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T cell receptor v repertoire of double-negative ╨Ю┬▒ t cells in patients with systemic sclerosis.

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944
T CELL RECEPTOR V, REPERTOIRE OF
DOUBLE-NEGATIVE Gulp T CELLS IN
PATIENTS WITH SYSTEMIC SCLEROSIS
AKEMI SAKAMOTO, TAKAY UKI SUMIDA, TOSHIRO MAEDA, MICHIHIRO ITOH,
TAKAYOSHI ASAI, HIDENORI TAKAHASHI, SHOUJI YOSHIDA, TAKA0 KOIKE,
HISAO TOMIOKA. and SHO YOSHIDA
Objective. To analyze the T cell receptor V, gene
on double-negative (DN) do T cells, which are increased in number, on peripheral blood lymphocytes
(PBL) from patients with systemic sclerosis (SSc).
Methods. The DN do T cells were sorted by flow
cytometry from PBL obtained from 3 patients with SSc.
The V, repertoire was analyzed by polymerase chain
reaction.
Results. Only 1 or 2 V, genes (VSl7, 5, or 17)
were predominantly expressed on DN do T cells from
these 3 patients.
Conclusion. The V, repertoire on DN do T cells
in PBL from patients with SSc is rather restricted.
-
_-
From the Second Department of Internal Medicine and the
Department of Blood Transfusion, School of Medicine, Chiba
University, the Department of Internal Medicine, Asahi General
Hospital, and the Department of Internal Medicine, School of
Medicine, Toho University, Chiba, Japan.
Akemi Sakamoto, MD: Second Department of Internal
Medicine, School of Medicine, Chiba University; Takayuki Sumida,
MD: Second Department of Internal Medicine, School of Medicine,
Chiba University; Toshiro Maeda, MD: Second Department of
Internal Medicine, School of Medicine, Chiba University; Michihiro
Itoh, PhD: Department of Blood Transfusion, School of Medicine,
Chibd University; Takayoshi Asai, MD: Department of Blood
Transfusion, School of Medicine, Chiba University; Hidenori Takahashi, PhD: Department of Internal Medicine, Asahi General Hospital; Shouji Yoshida, MD: Department of Internal Medicine, Asahi
General Hospital; Takao Koike, MD: Second Department of Internal Medicine, School of Medicine, Chiba University; Hisao Tomioka, MD: Department of Internal Medicine, School of Medicine,
Toho University; Sho Yoshida, MD: Second Department of Internal
Medicine, School of Medicine, Chiba University.
Address reprint requests to Takayuki Sumida, MD, Second
Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260, Japan.
Submitted for publication June 21, 1991; accepted in revised form March 18, 1992.
Arthritis and Rheumatism, Vol. 35, No. 8 (August 1992)
T cell receptor (TCR) alppositive T cells are
usually associated with either CD4 or CD8 molecules
involved in major histocompatibility complex class IIand class I-restricted T cell recognition (1,2). A subset
of TCR d p T cells that do not express the CD4 and
CD8 molecules has been detected in mice (3). In
autoimmune-prone mice such as MRL-lprDpr and
C3H/HeJ-gld/gld, many double-negative (DN) d p T
cells accumulate in peripheral lymph nodes. The biological function of these cells, however, has not been
clarified (4). DN T cells predominantly express the
V,8 gene in MRL-lpr/lpr mice, while the TCR repertoire of CD4- or CD8-positive T cells is diverse (3).
In humans, DN d p T cells were found to be
markedly expanded in patients with systemic lupus
erythematosus, and some established T cell clones
augmented the production of anti-DNA autoantibodies
( 5 ) . This supported the notion that these cells may play
a significant role in the progression of autoimmunity.
We carried out a quantitative analysis of DN d p T
cells in peripheral blood lymphocytes (PBL) from
patients with systemic sclerosis (SSc) and found that
these cells were increased in number. To analyze the
nature of DN d p T cells in SSc patients, the repertoire
of TCR V, genes from DN d p T cells in SSc patients
was analyzed using the polymerase chain reaction
(PCR). We obtained evidence that the limited V, genes
were preferentially expressed on these cells.
PATIENTS AND METHODS
Study subjects. Nineteen patients with SSc were
diagnosed according to established criteria (6). Four patients
were evaluated during the swelling phase of SSc; the other
15 patients were evaluated during the sclerosis phase. Nine
TCR V, REPERTOIRE IN SSc
945
n
00
P,
1
-I
,
99.5%
Figure 1. Separationof double-negative (DN) alp T cells on peripheral blood lymphocytes (PBL) from patients with
systemic sclerosis (bottom panels). PBL were stained with phycoerythrin-coupled antLCD4 plus anti-CD8
monoclonal antibodies (MAb) and with fluorescein isothiocyanate-conjugated WT-31 MAb (left panel). By
fluorescence-activatedcell sorting, the DN d p T cells (right bottom panel) and CD4- and/or CD8-positive T cells
(right top panel) were collected (see Patients and Methods for details).
disease-free subjects were also examined; these subjects
served as controls. All patients and healthy subjects were
recruited from the Chiba University Hospital and were of
Japanese ancestry. The TCR V, gene repertoire on DN dB
T cells was analyzed in 3 of the patients whose SSc was in
the swelling phase.
Flow cytometry. Lymphocytes (1 x 10’) from 20 ml
of peripheral blood obtained from all study subjects were
isolated by Ficoll-Paque separation (Pharmacia, Piscataway,
NJ). For 2-color staining, PBL were incubated for 1 hour on
ice with phycoerythrin ( P E k o u p l e d anti-CD4 (Leu-3a) plus
antLCD8 (Leu-2a) monoclonal antibodies (MAb) (Becton
Dickinson, Mountain View, CA) and fluorescein isothiocyanate (FITCkonjugated MAb to d p TCR (MAb WT-31;
Becton Dickinson).
After incubation, the cells were washed and resuspended at a concentration of lo6 cells/ml and then analyzed
by FACScan (Becton Dickinson). To enrich for DN d p T
cells, 1 x lo5 DN d p T cells and lo6 CDCpositive and/or
CD8-positive T cells were sorted with FACStar (Becton
Dickinson) and PE-conjugated anti-CD4 plus anti-CD8
MAb, and these were used for further experiments.
Preparation of RNA and the PCR. Total RNA ( 1 0 4 . 1
pg) were prepared using RNAzol solution (Biotecx, Houston, TX) from sorted DN d/3or CD4- and/or CD8-positive T
cells. Complementary DNA (cDNA) were synthesized in a
20-pl reaction mixture containing oligo(dT) primer by avian
myeloblastosis virus (AMV) reverse transcriptase from
0.1-1 pg of total RNA.
Amplification was performed with Taq polymerase in
50 pl of standard buffer, using 0.2 pl of cDNA (corresponding to 1-10 ng of total RNA). Twenty different V, and C,
primers or 5’-C, and 3’-C, primers (as a control) were used
for PCR. The sequences of V and C, primers were from
previously published data (7). dligonucleotides were synthesized in a DNA synthesizer (Applied Biosystems, Foster
City, CA). Denaturing was done at 94°C for 1.5 minutes, the
annealing at 60°C for 1 minute, and the extension step at
946
SAKAMOTO ET AL
72°C for 1 minute, for 30 cycles on a DNA Thermal Cycler
(Zymoreactor V2; Atto Co., Tokyo, Japan). One-Mth of
each sam le was loaded on a 2% agarose gel and hybridized
with the 'P-labeled Psr I fragment of the JUR-p2 gene (C,J
(8). The intensities of the autoradiographic bands were
quantitated by densitometer (Fujix BAS 2000; Fujifilm I & I
Co., Tokyo, Japan), and the relative V, gene expression was
calculated as the V,:Cg ratio.
In some expenments, 0.2 pl of the same cDNA
(corresponding to 1-10 ng of total RNA), as described
above, from sorted DN d p T cells was used for PCR with
5'-CD4 (5'-GACCAAGGAAACTTCCCCCTG-3') and 3'CD4 (5'-TTGCACTGAGGGGCTACTACC-3')
or 5'-CD8a
(5'-GAGACAGTGGAGCTGCTGAAGTGC-3')
and 3'CD8a (5'-CGCCTTGGGCTTGTTTTGGGA-3')
primers.
The PCR was performed under the same conditions as
above. These PCR products, including CD4 and CD8a
genes, yielded a band of 200 basepairs and 134 bp, respectively. Amplified DNA were separated by agarose gel and
were hybridized with the '*P-labeled Eco RI fragment of the
human CD4 gene (9) or the Pst IIHinc I1 fragment of the
human CD8a gene (10). The cDNA (1 ng) encoding CD4 or
CD8 genes were used for PCR as a positive control.
Statistical analysis. To examine the statistical significance, we used Student's r-test.
RESULTS
Double-negative (Jp T cells on PBL from patients with SSc. The DN alp T cells on PBL from 19
patients with SSc and from 9 healthy subjects were
analyzed by flow cytometry using PE-coupled antiCD4 plus anti-CDS MAb and FITC-conjugated antiTCRdp MAb. DN alp T cells from the PBL of
patients with SSc could be clearly distinguished from
CD4-positive and from CDS-positive d p T cells (Figure 1, left panel). DN a/p T cells in the 4 patients with
Figure 3. Analysis of V, gene usage in double-negative (DN) alp T
cells from patients with systemic sclerosis. The complementary
DNA from sorted DN alp T (b, d, and fJand CDepositive andor
CD8-positive cells (a, c, and e) from 3 patients were used for the
polymerase chain reaction. Protein markers are shown to the right
(in basepairs); V, markers are shown across the top.
swelling-phase SSc (6.35
Figure 2. Expression of CD4 and CD8 genes in sorted double-
negative (DN) cells. The complementary DNA from sorted DN dB
T cells from 3 patients with systemic sclerosis (b and f, c and g, and
d and h, respectively), a CD4 clone (a), and a CDS clone (e) were
used for the polymerase chain reaction, with primers specific for
CD4 gene (A)
and CD8 gene (e-h). Protein markers are shown to
the right (in basepairs).
?
2.76% mean
?
SD; P <
0.005) and the 15 patients with sclerotic-phase SSc
(2.39 k 1.56%; P C 0.01)were inCEaSed in number
compared with those in the 9 healthy individuals (0.84
0.69%).
Enrichment of DN rJP T cells on PBL from SSc
Patients. To separate DN d p T ceus from CD4positive and/or CDS-positive T cells of patients with
947
TCR V, REPERTOIRE IN SSc
swelling-phase SSc, PBL were stained with PEcoupled anti-CD4 plus anti-CD8 MAb and FITCconjugated WT-31 MAb, and then CD4-positive andor
CD8-positive T cells were sorted out. A representative
sort is shown in Figure 1. DN d p T cells (1 X lo5)
were enriched from 6.5% to 28.1%, and CD4 andlor
CD8 T cells numbered <0.1%.
To elucidate whether the sorted DN dp T cells
included CDCpositive and/or CD8-positive T cells, the
transcripts encoding CD4 and CD8 genes in the separated DN cell population were examined by PCR
methods and by Southern blot analysis. As shown in
Figure 2, the expression of both CD4 and CD8 genes in
this fraction was not detected, even with a longer
exposure, whereas the CD4 and CD8a cDNA clones
were amplified. These data indicate that sorted DN
cells include neither CDCpositive nor CD8-positive T
cells.
Analysis of Vp gene usage on DN dj?T cells from
patients during the swelling phase of SSc. To examine
V, gene usage on DN d p T cells in SSc, total RNA
was prepared from sorted DN d p T cells and sorted
CD4-positive and/or CD8-positive T cells, and was
then used for synthesis of cDNA. The cDNA encoding
VDJC p genes were amplified with primers specific for
20 different V, families and the primer for the C, gene.
The V,7 gene (V,:C, ratio 0.68) and V$ gene
(V,:C, 0.31) in the first patient (Figure 3b), the V,5
gene (V,:C, 0.36) in the second patient (Figure 3d),
and the V,17 gene (V,:C, 0.21) in the third patient
(Figure 3f) were predominantly expressed on DN dj3
T cells from patients with SSc. The other V, genes
were not detected, even with a longer exposure. By
contrast, the usage of V, genes on CDCpositive andor
CD8-positive sorted cells in the same patients was
heterogeneous and diverse (Figures 3a, c, and e).
DISCUSSION
We analyzed the proportion of double-negative
d p T cells on PBL from 19 patients with SSc by flow
cytornetry and found that DN d p T cells were increased in number in SSc patients compared with
healthy subjects. To analyze the nature of DN d p T
cells, the usage of TCR V, genes on DN d p T cells in
these patients with SSc was examined by the PCR. We
obtained evidence that the repertoire of TCR V, genes
on DN d p T cells in SSc patients was rather restricted.
In autoimmune-prone mice, such as the MRLlprllpr mouse, DN T cells have been found in large
numbers in lymph nodes, and have shown a strikingly
limited TCR V, repertoire, in which V, chains encoded by the V&3 gene family are expressed at an
unusually high frequency in these species (3,11,12).
DN dj3 T cells in these mice might function as
autoreactive T cells, in collaboration with CD4positive T cells, which could produce interleukin-4 and
interferon-y (13).
With regard to human autoimmune diseases,
the TCR repertoire of DN d p T cells has not heretofore been reported. We clarified that only 1 or 2 V,
genes (V,5/7, V G , and V,17) were preferentially expressed on DN d p T cells in 3 SSc patients, although
the repertoire of V, genes on CD4-positive andor
CD8-positive T cells from the same patients was
heterogeneous and diverse. Therefore, the V, gene
usage on DN Culp T cells of PBL from SSc patients was
limited and was not diverse.
Groh et al (14) established two DN d p T cell
clones from normal human skin and believed that they
shared one predominant TCR V, gene rearrangement.
This, along with our current observations, may support the idea that the limited heterogeneity of the TCR
repertoire in both murine and human DN d p T cells
suggests that DN d p T cells might be selected or
restricted in ontogeny. Ohteki et a1 (15) showed that
nonparenchymal mononuclear cells from the liver of
diseased MRL-lpr/lpr mice were increased in number,
proliferated in vitro, and consisted of DN d p T cells.
They concluded that the liver was a possible site for
the proliferation of DN d p T cells in MRL-lpr/lpr
mice. The environment in which DN d p T cells
proliferate in humans remains the subject of ongoing
study.
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