGA AGT 3'.
Phenotyping
Cytofluographic analysis of T cells was performed by means
of direct immunofluorescence with fluorescein-conjugated
anti-CD3 monoclonal antibody (mAb), anti-CD4 mAb, and
phycoerythrin-conjugated anti-CD8 mAb at a dilution of
1 : 20 (kindly supplied by Coulter Immunology, Hialeah, FL)
as previously reported {IS]. Flow cytometric analysis was
performed by using an Epics C flow cytometer (Coulter Electronics, Hialeah, FL).
Results
T cells were directly cloned before other in vitro manipulation and stimulated with mitogen and growth
factors. By using this technique, a very high cloning
efficiency could be obtained with T-cell clones that
were representative of t h e original populations of cells
in the CSF or blood [S, 161. In these experiments, the
Lee et al: T-cell Receptors in Multiple Sclerosis 35
cloning efficiency of the plated T cells was always
greater than 78%. A total of 1,017 independent longterm (> 2 months) T-cell cultures were derived from
the different patient groups. The pattern of TCR p and
y chain gene rearrangements for each of the cultures
was determined as previously described by Southern
blot analysis with D N A probes specific for the Jpl,
Jp2, and J, gene segment clusters {8]. Nine hundred
ten T-cell clones had 2 or fewer different rearranged
fragments hybridizing to either the Jpl or Jp2 probes,
and these were used for analysis of common T-cell
clonality. The other cultures were assumed to have had
more than one T cell originally plated and thus were
not examined.
The pattern of p chain and y chain gene rearrangements of the 910 T-cell clones were determined
by Southern blotting from a total of 9 patients with
demyelinating disease, 8 patients with other neurological diseases, and 4 normal subjects. Three hundred
eighteen of these clones were presented in an initial
report IS] and are combined here for analysis. The
genomic DNA isolated from a number of T-cell clones
among some patients with demyelinating disease had
identical restriction fragment patterns after digestion
with the enzymes EcoRI or Hind111 followed by hybridization in separate experiments with the TCR gene
probes J p l , Jp2, or J,. Representative Southern blots
after an initial screening of genomic DNA digested
with EcoRI and probed with a combination of Jpl and
Jp2 probes is shown in Figures 1A and 2A. Each lane
represents a different T-cell clone; experimentally, the
restriction fragment pattern of each clone was compared with the pattern produced by the other clones.
As is seen, the majority of clones have different restriction fragment patterns. In contrast, some clones,
such as Po.B, Po.Q, and Po.1 in Figure lA, and Re.2
and R e . 0 in Figure 2A had similar restriction fragment
patterns and were selected for further analysis with
other enzyme and TCR probe combinations. For two
clones to be considered identical, they had to have the
same patterns of non-germ line rearrangements after
digestion with both EcoRI and HzndIII restriction enzymes and probing with J p l , Jp2, and J, gene probes
(Figs lB,C,2B,C,D). The data from 15 patients in the
present study and 6 subjects from the initial report are
presented in Table 1. Specifically, subject Pw had 3
clones with identical restriction fragment patterns of
which 2 were from the blood and 1 was from the CSF.
Patient Re had 2 pairs of clones with identical restriction fragment patterns; each pair comprised of 1 clone
from the CSF and the other from blood. Patient Au
with childhood demyelinating disease had 1 pair of
identical clones in the CSF. Patients Ka and G r have
been previously reported; there was 1 clone in the
blood of Patient G r with identical restriction fragment
patterns in the CSF. None of the T-cell clones from
36 Annals of Neurology
Vol 29
No l January 1991
Fig 1 , (A)A n EcoRl digest of genomic D N A from clones of
Patient Po, who has multiple slcerosis, probed with both J p l and
J$ probes (Repeat individual Southern blots with these and J.,
probes are not shown). D N A from these clones was also digested
with HindIII and probed with J p l , Jp2, and J y probes; shown
are Southern blots (B and Cj aftev a HindIII digest of genomic
D N A with Jp2 andJ, probes. Clones P0.B. Po.Q, and Po.1
have the same restriction fragment rearrangement patterns.
patients with other neurological diseases and none of
the normal controls had common restriction fragment
patterns.
Oligoclonal T-cell populations derived from 3 patients with chronic progressive multiple sclerosis (Po,
Ka, Re) were further analyzed for TCR Vp gene usage.
PCR amplification was performed by use of a panel of
TCR Vp primers, which were specific for published Vp
families (Table 2). This analysis confirmed that T-cell
clones, which shared the same TCR rearrangement
pattern, were indeed clonally related by demonstrating
usage of the same Vp gene segment. When TCR Vp
gene usage was compared among 4 oligoclonal T-cell
populations derived from 3 patients with chronic progressive multiple sclerosis, common usage of the Vp12
gene segment was found among all T-cell clones (Fig
3). Also, T-cell clones derived from Patients Po and
Ka could be examined by PCR amplification of genomic DNA for assessment of rearrangement patterns.
Both oligoclonal T-cell populations from these 2 pa-
Fig 2. (A) An EcoRl digest of genomic D N A from clones of
patient Re, who has multiple sclemis, p d e d with both,JB1and
probes (repeat indizidual Southern blots with these and J ,
probes are not shown). D N A from these clones was also digested
with HindIII and probed with J p l and Jp2, and J , probes:
shown are Southern blots after a Hind111 digest of genomic
D N A with J$ (B),J$ ICj, and.Jy ID) probes. Clones Re.2
and Re.0 have the same restriction fragment reawangemeizt patterns.
tients shared a similar rearrangement to the Jp2 gene
cluster.
T-cell clones were phenotyped for the expression of
CD4 and CD8 cell surface molecules. Eighty percent
of the blood clones expressed high densities of CD4+
with either low density or no expression of CD8 determinants, whereas 209% were high density CD8+ and
CD4 [12). In the CSF, 9 195 of the clones expressed
high densities of CD4+ with either low density or no
expression of CD8 determinants, whereas 99h were
high density CD8+ and CD4K. All of the oligoclonal
T cells expressed the CD4 determinant. None of the
oligoclonal T cells proliferated in standard proliferation assays to myelin basic protein, tetanus toxoid,
measles virus, or mumps virus.
Table 2. T-cell Receptor Vp Gene Usage among Oligoclonal
T Cells from Patients with Multiple Sclerosis
Oligoclonal Pair
Vp Gene Usage
Po.1 (CSF)
Po.B (blood)
Ka.1 (CSF)
Ka.3 (CSF)
Re.18 (CSF)
Re.Q (blood)
Re.2 (CSF)
R e . 0 (blood)
Vp 12-Jp2
Vpl 2-Jp2
Vp12-Jp2
Vp12-Jp2
vp12
vp12
ND
vp12
T-cell receptor V, gene usage was determined by polymerase chain
reaction of genomic DNA (Patients Po and Ka) or complementary
DNA (cDNA) (Patient Re) by use of a panel of T-cell receptor V,
primers in combination with a C , primer (cDNA) or a Jgl and Jp2
primer (Renomic DNA). Amplified DNA was separated on agarose
gels, and Southern blots were hybridized with internal probes. N D
= nor done.
+
Discussion
T cells were directly cloned from the CSF and blood of
patients with multiple sclerosis and other neurological
diseases before other in vitro manipulation and were
compared by Southern blot analysis of the rearrangement patterns of their TCR f3 chain and y chain
genes. This allowed the determination of whether two
T-cell clones shared the same TCR and thus arose
from identical, clonally expanded progenitor T cells.
As an extension of previous studies, oligoclonal T-cell
clones representing restricted, clonally expanded Tcell populations were identified among both blood and
CSE; populations in 5 of 9 patients with demyelinating
disease. Moreover, identical clones between blood and
CSF were observed in 3 of those 9 patients. In contrast, oligoclonal T cells were not observed in 8 patients with other neurological diseases or in the blood
of 4 normal control patients.
Lee et al: T-cell Receptors in Multiple Sclerosis
37
Fig 3.Southern blot anahisis of7'-cell receptor i'TCR) Vp gene
usage for oligoclonal T celh derivedfrnm 3 patients with multiple sclemszs. Pohimerase chain veclction of genomzc D N A (Patients Po and Ka) and of complementaary DNAs (cDNAs)(Patient Re) zoas done by using a panel of TCR V, primers
(Vpl-l 6)in combination uith,Jplandj$ primen (geriomic
D N A ) or a Cpprimer (cDNA).Oligoclonal T' ceflr from Patients Po and Ka were all found t o have a Ve12d$ rear-
rangement, whereas no amplification products were obtained
using VB-Jpl primers (data not shown). Southern blots were bybridzzed by using internal TCK probes and autoradiographed.
Data from these Soathern blot.( are summarized in Table 2.
bji
Only a minority of T cells were clonally expanded in
either blood or CSF of some but not all patients with
multiple sclerosis. Nevertheless, this appears to represent an extraordinary degree of clonal expansion. That
is, it is estimated that there are over lo6 different TCR
gene rearrangements, and of these, we estimate that
this methodology allows us to determine if 2 T cells
are identical with a very high level of confidence (p >
0.001) [S}. This is based on the resolution of Southern
blotting, with the use of different probe and restriction
enzyme combinations, each of which gyves an independent assessment of clonality. Thus, considering there
are likely to be at least lo4 different T-cell clones, this
methodology could detect IS], finding two identical Tcell clones among 20 to 60 randomly selected clones
represents a significant expansion of the circulating T-
38 Annals of Neurology Vol 29 No 1 January 1991
cell population. Moreover, direct single cell cloning
will only detect high degrees of clonal expansion. Patients and controls negative for oligoclonal T cells may
have more subtle clonal expansion that could only be
detected with the investigation of an order of magnitude of more T-cell clones. Nevertheless, the present investigation extends our initial work, which was
the first to demonstrate that clonally expanded T cells
can exist in an immune compartment in an autoimmune disease. Recent reports indicate that oligoclonal
T cells may be found in other immune compartments,
such as the synovial fluid in rheumatoid arthritis [9]
and the liver in primary biliary cirrhosis {lo] during
the course of an inflammatory autoimmune disease
process. As T cells regulate immunoglobulin synthesis,
it is also possible that these oligoclonal T-cell populations are directly responsible for the oligoclonal bands
observed in multiple sclerosis, and this is presently
under investigation.
We have previously demonstrated oligoclonal T-cell
populations in the CSF of 2 patients with chronic progressive multiple sclerosis (Patients Gr and &),
whereas a patient with acute fatal multiple sclerosis
(Patient Mu) did not show oligoclonal T-cell populations [8). In the present study, an additional 3 patients
with well-defined chronic progressive disease were
studied and 2 of the patients had oligoclonal T cells.
These patients did not have the same degree of clonally expanded CSF T cells as observed in Patient Gr.
As all the clones were established by using essentially
the same technique, it seems unlikely that this difference is due to a methodological problem. The patient
groups we studied were also similar. The possibility
that cross-contamination of T-cell clones occurred at
an early stage of the cloning procedure in the first
patient we studied (Gr), however, cannot be totally
excluded.
It was important to determine if clonal T-cell expansion is an early or late event in the disease. Two patients with relapsing remitting multiple sclerosis and l
patient (Au) with a monophasic demyelinating episode
were studied. Patient Au, with biopsy-confirmed early
inflammatory demyelinating disease, had oligoclonal T
cells in the CSF. This patient is potentially instructive
in suggesting that clonal expansion of T cells may
occur early in the course of a demyelinating disease
episode. The relation between this potentially single
episode of demyelinating disease (acute penvenous
encephalomyelitis) and adult multiple sclerosis, however, is as yet unknown.
Analysis of TCR V, gene usage among oligoclonal
T-cell populations from 3 patients with multiple sclerosis demonstrated a common usage of the V,12 gene
segment among 4 sets of oligoclonal T cells. These
data raise the possibility that oligoclonal T cells may
share similar antigenic specificities and that the clonal
expansion of these cells may be the result of specific
stimulation with a myelin antigen, although an antigen
reactivity could not be found for these clones. This is
particularly interesting because of recent observations
indicating that myelin basic protein reactive T-cell
clones that induce an experimental model of multiple
sclerosis, experimental allergic encephalomyelitis, use
restricted TCR Vp and V, genes in both mice and rats
{17-191. Furthermore, we have recently deSCrlbed a
shared usage of TCR Vp genes among myelin basic
protein reactive T cells in patients with multiple sclerosis, and these T cells frequently use VP17 or V,12
1141. The TCR VP12 gene segment identified among
both MBP (84-102) reactive T-cell lines and oligoclonal T cells is homologous to the Vp8.2 gene segment described among the majority of encephalitogenic, MBP-reactive T cells in both mice and rats.
Examination of a large series of T-cell clones in the
present study demonstrates common T-cell clonotypes between the blood and CSF of patients with
chronic progressive multiple sclerosis, suggesting the
presence of an equilibrium between T cells in the
blood and CSF immune compartments. This is consistent with previous studies using monoclonal antibodies
to label circulating T cells in vivo, which indicated
there is rapid traffic of T cells from the blood to CSF in
patients with multiple sclerosis {20]. Other investiga-
tors have suggested there can be rapid movement of
immune B cells into the CNS comparcment after systemic immunization. Specifically, Sandberg-Wollheim
and co-workers [21} demonstrated that CSF B cells
stimulated with T cells/monocytes plus pokeweed
mitogen synthesized anti-tetanus toxoid antibodies
several weeks after a booster injection, but CSF cells
stimulated before the booster did not. In total, it appears that unlike immunoglobulin, which is locally synthesized in the CNS and generally does not cross the
blood-brain barrier, close relations exist among certain
clonally expanded T-cell populations and B-cell populations between blood and CSF.
In summary, clonal T-cell expansion was observed
among T cells cloned from blood ,and CSF of patients
with multiple sclerosis. Common T-cell clones found
between blood and CSF provides further evidence for
a close relation between T-cell populations in blood
and CSF immune compartments in patients with multiple sclerosis. Common TCR V, gene usage among
oligoclonal T cells between different patiems suggests
that these cells may share similar specificities and that
clonally expanded T cells bearing specific TCRs play
an important role in the inflammatory response leading
to CNS demyelination.
Supported by National Institutes of Health (NIH) Grants NS17182 and NS-00981, granrs from the National Multiple Sclerosis
Society, and the Albert J. and Diane E. Kaneb Charitable Lead
Trust. S.J.L. and S.A.B. are the recipienrs of N I H National Research Service Awards. K.W.W. is the Susan Furbacher Conroy
Fellow in Multiple Sclerosis of the Center for Neurologic Disease,
Brigham & Women’s Hospital, Boston, MA.
We acknowledge the helpful comments of Dr J.G. Seidman and the
technical assistance of Ms Meghan Purvee. Dr Cynthia J. Rutherford
and t h e Blood Bank of the Brigham and Women’s Hospital are
gratefully appreciated for rheir assistance in supplying blood products.
References
1. McFarlin DE, McFarland H . Multiple sclerosis. N Engl J Med
1983;307:1181-1188
2. H d e r DA, Weiner HL. MS: a CNS and systemic autoimmune
disease. lmmunol Today 1989;lO:104-107
3. Hofman FM, von Hanwehr RI, Dinarello CA, et al. Immunoregulatory molecules and IL 2 receptors identified in multiple
sclerosis brain. J Immunol 1986;136:3239-3245
4. Ota K, Matsui M, Milford E, et al. T cell recognition of an
immunodominant myelin basic protein epitope in multiple sclerosis. Nature 1990;346:183-187
i. Martin R, Jarayuemada D, Flerlage M, et al. Fine specificity and
HLA restriction of myelin basic protein-specific cytotoxic T cell
lines from multiple sclerosis patients and healrhy individuals. J
Immunol 1990;145:540-548
6. Toyonaga B, Ytoshikai Y, Vdasz V, et al. Organization and
sequences of the diversity, joining, and constant region genes of
the human T cell receptor beta chain. Proc Narl Acad Sci USA
1985;82:8624-8628
7 Waldman TA, Davis MM, Rongivanni KF, Korsmeyer SL. Rearrangements of genes for the antigen receptor on T cells as
Lee et al: T-cell Keceptors in Multiple Sclerosis 39
8.
9.
10.
11.
12.
13.
14.
15.
markers of lineage and clonality in human lymphoid neoplasms.
N Engl J Med 1985;313:776-783
Hafler DA, Duby AD, Lee SJ, et al. Oligoclonal T lymphocytes
in the cerebrospinal fluid of patients with multiple sclerosis. J
Exp Med 1988;167 :I 3 13-1 322
Sramenkovic I, Stegagno M, Wright KA, et al. Clonal dominance among T-lymphocyte infiltrates in arthritis. Proc Natl
Acad Sci USA 1988;85:1179-1183
Mosbius U, Meuer SC, Hess G, er al. T ccll receptor gene
rearrangements of T cells infiltrating the liver in chronic active
hepatitis and primary biliary cirrhosis. Proc 7th lnt Congress
Imunol 1989;7:567 (Abstract)
Kurtzke JK. Course of exacerbations of multiple sclerosis in
hospitdzed patients. Arch Neurol Psychiatry 1956;76:175184
Brad SA, Purvee M, Hafler DA. IL-4 induces CD8 expression
on human T cell clones. Cell Immunol 1990;125:426-436
Southern EM. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol 1975;
981503-511
Wucherpfennig KW, Ota K, Endo N, et al. Shared human T cell
receptor V, usage to immunodominant regions of myelin basic
protein. Science 1990;248:1016-1019
Hafler DA, Fox D, Schlossman SF, et al. In vivo activated Tlymphocytes in the peripheral blood and cerebrospinal fluid of
40 Annals of Neurology Vol 29 No 1 January 1991
patients with multiple sclerosis. N Engl J Med 1985;312:14051411
16. Moretta A, Pantaleo G, Moretta LJ, et al. Direct demonstration
of the clonogenic potential of every human peripheral blood
cell. J Exp Med 1983;157:743-753
17. Urban JL,Kumar V, Kono DH, er al. Restricted use of T cell
receptor V genes in murine autoimmune encephalomyeliris
raises possibilities for antibody therapy. Cell 1988;54:577-592
18. Acha-Orbea H , Mitchell DJ, Timmermann L, et al. Limited
heterogeneity of T cell receptors from lymphocytes mediating
autoimmune encephalomyelitis allows specific immune intervention. Cell 1988;54:263-273
19. Burns FR, Li X, Shen N , et al. Both rat and mouse T cell
receptors specific for the encephalitogenic determinant of myelin basic protein use similar V, and V, chain genes even
though the major histocompatibility complex and encephalitogenic determinants being recognized are different. J Exp Med
1989;169:27-39
20. Hafler DA, Weiner HL. In vivo labeling of blood T cells: rapid
traffic into cerebrospinal fluid in multiple sclerosis. Ann Neurol
1987;21:89-93
21. Sandberg-Wollheim M, Zweiman B, Levinson AI, Lisak RP.
Humoral immune responses withn the human central nervous
system following systemic immunization. J Neuroimmunol
1986;11:205-214
Документ
Категория
Без категории
Просмотров
4
Размер файла
814 Кб
Теги
patients, common, usage, fluid, blood, cells, cerebrospinal, oligoclonal, sclerosis, multiple, receptov, derived, lymphocytes
1/--страниц
Пожаловаться на содержимое документа