close

Вход

Забыли?

вход по аккаунту

?

Mycobacteria and human heat shock protein В Эspecific cytotoxic t lymphocytes in rheumatoid synovial inflammation.

код для вставкиСкачать
MYCOBACTERIA AND HUMAN
HEAT SHOCK PROTEIN-SPECIFIC
CYTOTOXIC T LYMPHOCYTES IN
RHEUMATOID SYNOVIAL INFLAMMATION
SHU GUANG LI, ALISON J. QUAYLE, YAMIN SHEN, JENS KJELDSEN-KRAGH,
FREDRIK OFTUNG, RADHEY S. GUPTA, JACOB B. NATVIG, and OYSTEIN T. F@RRE
Objective. To study the cytotoxic capacity of
mycobacteria-specific T lymphocyte lines and clones
from sites of inflammation in patients with rheumatoid
arthritis (RA). We also studied antigen specificity, surface phenotype, expression of T cell receptors (TCR),
and HLA restriction.
Methods. Autologous macrophages (M+) from
the synovial membrane (SM), synovial fluid (SF), or
peripheral blood (PB) were used as target cells in
cytotoxicity assays.
Results. All SM and SF cell lines tested thus far
have shown specific lysis of the autologous M+ from SM
or PB that had been pulsed with BCG (bacillus CalmetteGuerin), but no cytotoxicity when the targets were
-
From the Institute of Immunology and Rheumatology,
University of Oslo, the Laboratory of Immunology, Institute for
Cancer Research, the Norwegian Radium Hospital, and the Oslo
Sanitetsforenings Rheumatism Hospital, Oslo, Norway.
Supported by the Norwegian Women’s Public Health Organization, the Grethe Harbitz Legacy. the Leon and Norma Hess
Legacy for Rheumatic Research, and by Hafslund-Nycomed, Oslo.
Shu Guang Li. MD. PhD: Institute of Immunology and
Rheumatology. University of Oslo (current address: Specialty Laboratories Inc.. Santa Monica, CA); Alison J. Quayle, MSc, PhD:
Institute of Immunology and Rheumatology, University of Oslo;
Yamin Shen, MD: Institute of Immunology and Rheumatology,
University of Oslo; Jens Kjeldsen-Kragh. MD: Institute of Immunology and Rheumatology, University of Oslo; Fredrik Oftung,
PhD: Laboratory of Immunology, Institute for Cancer Research, the
Norwegian Radium Hospital; Radhey S. Gupta, PhD: Professor of
Biochemistry, Department of Biochemistry, McMaster University,
Hamilton. Ontario, Canada; Jacob B. Natvig, MD, PhD: Professor
of Medicine, Institute of Immunology and Rheumatology, University of Oslo; gystein T. Fqirre, MD, PhD: Professor of Medicine,
Oslo Sanitetsforenings Rheumatism Hospital.
Address reprint requests to Shu Guang Li, MD, PhD,
Specialty Laboratories Inc., 221 1 Michigan Avenue, Santa Monica,
CA 90404-3900.
Submitted for publication May 17, 1991;accepted in revised
form November 6, 1991.
Arthritis and Rheumatism, Vol. 35, No. 3 (March 1992)
pulsed with irrelevant antigens such as tetanus toxoid
and Chlamydia. Both CD4+ and CDS+ cells were
shown to be involved in the specific cytolysis. The majority of the cytotoxic T lymphocyte (CTL) lines were TCRaI
@+ cells. However, both TCRaIP+ and TCRy/S+
clones (TCR 61+)from one RA patient showed antigenspecific lysis. Antigen-specific recognition by a number
of CTL lines and clones generated from SF and SM was
restricted by HLA-DR molecules. Two Mycobucterium
bovis 65-kd heat shock protein (HSP)-specific TCRcul
@+ SF T cell clones also lysed M + that had been pulsed
with a recombinant human 65-kd HSP.
Conclusion. Joint inflammation and destruction
might be partly attributable to a cross-reaction of
mycobacteria-induced cytotoxic T cells with self HSP.
Although the cause of rheumatoid arthritis (RA)
is not known, activated T lymphocytes in the synovial
fluid (SF) and synovial membrane (SM)of RA patients
may contribute to the pathogenesis of the disease.
Evidence has accumulated which indicates that T cells
specific for mycobacterial antigens may play a role in
chronic arthritis. Studies of the adjuvant arthritis
model (experimentally induced by immunization of
Lewis rats with Mycobacterium tuberculosis in oil)
indicate that autoreactive T cells triggered by mycobacterial antigens can mediate persistent, chronic arthritis or can provide protection from the development
of arthritis (1,2). This disease can be transferred to
irradiated naive rats by T cells reactive both with an
epitope on the 65-kd heat-shock protein (HSP) of M
tuberculosis and with cartilage proteoglycans (3,4).
Since HSPs may be produced at sites of inflammation (9,it has been speculated that T cells activated
by bacterial antigens might conceivably mediate auto-
HSP-SPECIFIC T CELLS IN RA
immune disease. It has been observed that mononuclear cells (MNC) derived from the SF of RA
patients reacted more with the purified protein derivative of tuberculin (PPD) (6), with an acetoneprecipitable fraction of M tuberculosis (7), and with a
recombinant preparation of the Mycobacteriurn bovis
65-kd HSP (8) or Mycobacterium leprae HSP (9,lO)
than did MNC from RA peripheral blood (PB). The
HLA-DR4-associated regulation of the immune response to M tuberculosis in RA has been demonstrated (11). However, the 65-kd HSP reactivity was
more pronounced in patients with reactive arthritis
than in those with RA (8,9), and the enhanced reactivity was not confined to mycobacteria, but was also
directed against other bacteria (8,12). Recently, Res et
a1 (13) reported that the enhanced reactivity against
mycobacterial antigens was not a specific feature of
MNC from chronically inflamed joints but might be a
general characteristic of chronic inflammation.
It has been postulated that when autoantigenreactive cytotoxic T cells encounter their specific
antigen on the membrane of autologous macrophages
(M+) or other cells, lysis of such targets may occur,
(possibly) resulting in tissue damage (14). In humans,
CD4+ T cell clones reactive with BCG (bacillus Calmette-Guerin) were reported to display cytotoxic activity toward antigen-presenting cells (APC) from
BCG-vaccinated healthy subjects (15). In addition,
BCG- and 65-kd HSP-induced cytotoxic T lymphocytes (CTL) directed toward the antigen-pulsed Mc$
have been isolated from healthy individuals (1620)
and from leprosy patients (16,18) and tuberculosis
patients (20). In studies of mice, accumulating evidence has suggested an important role for CTL in the
immune response to mycobacteria and other intracellular parasites (21-23). Thus, the involvement of
mycobacteria-specific CTL might be a general phenomenon in chronic inflammation.
We present here the results of our studies of
antigen-specific cytotoxicity displayed by CTL lines
and clones derived from inflamed tissues of RA patients. Their antigen specificity, surface phenotype,
expression of T cell receptor (TCR), and HLA restriction are described. We also discuss the possible immunopathologic roles of mycobacteria-activated CTL
in the synovial inflammation of RA.
MATERIALS AND METHODS
Patients. SM, SF, and PB samples were obtained
from patients with RA who were hospitalized at Oslo Sani-
27 1
tetsforenings Rheumatism Hospital. Nine patients (2 males
and 7 females) were classified as having RA, according to the
1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (24).
The average age of the study population was 56 years. and
the average disease duration was 12 years.
Antigens. BCG and Mycobacterium kansasii (MK)
that had been cultured in synthetic Sauton medium were
kindly provided by Dr. H. G. Wiker (Oslo, Norway) and Dr.
Sadadu Nagai (Osaka, Japan). The purified recombinant M
bovis 65-kd HSP was provided by Dr. Ruurd van der Zee
(supported by the United Nations Development Project/
World BanMWorld Health Organization special program for
research and training in tropical diseases; the National
Institute of Public Health and Environmental Protection,
Bilthoven, The Netherlands). Recombinant P1 protein, a
human (Hu) 65-kd HSP, was produced by cloning complementary DNA (cDNA) for the mature form of this protein at
the Nco I site in the prokaryotic expression vector
pKK233-2, as described previously (25.26). The protein
preparation was obtained by electroelution from sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The
protein and a control preparation from a culture supernatant
of Escherichia coli were further purified on nitrocellulose by
Dr. M. Sioud (27). Tetanus toxoid (TT) and Chlamydia were
kindly provided by the Statens Institutt for Folkehelse (Oslo,
Norway).
Generation of BCG-stimulated T cell lines from PB,
SF, and SM from RA patients. MNC were isolated as
previously described from PB, SM, and SF obtained from
RA patients (28). Throughout these studies we used RPMI
1640 supplemented with 2 mM glutamine, 100 unitslml
penicillin, 100 pdml streptomycin, and 10% human AB
serum (complete RPMI). MNC were cultured at lo6 cells/
well for 5 days in 24-well tissue culture plates (Falcon 3047;
Becton Dickinson, Mountain View, CA), with 10 pg/rnl
BCG, at 37°C in a fully humidified 5% CO, incubator. On day
4, medium containing 20% (volume/volume) interleukin-2
(IL-Zjrich supernatant made from a 48-hour culture of
phytohemagglutinin (PHA)-stimulated PBMC from 8
healthy blood donors was added to the cultures. After an
additional 4-7 days, the cultures were restimulated with the
antigen and irradiated allogenic PB MNC mixture as feeders
for the enrichment of the cells.
T cell cloning. The BCG-specific TCRa/P+ T cell
clones were generated in the following way. SF cells from
patient H were stimulated for 7 days with 10 pdml of BCG
and then cloned by limiting dilution in 96-well plates, using a
mixture of autologous irradiated S F and PB MNC as APC,
20 pg/ml of BCG, and 50 unitslml of recombinant IL-2
(rIL-2). Fresh complete RPMI and rIL-2 were added to the
cloning plates after 7 and 12 days. Between days 14 and 20,
each clone was transferred to 1 well of a 24-well plate and
restimulated with allogeneic PBMC, PHA, and 10% (vlv)
IL-2-rich supernatant. Clones were expanded by a twiceweekly addition of complete RPMI and 1L-2 supernatant,
and restimulation with allogeneic PBMC and PHA every 12
days (29). None of the clones generated in this way responded to the 65-kd HSP.
TCRaIP+ clones specific for 65-kd HSP were generated from patient H according to the same protocol, but with
272
LI ET AL
Table 1.
Monoclonal antibodies used
Specificity
Name
IgG isotype
Source
CD3
CD4
CDX
CD19
CD45RA
CD45RO
TCRaIP
TCR V,5 family
TCR V& family
TCR V,8 family
TCR V,12 family
TCRyI6
HLA class 1
HLA-DR
HLA-DQ
H LA-DP
IgD
IOT3
Anti-Leu-3a
Anti-Leu-2a
HD237
2H4-RDI
UCHLI
TCR- I
,V5 (a)
,V6 (a)
,V8 (a)
,V12 (a)
TCR-61
B9.12.1
B8.11.2
Anti-Leu-I0
Anti-human HLA-DP
TI B96
IgG I
IgG I
IgG I
IgGZb
IgG 1
IgG2a
IgG I
IgG 1
IgG I
lgG2b
lgG2a
IgG I
lgG2a
lgG2b
IgG I
IgG I
IgG I
Imrnunotech, Paris, France
Becton Dickinson. Mountain View, CA
Becton Dickinson, Mountain View, CA
Ref. 31
Coulter. Hialeah, FL
Dakopatt s, Copen hagen, Denmark
Becton Dickinson, Mountain View, CA
T Cell Sciences, Cambridge, MA
T Cell Sciences, Cambridge, MA
T Cell Sciences, Cambridge, MA
T Cell Sciences, Cambridge, MA
T Cell Sciences, Cambridge, MA
Ref. 32
Ref. 32
Becton Dickinson, Mountain View, CA
Becton Dickinson, Mountain View, CA
Ref. 33
15 Fglml65-kd HSP for the 7-day stimulation of S F cells, and
then 20 pg/ml of BCG and 2 pglml65-kd HSP in the cloning.
The 65-kd HSP-specific TCRy/G+ clones were generated
from the synovial fluid of patient I. First, a 65-kd-specific T
cell line was established. The cell line was depleted of CD4+
cells and TCRaIP+ cells by use of magnetic beads (30), and
the remaining cell population was then cloned by limiting
dilution (Kjeldsen-Kragh J et al: unpublished observations).
Depletion of lymphocyte subsets using magnetic beads.
Commercial magnetic beads (Dynabeads; Dynal, Oslo, Norway) precoated with anti-CD4 and anti-CD8 monoclonal
antibodies (MAb) were used for depletion experiments according to the method of Lea et al (30).
Monoclonal antibodies. The MAb used in fluorescence-activated cell sorter (FACS) analysis and for inhibition of antigen-specific cytotoxicity are listed in Table 1.
Cytotoxicity assay. The use of adherent M d as target
cells has been described by Ottenhoff et al (16). Briefly,
MNC from PB andlor from SM or SF (10' cells/well) were
plated in 96-well U-bottom tissue culture plates (Nunc,
Raskilde, Denmark). Approximately 10% of the cells adhered. This number was used for calculating effector:target
(E:T) cell ratios. Cells were cultured for 6-14 days, and
during the last 18 hours, were pulsed with various antigens
(see Results) and labeled with 2 pCi/well of "Cr. Cells were
washed 3 times with preheated medium, and the effector
cells were added to the target cells (150 pllwell) in duplicate
or in triplicate. One triplicate of target cells in medium
without effector cells was used to determine the level of
spontaneous release. After 16 hours, the content of each well
was transferred to a detachable counting tube, and 100 pl of
1% Triton X-100 (no. T-6878; Sigma, St. Louis, MO) was
added to the original wells to lyse the remaining adherent
cells. After 3 hours, the total volume of Triton was transferred to similar tubes, and the samples were counted in a
gamma counter (model 1275 minigamma; LKB, Stockholm,
Sweden).
The percentage of specific "Cr release for each well
was calculated as follows: 3' 6 specific lysis = [test counts per
minute/(test cpm + cpm after Triton X-100treatment of the
same well)] x 100% - % spontaneous release. The percent-
age of spontaneous release was calculated as follows: cpm in
spontaneous release well/(cpm in spontaneous release well
+ cpm after Triton X-100treatment of the same well) x 100%.
Phenotypic analysis of effector cells. T cell lines and
clones were stained by using a standard direct andlor indirect immunofluorescence technique, and were analyzed by a
FACScan flow cytometer (Becton Dickinson) (34).
RESULTS
Cytotoxicity of BCG-specific T cells from SF and
SM. As shown in Figure 1, BCG-stimulated lymphocytes from the SM (Figure 1A) and the SF (Figure 1B)
of RA patients lysed BCG-pulsed autologous M 4 in a
dose-dependent manner. The antigen-dependent lysis
was specific; it was not accompanied by lysis of
nonpulsed target cells or of target cells pulsed with
irrelevant antigens (TT or Chlamydia). The background killing of nonpulsed M 4 was between 2% and
35%, depending on the individual; this level is consistent with those in other published studies (16-18,20). A
panel of antigens was used in this cytotoxicity assay to
test for antigen specificity. A BCG-stimulated SF T
cell clone from patient H, USSF2B9 (Figure IB),
showed cross-reactivity between BCG and MK, but
not with unrelated antigens (TT or Chlamydia) or with
a recombinant M bovis 65-kd HSP.
To detect false lytic activity, we included both
positive and negative controls in our assay system.
The positive lysis control was a human HLA-DR4/
Dw4-specific CTL cell line generated from a random
donor against an HLA-DR4/Dw4 positive mouse
transfectant (Shen Y et al: unpublished observation)
lysed both BCG-pulsed and unpulsed targets (HLA-
273
HSP-SPECIFIC T CELLS IN RA
*O/
40
A
t
0
7 7
t
10
20
30
c
-
2ol'O
0
5
10
loor D
t
30t
40
I
Figure ID shows such an example by using a T cell
clone from patient H. This was used as the negative
lysis control.
To confirm the above observations, more RA
patients were tested. Figure 2 shows that SM and SF T
lymphocyte lines and clones from 9 RA patients,
which were raised after stimulation with BCG for
short-term (7 days to 1 month) or long-term (>12
months) culture, significantly lysed BCG-pulsed M+
from SM, SF, or PB as compared with antigenunpulsed and TT-pulsed targets.
Furthermore, all BCG-induced T cell lines
tested from patients A 4 were cytotoxic neither for
K562 cells (the conventional natural killer [NK] target)
nor for Daudi cells (resistant to NK cells but sensitive
to activated killer [AK] activity target) (data not
shown). The results suggest that the mycobacteriainduced cytotoxicity is not an NWAK-like activity.
Phenotype of specific CTL originating from SM
and SF. The phenotyping data from 7 BCG-stimulated
SM CTL lines from 7 RA patients are shown in Table
2. Two well-defined 65-kd HSP-specific TCRaIP+ SF
T cell clones derived from patient H and 1 TCRyIG+
60
0
10
20
30
0
1.5
15
E/T ratio
Figure 1. Antigen-specific cytotoxicity of bacillus Calmette-Guerin
(BCGtstimulated T effector cells from the synovial membrane (SM)
and the synovial fluid (SF) of rheumatoid arthritis (RA) patients,
against antigen-pulsed autologous macrophages (MI$). A, BCGstimulated SM T cell line from patient A, cytotoxic against BCGpulsed (O),but not tetanus toxoid ('ITtpulsed (A) or unpulsed (O),
autologous M 4 from SM. B, BCG-stimulated S F T cell clone
(USSF2B9) from patient H, cytotoxic to autologous MI$ from
peripheral blood (PB) pulsed with BCG (0)and Mycobacterium
kansasii (0),but not TT (A),Chlamydia (A), recombinant Mycobacterium bovis 65-kd heat-shock protein (HSP) (W), or unpulsed
(0)cells. C, A human cytotoxic T lymphocyte (CTL) line against
mouse HLA-DR4/Dw4 positive transfectant generated from a random donor (HLA-DR7, 10; DQI, 2) lysed PB M 4 from patient H
(HLA-DR4/Dw4, DR5), both when pulsed with BCG (0)and when
unpulsed (without antigen) (0).D, Antigen-specific cytotoxicity of
M bovis 65-kd HSP-stimulated SF T cell clone (USSF2C6 from
patient H; HLA-DR4,5/Dw4) against BCG-pulsed autologous MI$
(O),but not unpulsed autologous MI$ (0)
from
. PB. It did not lyse
BCG-pulsed (Vand +) or unpulsed (V and 0)allogeneic MI$from the
PBofdonor I (VandV)(HLA-DRI, 1; DQ1, w5,6)ordonor2(+and
0 )(HLA-DRS, w6, DRw52; DQI. 3, w6, 7). Em = effectorltarget.
DR4Dw4 and DR5) equally (Figure 1C). In contrast,
BCG-stimulated SM as well as SF effector cells did not
lyse BCG-pulsed, HLA-mismatched allogeneic M+.
-a?
>.
.0
.-
50
40
c
x
0
0
30
c
20
0
ia
a
A
B
C
D
E
F
G
H
I
RA patient
Figure 2. BCG-activated SM T cell lines (A-G) and SF T cell clones
(H-I) of 9 RA patients lyse BCG-pulsed, but not TT-pulsed or
unpulsed autologous M+ from PB (A, B, E, H, and 1) or SM (C, D,
F, and G). Values are the percentage of mean 5'Cr release in
triplicate cultures; the SEM 5'Cr release did not exceed 15%. The
E m ratio was 7:l for patient 1, 15:l for patient G, 20:l for patients
C-E, 301 for patient A and H, 40:l for patient F, and 50:1 for
patient B. Pulsing of MI$ did not affect viability of the M 4 in the
absence of effector cells. Thus, the spontaneous "Cr release of
antigen-unpulsed M 4 from PB was 24.6% (n = 5 ; SEM 2.2%) and
that of M 4 from SM was 28% (n = 4; SEM 2.1%) after 18 hours of
incubation, release of BCG-pulsed MI$ from PB was 24.6% (SEM
2.6%) and that of M 4 from SM was 28.3% (SEM 3.3%), release of
'IT-pulsed M4from PB was 27.2% (SEM 2.5%) and that of M4frorn
SM was 28.8% (SEM 3.3%). Ag = antigen (see Figure 1 for other
definitions).
LI ET AL
274
Table 2. Phenotypes of BCG-stimulated T cell lines (A-G) and 65-kd HSP-induced clones (H and 1) derived from 9 RA patients*
% labeling of TCR types
9% labeling of CD MAb
Patient
3
4
8
45RO
alp
V5
VB6
V$
3
3
3
5
3
5
2
83
95
84
95
76
92
93
2
94
6
I
7
I
6
2
3
11
94
91
89
9
2
3
2
2
5
9
45RA
% labeling of
V012
Y/6
HLA-DR
15
1
6
7
12
21
~~
A
B
C
D
E
F
G
H
98
96
90
91
91
89
84
59
63
62
Clone I
Clone 2
97
99
90
96
2
<o.s
1
C0.5
9.5
92
96
98
Clone I
96
I
1
4
93
1
81
19
72
59
20
20
21
12
54
12
20
85
90
82
~0.5
1
95
2
4
4
S
2
95
98
95
90
93
92
92
NT
2
C0.5
<0.5
1
93
96
CO.5
<0.5
94
93
4
22
3
3
8
4
2
1
3
I
I
4 . 5
~ 0 . 5
* The monoclonal antibodies (MAb) were used at a final dilution of 1: LO, except for CD3 and CD45RA, which were at 150 dilution. Clones from
patients H and I, respectively, were as follows: TCRalPclone USSF2C6 (clone I); sequence analysis showed the TCR usage was V& (ref. 29).
TCRalP clone USSFIC6 (clone 2); sequence analysis showed the TCR usage was V,I (ref. 29). TCRy/G clone BSSFD9 (clone I). BCG =
bacillus Calmette-Guerin; HSP = heat-shock protein; RA = rheumatoid arthritis; TCR = T cell receptor; NT = not tested.
SF T cell clone derived from patient 1 were used as
staining controls for the MAb. CD4+ cells were the
dominant population in 6 of 7 SM CTL lines, whereas
the majority of cells from another SM CTL line
primarily expressed the CD8+ phenotype. All
TCRaIP+ T cell clones generated from the SF of 2 RA
patients (patients H and I) expressed the CD4+ phenotype (data not shown). All cell lines and clones were
memory cells, since they were positive for CD45RO
but negative or weakly positive for CD45RA. Table 2
also shows the heterogeneity of the TCR V, usage by
the different cell lines. The cells were activated, since
they all strongly expressed HLA-DR molecules on
their cell surface.
To test whether the BCG-induced cytotoxicity
was exerted by CD4+ or CD8+ cells, depletion experiments were performed by using MAb-coated magnetic beads. Table 3 shows one representative experiment with cells from patient D. Both CD4+ and CD8+
cells lysed BCG-pulsed M+, but to a lesser extent than
did the mixed population. Furthermore, 20 of 23
mycobacteria-specific CD4+ T cell clones from both
the SF and PB of patient H lysed autologous M 4 from
PB that had been pulsed with BCG but not with
unrelated antigen (TT and Chlamydia) (data not
shown). These results, combined with the phenotype
data in Table 2 and depletion data in Table 3, indicated
that both CD4+ and CD8+ cells in the inflamed SM
and SF of RA patients were responsible for BCGinduced cytotoxicity.
Table 3 also shows that both BCG-specific CTL
lines from the SM and PB of patient D could lyse only
BCG-pulsed autologous M 4 from SM but not BCG-
pulsed autologous M+ from PB. This finding may
indicate that in some cases, M 4 from SM may be more
sensitive targets than M4 from PB.
Table 3. Lysis of M 4 by undepleted and depleted BCG-stimulated
S M and PB T cell lines*
% s'Cr release from tar-
gets pulsed with
Purity
Effector cells
(%)
Synovial membrane
CD4+ plus CD8+
-
cD4t
CD8-t
Peripheral blood
CD4+ plus CD8+
CD4+
CD8+
96
95
94
97
E:T
ratio
MC#Ifrom
SM
M 4 from
PB
BCG
TT
BCG
TT
IS: 1
5 :1
2: 1
15: 1
5:l
2: I
15: 1
5:I
38
29
13
20
2
7
I
2
I
0
0
3
3
2
NT
1
0
NT
6
3
NT
2
2
NT
1
0
0
1
2: I
IS: 1
5:1
2: 1
IS: I
5 :1
2: 1
IS: 1
5:l
2: 1
6
33
1
NT
0
0
2
2
NT
3
2
4
4
18
6
12
10
I5
4
22
10
5
17
10
11
0
4
2
1
1
1
5
2
I
NT
NT
1
0
0
0
NT
1
0
NT
* Effector cells were a bacillus Calmette-Guerin (BCG)-stimulated
cytotoxic T lymphocyte line from the synovial membrane (SM)of
patient D. CD8+ cells were depleted from the CD4+ effector
population, and vice versa. The final concentration of antigens used
in the "Cr release assay was 20 &ml for BCG and 1 :120 dilution for
tetanus toxoid (TT). M 4 = macrophage; PB = peripheral blood;
E:T = effect0r:target; NT = not tested.
275
HSP-SPECIFIC T CELLS IN RA
Both TCRa/P+ and TCRy/G+ T cells are involved in antigen-specificcytotoxicity. As demonstrated
above, mycobacteria-specific TCRa/P+ cells bearing
CD4 or CDS, and of various TCR V, chains, have a
cytotoxic capacity. Recently, T cell clones that used
TCR yIG in their recognition of mycobacterial antigens
and 65-kd HSP antigen were isolated from the SF of a
patient with RA (35). It has also been shown that
selective expansion of a specific population of TCRy/S
cells in the SM of patients with RA can occur (36).
Since the line from patient B (Table 2) and that from
patient I (data not shown), in addition to TCRalP
cells, also contained a great percentage of TCRy/G+
cells (Table 2), we wanted to compare the function of
these 2 subsets. Table 4 shows the antigen-specific
lysis of M+ by a representative TCRa/P+ T cell clone
and 2 TCRy/G+ T cell clones raised from patient I.
Recognition of mycobacterial and human 65-kd
HSP by T cell clones from RA patients. The mycobacterial 65-kd HSP has been demonstrated to be an
important target antigen for CD4+ cytotoxic lymphocytes (16,17,19). We have recently found that two of
our 65-kd HSP-specific clones proliferated to synthetic peptides representing sequences which have a
very high degree of homology to the human 65-kd HSP
(29). In Table 5, we demonstrate that these two clones
(USSF2C6 and USSFlFl 1) are capable of lysing autologous macrophages pulsed with BCG, with M bovis
65-kd HSP, and with a recombinant preparation of the
human 65-kd HSP (25). These clones did not, however, lyse M+ which had been pulsed with a control
preparation of supernatant from E coli. This preparation does not contain the 69-kd HSP of E coli (Sioud
M: personal communication). These results therefore
suggest that joint inflammation might to some degree
Table 4. Specific lysis of M4 from PB by T cell clones bearing
both TCRaIP+ and TCR y/S+ *
% "Cr release from
with
T cell clone,
TCR type
E:T
ratio
BSSFB8, alp+
Expt. 1
None
TT
6
0
0
11
6
0
50: 1
25: I
12.5: I
16
14
19
13
10
15: 1
12
8
5
10
11
40: 1
20: 1
10: 1
Expt. 2
7: 1
3: 1
BSSGD9, y/S+
Expt. 1
Expt. 2
7: 1
3: I
BSSPCS, y/S+
Expt. 1
IS: 1
Expt. 2
65-kd
HSP
BCG
17
18
9
32
16
18
20
14
18
8
47
30
23
22
18
5
34
22
20
33
25
16
18
13
6
25
12
18
9
8
24
1
0
6
4
0
0
0
16
8
8
7.5: 1
3.75: 1
20: 1
5:I
2.5: 1
M 4 pulsed
2
0
6
0
0
12
6
7
19
10
12
*
The final concentration of antigens used was I : 120 dilution for TT,
and 20 pdml for Mycobacterium bovis 65-kd heat-shock protein
(HSP) and BCG. Experiments 1 and 2 represent 2 different experiments using the same clone. For BSSFB8, experiment 2, it was not
possible to expand the clones to such an extent that a higher
effector:target (E:T) cell ratio could be tested. TCR = T cell
receptor (see Table 3 for other definitions).
be attributed to bacteria-induced T cell clones crossreacting with the self HSP.
Class I1 major histocompatibility complex
(MHC) restriction of mycobacteria-induced CTL from
SM and SF. The MHC restriction of mycobacteriainduced CTL lines and clones from SM and SF was
Table 5. Recognition of mycobacterial 65-kd HSP and a recombinant human 65-kd HSP by mycobacterial 65-kd HSP-induced CTL clones
raised from the S F of patient H*
% "Cr release of
Clone
E:T
ratio
None
BCG
(20 @ml)
Mycobacrerium
bovis
65-kd HSP
(20 pdml)
Mc$from PB pulsed with
Recombinant
human 65-kd HSP
(pdml)
5
20
10
~
Experiment 1
USSF2C6
Experiment 2
USSFlFllt
Supernatant of
Escherichia coli
(pdml)
TT
5
10
20
(1:120)
~~~~~~
10: 1
24
73
81
-
-
49
-
-
24
25
20: 1
26
-
-
44
47
-
30
28
-
21
* HSP = heat-shock protein; CTL = cytotoxic T lymphocyte; SF = synovial fluid; M4 = macrophage; PB = peripheral blood; E:T = effector:
target; BCG = bacillus Calmette-Guerin; TT = tetanus toxoid.
t In a separate experiment, the 65-kd HSP-induced clone USFlFl I strongly lysed M 4 pulsed with M bovis 65-kd HSP, but did not lyse M 4
pulsed with BCG.
LI ET AL
276
Table 6. Determination of class I1 MHC restriction by the mycobacteria-specific T cell line and T cell clones derived from RA patients, using
a panel of allogeneic M& from PB as targets*
Effector
Allogeneic target
(HLA-DR)
pulsed antigen
USSFIC6,
(HLA-DRU5).
E:T ratio
BCG line
(HLA-DR4/5),
E:T ratio
1O:l
5:l
20:l
10: 1
0
0
0
0
0
0
0
0
0
17
30
5
NT
0
1
NT
0
0
NT
0
0
NT
33
0
27
12
2
26
9
2
23
2
0
4
13
14
7
7
17
0
15
2
4
4
0
4
20:1
USS FIC6
(HLA-DR4w4/5),
E:T ratio
USSF2DI 1,
(HLA-DR4/5),
E:T ratio
2.5:1
1.25: 1
5:l
5:1
13
2
27
4
19
0
6
3
0
0
1
1
0
0
0
NT
37
0
NT
22
0
14
28
2
7
8
2
4
2
0
1
4
4
5
1
4
8
I
2
5
0
I
0
1
0
4
0
0
0
0
0
0
0
0
2
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
8
3
4
6
6
I
3
0
0
0
0
0
I
0
7.5: I
IS: 1
22
28
3
12
0
1 (DR4/7)
BCG
65-kd HSP
IT
2 (DR2/4)
BCG
65-kd HSP
TT
3 (DK5/7)
BCG
65-kd HSP
TT
4 (DR5/6)
BCG
65-kd HSP
TT
5 (DR1/2)
BCG
65-kd HSP
TT
6 (DR4w4, DR2)
7 (DR2, DR211)
8 (DR4w4, DR4w13)
9 (DR5, DR7)
2
3
2
0
0
5
I
1
1
1
5
4
0
* The BCG line of effector cells was a T cell line derived from patient F; USSFIC6 and USSF2Dl 1 were T cell clones derived from patient H.
See Table 4 for the final concentrations of antigens used. Targets 6-9 were pulsed with BCG. Values are the 3' 6 "Cr release from M&targets
after subtraction of ?6 5'Cr release from nonpulsed M& targets. MHC = major histocompatibility complex; RA = rheumatoid arthritis; NT =
not tested (see Table 5 for other definitions).
investigated using a panel of class I1 MHC-matched
and mismatched allogeneic M4 as targets (Table 6).
One SM CTL line from patient G and 2 CTL clones
from patient H all showed specific lysis of autologous
M 4 when pulsed with relevant antigens (see above and
below). Table 6 shows that the BCG line specifically
lysed only DR5-matched allogeneic M4, whereas 2
clones specifically lysed DRCmatched allogeneic M4.
The results indicate that HLA-DR5-restricted cells
are dominant in the BCG line. It is also possible that
the allogeneic M4 used as targets for the cell line
lacked the right DR4/Dw type for DRCrestricted cells
in this line. Clone USSFlC6 lysed DR4-matched allogeneic M+ pulsed with BCG and/or 65-kd HSP (Table
6). Table 6 also shows that this clone specifically lysed
DRWDw4-matched allogeneic M4 when pulsed with
BCG. Clone USSF2D1 I lysed only one DRCmatched
target. The reason it did not lyse another DR4matched target might be due to a too low E:T cell ratio
or a mismatched target at the subtype level. None of
the clones or the cell line lysed HLA-DR-mismatched
allogeneic M 4 targets. These data suggest that the
cytotoxicity of SM and SF CTL specific for BCG
and/or 65-kd HSP is restricted by HLA-DR molecules.
Inhibition studies were also performed by using
HLA-specific MAb. As shown in Table 7, an HLADR-specific MAb inhibited the lysis of BCG-pulsed
M 4 by 2 representative BCG-specific SM T cell lines,
and the lysis of M hovis 65-kd HSP-pulsed M 4 by a
65-kd HSP-specific SF T cell clone. The lysis could
not be inhibited by HLA-DQ, HLA-DP, HLA class
I-specific MAb, or control antibodies with the same
IgG isotype.
DISCUSSION
In the present study, a cytotoxicity assay was
used to study the in vitro capacity of mycobacteria-
HSP-SPECIFIC T CELLS IN RA
277
Table 7. Inhibition of specific cytotoxicities by an anti-HLA-DR monoclonal antibody (MAb)*
% specific lysis of M+
(pulsed antigen; effector cells)
BCG;
BCG line of
patient A,
E:T ratio
MAb added,
dilution
None
Anti-HLA class 1
1 :2oo
1: loo
Anti-HLA-DR
1:200
1:loo
Anti-HLA-DQ
1:2oo
1 : 100
Anti-HLA-DP
1:200
1 : 100
Control IgG I a
1 :200
1:lOO
Control IgG2b
1 :200
1 : 100
M b & ~ 65-kd HSP;
65-kd HSP S F clone
USSFIC6,
E : T ratio
10: 1
15: 1
7: 1
BCG;
BCG line of
patient F,
E:T ratio
15: 1
41
30
48
36
38
34
31
32
47
49
NT
NT
15
5
II
2
23
2
8
NT
33
39
30
29
49
51
31
NT
37
46
33
36
50
47
36
NT
42
41
33
35
NT
NT
NT
NT
39
35
30
32
NT
NT
NT
NT
*The final concentration of the pulsed antigens used was 20 pg/ml for BCG and 10 pghl for
Mycobucferium bovis 65-kd HSP. The IgG isotype control MAb used were TIB96 for 1gGla and
HD237 for IgG2b. Background lysis has been subtracted from the values shown. The background lysis
for BCG line of patient A at IS: 1 E:T ratio was 30% for medium and 31% for BCG, and at 7: I was
25% for medium and 24% for BCG. The background lysis for BCG line of patient F at 15: 1 E :T ratio
was 35% for medium and 28% for BCG. The background lysis for 65-kd HSP SF clone USSFIC6 at
10: 1 E:T ratio was 26% for medium and 27% for 65-kd HSP. NT = not tested (see Table 5 for other
definitions).
induced T lymphocyte lines and clones derived from
rheumatoid SM and SF to kill human autologous
andlor allogeneic M 4 from SM, SF, or PB from RA
patients. We demonstrated that long-term cultured SM
and SF T lymphocytes specific for BCG and 65-kd
HSP, derived from inflamed tissues of RA patients, are
capable of lysing relevant antigen-pulsed autologous
MC#Ifrom both PB and SM, but not M 4 pulsed with
irrelevant antigens. The 65-kd HSP molecule has been
reported to be an important antigen for restimulating
BCG/PPD-specific cytotoxic precursor cells in vitro
(17). It has also been shown that CD8+ T cells from
animals immunized with the mycobacterial65-kd HSP
could lyse bone marrow MC#Iactivated with interferon-y
or infected with either murine cytomegalovirus or M
bovis (14). The 65-kd HSP is highly conserved (25),
and the protein is present in inflamed human joints
(37). Due to the high conservation, certain epitopes on
65-kd HSP could become immunogenic during stages
of a mycobacterial infection, and subsequently activate autoimmune T cells.
Lamb et al(38) have proposed that autoimmune
disease could occur if lymphocytes activated by microbial stress proteins during infection also included
cells reactive with conserved cross-reactive determinants. Consistent with this, we have observed that a
few mycobacterial65-kd HSP-specific SF CTL clones
could specifically lyse MC#Itargets pulsed with a recombinant human 65-kd HSP preparation. We also observed that TCRy/S+ cells could specifically lyse M+
targets pulsed with BCG and M hovis 65-kd HSP.
Recently, it has also been demonstrated that a mycobacterial 65-kd HSP-reactive TCRyIS+ cell clone
responded to the homologous human HSP (26). It is
possible that the killing of M+ pulsed with mycobacterial antigens could reflect an immune response specific to stress proteins of the host (14). Thus, local
accumulation of self stress proteins at a site of inflam-
278
mation might trigger autoreactive T cells and result in
a cycle of events that include cytotoxicity leading to
the pathology associated with autoimmune disease.
The association between RA and HLA-DR4
has been firmly established. In most populations, RA
is associated with the HLA-DR4 phenotype (39-42).
Recently, it has also been shown that DR4 is associated with a more severe disease course (43). The
biological significance of class I1 MHC molecules is
that they function as antigen-presenting structures of
peptides for T cells. Both in vivo and in vitro observations have indicated a role for HLA-DR4 in controlling the T cell response to mycobacterial antigens
( I I ,44). Recently, HLA-DR4/Dw4-restricted T cells
which recognized self antigen(s) in the rheumatoid
synovial compartment have been demonstrated (45). It
is therefore significant that the recognition of BCG
and/or 65-kd HSP by some CD4+, TCRaIP+ T cell
clones is restricted by HLA-DR4. We have demonstrated that the recognition of BCG by our clone
USSFIC6 was indeed restricted by DR4/Dw4. We
have also shown that the proliferation by 3 of 4 65-kd
HSP-specific SF T cell clones could be inhibited by
the anti-HLA-DR MAb (29). The data suggest that the
T cell repertoire for recognizing 65-kd HSP in the
context of DR4 is present in the inflamed joint. This is
different from the HLA-DP-restricted 65-kd HSPreactive RA synovial T cell clones reported by Gaston
et al (46).
The presentation of T cell epitopes on the 65-kd
HSP by HLA-DR5 molecules has also been reported
previously (for review, see ref. 47). Here, we show
that recognition of the 65-kd HSP by T cells from 1
mycobacteria-specific T cell line is restricted primarily
via HLA-DR5 molecules. So far, we do not know the
possible restriction element(s) for our mycobacteriaspecific TCRy/G+ RA T cell clones. It has been
suggested, however, that the recognition of mycobacterial antigens by TCRy/S+ T cells is non-MHCrestricted (35,48-50), and these cells have also been
implicated in the pathogenesis of RA (35,3631). Nevertheless, the heterogeneity of T cells directed against
mycobacterial antigens infiltrating the inflamed joints
may reflect the impact of HLA class I1 molecules on the
disease, and analysis of the role of HLA molecules may
further elucidate the pathogenetic mechanisms of RA.
The majority of specific CTL cells described here
are TCRcrIP positive. The expression of V, families by
the BCG CTL lines was heterogeneous. We also demonstrated that both TCRaIP and TCRylGbearing CTL
clones generated from 1 RA patient displayed similar
LI ET AL
antigen-specific lysis. These findings suggest that individual responses to BCG andor 65-kd HSP will show
differences with respect to HLA class I1 restriction,
peptide specificities, and TCR usage.
The major issue to be addressed is whether
mycobacteria-specific cytotoxic T cells in inflamed
tissues contribute to protective immunity or to tissue
damage in focal immune reactions. Regarding protection, Kaufmann (52) has demonstrated that mycobacterial antigens induced the generation of both CD4+
and CD8+ CTL from mice. It is assumed that these
cells contribute to protective immunity by eliminating
the reservoir of mycobacteria by killing the infected
M4. A recent observation in cells from humans has
shown that 65-kd HSP-stimulated effector cells
strongly inhibited colony-forming unit formation by
live BCG-infected autologous M$J(1 7). Regarding immunopathology, one of the main features of leprosydamage to tissues surrounding the lesions-may be
caused by nonspecific effector mechanisms that come
into play during the immune response to M lepraeinfected cells, such as Schwann cells, which express
class I and class I1 MHC antigens and can present
antigens to T cells (53).
There are a number of reasons for considering a
role for mycobacteria-specific CTL in the immunopathology of rheumatoid lesions. In the experimental
rat model, adjuvant arthritis can be induced by immunization with M tuberculosis (1,2), and T cells reactive
with 65-kd HSP can transfer arthritis to naive rats
(3,4). In humans, strong antimycobacterial activity is
found in the synovial compartment (&lo), and some
cancer patients undergoing BCG immunotherapy develop arthritis (54). We have demonstrated that both
CD4+ and CD8+ CTLs, as well as TCRaIp-t and
TCRy/G+ CTLs, were involved in the mycobacterialspecific lysis of autologous M4. A few mycobacterial
65-kd HSP-specific CTL clones also recognize self
HSP. We have also demonstrated that the APC from
some synovial compartments have a much greater
capacity for triggering mycobacteria-specific CTL
than those from PB. Furthermore, mycobacterial
antigen-induced CTL can also lyse antigen-pulsed M4
from healthy individuals (16-20), leprosy patients
(16,18), tuberculosis patients (20), and mice (21-23).
This suggests an important role for CTL in the immune
response to mycobacteria and other intracellular parasites. Thus, the involvement of mycobacteria-specific
CTL in local inflammation might be a general characteristic of chronic inflammatory diseases including
rheumatoid arthritis.
HSP-SPECIFIC T CELLS IN RA
ACKNOWLEDGMENTS
We thank the HLA typing laboratories of the Department of Immunohaematology and Bloodbank, Leiden University, The Netherlands, and the Institute of Transplantation Immunology, University of Oslo, Norway, for HLA
typing. Zhang Yan is also acknowledged for technical assistance, and Suzanne Garman-Vik and Bente Brenna for
typing the manuscript.
REFERENCES
1. Holoshitz J, Haparstek Y , Ben-Nun A, Cohen IR: Lines
of T lymphocytes induce or vaccinate against autoimmune arthritis. Science 219:5658, 1983
2. Cohen IR, Holoshitz J, van Eden W, Frenkel A: T
lymphocyte clones illuminate pathogenesis and affect
therapy of experimental arthritis. Arthritis Rheum 28:
841-845, 1985
3. Van Eden W, Holoshitz J , Nevo Z, Frenkel A, Klajman
A, Cohen IR: Arthritis induced by a T-lymphocyte clone
that responds to Mycobacterium tuberculosis and to
cartilage proteoglycans. Proc Natl Acad Sci USA 82:
5117-5120, 1985
4. Van Eden W, Thole JER, van der Zee R, Noordzij A,
van Embden JD, Hensen EJ, Cohen IR: Cloning of the
mycobacterial epitope recognized by T lymphocytes in
adjuvant arthritis. Nature 331: 171-173, 1988
5. Polla BS: A role for heat shock proteins in inflammation? Immunol Today 9: 134-137, 1988
6. Abrahamsen TG, Fr#land SS,Natvig JB: In vitro rnitogen stimulation of synovial fluid lymphocytes from rheumatoid arthritis and juvenile rheumatoid arthritis patients: dissociation between the response to antigen and
polyclonal mitogens. Scand J Immunol7:81-90, 1978
7. Holoshitz J , Druker I, Yaretzky A, van Eden W, Klajman A, Lapidot Z, Frenkel A, Cohen IR: T lymphocytes
of rheumatoid arthritis patients show augmented reactivity to a fraction of mycobacteria cross-reactive with
cartilage. Lancet II:305-309, 1986
8. Res PCM, Schaar CG, Breedveld FC, van Eden W, van
Embden JDA, de Vries RRP, Cohen IR: Synovial fluid T
cell reactivity against 65 kD heat shock protein of
mycobacteria in early chronic arthritis. Lancet II:478480, 1988
9. Gaston JSH, Life PF, Bailey LC, Bacon PA: In vitro
responses to a 65 kilodalton mycobacterial protein by
synovial T cells from inflammatory arthritis patients. J
Immunol 143:2494-2500, 1989
10. Gaston JSH, Life PF, Jenner PJ, Colston MJ, Bacon
PA: Recognition of a mycobacteria-specific epitope in
the 65-kD heat-shock protein by synovial fluid-derived T
cell clones. J Exp Med 171:831-841, 1990
11. Ottenhoff THM, Torres P, de las Aguas JT, Fernandez
R, van Eden W, de Vries RRP, Stanford JL: Evidence
for an HLA-DR4-associated immune-response gene for
279
mycobacterium tuberculosis: a clue t o the pathogenesis
of rheumatoid arthritis? Lancet 11:310-313, 1986
12. Burmester GR, Altstidl U, Kalden JR, Emmrich F:
Stimulatory response towards the 65 kDa heat shock
protein and other mycobacterial antigens in patients
with rheumatoid arthritis. J Rheumatol 18: 171-176, 1991
13. Res PCM, Telgt D, van Laar JM, Pool MO, Breedveld
FC, de Vries RRP: High antigen reactivity in mononuclear cells from sites of chronic inflammation. Lancet
336:140&1408, 1990
14. Koga T, Wand-Wiirttenberger A, DeBruyn J, Munk
ME, Schoel B. Kaufmann SHE: T cells against a bacterial heat shock protein recognize stressed macrophages.
Science 245: 1112-1 115, 1989
15. Mustafa AS, Godal T: BCG induced CD4' cytotoxic T
cells from BCG vaccinated healthy subjects: relation
between cytotoxicity and suppression in vitro. Clin Exp
Immunol 69:255-262, 1987
16. Ottenhoff THM, Kale ABB, van Embden JDA, Thole
JER, Kiessling R: The recombinant 65 kD heat shock
protein of Mycobacterium bovis bacillus CalmetteGuerin/M. tuberculosis is a target molecule for CD4+
cytotoxic T lymphocytes that lyse human monocytes. J
Exp Med 168:1947-1952, 1988
17. Kaleab B, Kiessling R, van Embden JDA, Thole JER,
Kumararatne DS, Pisa P, Wondimu A, Ottenhoff THM:
Induction of antigen-specific CD4+ HLA-DR-restricted
cytotoxic T lymphocytes as well as nonspecific nonrestricted killer cells by the recombinant mycobacterial
65-kDa heat-shock protein. Eur J Immunol 20:369-377,
1990
18. Kaleab B, Ottenhoff T , Converse P, Halapi E, Tadesse
G , Rottenberg M, Kiessling R: Mycobacterial-induced
cytotoxic T cells as well as unspecific killer cells derived
from healthy individuals and leprosy patients. Eur J
Immunol 20:2651-2659, 1990
19. Munk ME, Schoel B, Modrow S, Karr RW, Young RA,
Kaufmann SHE: T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein. J
Immunol 143:2844-2849, 1989
20. Kumararatne DS, Pithie AS, Drysdale P, Gaston JSH,
Kiessling R, Iles PB, Ellis CJ, lnnes J, Wise R: Specific
lysis of mycobacterial antigen-bearing macrophages by
class I1 MHC-restricted polyclonal T cell lines in healthy
donors or patients with tuberculosis. Clin Exp Immunol
80:314-323, 1990
21. Kaufmann SHE, Flesch I: Function and antigen recognition pattern of L3T4+ T-cell clones from Mycobacterium tuberculosis-immune mice. Infect Immun 54:29 1296, 1986
22. DeLibero G , Kaufmann SHE: Antigen-specific Lyt-2'
cytolytic T lymphocytes from mice infected with the
intracellular bacterium Listeria monocytogenes. J Immunol 137:268&2694, 1986
LI ET AL
23. Kaufmann SHE, Hug E, DeLibero G: Listeria monocytogenes-reactive T lymphocyte clones with cytolytic
activity against infected target cells. J Exp Med 164:363368, 1986
24. Arnett FC, Edworthy SM, Bloch DA, McShane DJ,
Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang
MH, Luthra HS, Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL,
Hunder GG: The American Rheumatism Association
1987 revised criteria for the classification of rheumatoid
arthritis. Arthritis Rheum 31:315-324, 1988
25. Jindal S, Dudani AK, Singh B. Harley CB, Gupta RS:
Primary structure of a human mitochondria1 protein
homologous to the bacterial and plant chaperonins and
to the 65-kilodalton mycobacterial antigen. Mol Cell Biol
9:2279-2283, 1989
26. Haregewoin A, Singh B, Gupta RS, Finberg RW: A
mycobacterial heat-shock protein-responsive ysT cell
clone also responds to the homologous human heatshock protein: a possible link between infection and
autoimmunity. J Infect Dis 163: 156-160, 1991
27. Sioud M, Kjeldsen-Kragh J, Quayle A, Wiker HG,
Sdrskarr D, Natvig JB, Fdrre 0:Immune responses to
18.6 and 30-kDa mycobacterial antigens in rheumatoid
patients, and V, usage by specific synovial T-cell lines
and fresh T cells. Scand J Immunol 34:803-812, 1991
3 Waalen K, Fgrre $3, Teigland J, Natvig JB: Human
rheumatoid synovial and normal blood dendritic cells as
antigen presenting cells: comparison with autologous
monocytes. Clin Exp Immunol 7O:l-9, 1987
29. Quayle AJ, Wilson K, Li SG, Kjeldsen-Kragh J, Oftung
F. Shinnick TM, Fgrre $3, Capra JD, Natvig JB: Peptide
recognition, TCR usage and HLA restriction elements of
human shock protein (hsp) 60 and mycobacterial65-kDa
hsp reactive T cell clones from rheumatoid synovial
fluid. Eur J Immunol (in press)
30. Lea T, Vartdal F, Nustad K, Funderud S, Berge A,
Ellingsen T, Schmid R, Stenstad P, Ugelstad J: Monosized magnetic polymer particles: their use in separation
of cells and subcellular components, and in the study of
lymphocyte function in vitro. J Mol Recognition 1:9-18,
1988
31. McMichael AJ: Leucocyte Typing. 111. White Cell Differentiation Antigens. Oxford, Oxford University Press,
I987
32. Koning F: Identification and functional relevance of
epitopes on human lymphocytes (thesis). University of
Leiden, 's-Gravenhage, JH Pasmans, The Hague, 1986
33. Oi VT, Jones PP, Goding JW, Herzenberg LA, Herzenberg LA: Properties of monoclonal antibodies to mouse
Ig allotypes. H-2, and Ia antigens. Curr Top Microbiol
Immunol 81: 115-120, 1978
34. Kjeldsen-Kragh J, Quayle A, Kalvenes C, F@rre $3,
Sdrskaar D, Vinje 0, Thoen J, Natvig JB: Ty6 cells in
juvenile rheumatoid arthritis and rheumatoid arthritis: in
the juvenile rheumatoid arthritis synovium the TyS cells
express activation antigens and are predominantly
Val+, and a significant proportion of these patients have
elevated percentages of Ty6 cells. Scand J Immunol
32:651460, 1990
35. Holoshitz J, Koning F, Coligan E, DeBruyn J, Strober
S: Isolation of CD4-CD8- mycobacteria-reactive T
lymphocyte clones from rheumatoid arthritis synovial
fluid. Nature 339:22&229, 1989
36. Andreu JL, Trujillo A, Alonso JM, Mulero J, Martinez-A c: Selective expansion of T cells bearing the $8
receptor and expressing an unusual repertoire in the
synovial membrane of patients with rheumatoid arthritis. Arthritis Rheum 34:808-814, 1991
37. Karlsson-Parra A, Soderstrom K, Ferm M, Ivanyi J,
Kiessling R, Klareskog L: Presence of human 65 kD
heat shock protein (hsp) in inflamed joints and subcutaneous nodules of RA patients. Scand J Immunol3 1 :283288, 1990
38. Lamb JR, Bal V, Mendez-Samperio P, Mehlert A, So A,
Rothbard J, Jindal S, Young RA, Young DB: Stress
proteins may provide a link between the immune response to infection and autoimmunity. Int Immunol
I : 191-196, 1989
39. Watanabe Y, Tokunaga K, Matsuki K, Takeuchi F,
Matsuta K, Maeda H, Omoto K, Juji T: Putative amino
acid sequence of HLA-DRB chain contributing to rheumatoid arthritis susceptibility. J Exp Med 169:22632268, 1989
40. Wordsworth BP, Lanchbury JSS, Sakkas LI, Welsh K1,
Panayi GS. Bell JI: HLA-DR4 subtype frequencies in
rheumatoid arthritis indicate that DRBl is the major
susceptibility locus within the HLA class I1 region. Proc
Natl Acad Sci USA 86:10049-10053, 1989
41. Morel PA, Horn GT, Budd RC, Erlich HA, Fathman
CG: Shared molecular markers of genetic predisposition
to seropositive rheumatoid arthritis. Hum Immunol 27:
90-99, 1990
42. Lanchbury JSS, Hall MA, Welk KI, Panayi GS: Sequence analysis of HLA-DR4B 1 subtypes: additional
first domain variability is detected by oligonucleotide
hybridization and nucleotide sequencing. Hum Immunol
27:136-144, 1990
43. Van Zeben D, Hazes JMW, Zwinderman AH, Cats A,
Schreuder GMT, D'Amaro J, Breedveld FC: Association of HLA-DR4 with a more progressive disease
course in patients with rheumatoid arthritis: results of a
followup study. Arthritis Rheum 34:822-830, 199I
44. Palacios-Boix AA, Estrada GI, Colston MJ, Panayi GS:
HLA-DR4 restricted lymphocyte proliferation to a Mycobacterium tuberculosis extract in rheumatoid arthritis
and healthy subjects. J Immunol 140:18441850, 1988
45. Devereux D. O'Hehir RE, McGuire J, van Schooten
WCA, Lamb JR: HLA-DR4Dw4-restricted T cell recog-
28 1
HSP-SPECIFIC T CELLS IN RA
46.
47.
48.
49.
nition of self antigen(s) in the rheumatoid synovial
compartment. Int Immunol 3:635-640, 1991
Gaston JSH, Life PF, van der Zee R,Jenner PJ, Colston
MJ, Tonks S, Bacon PA: Epitope specificity and MHC
restriction of rheumatoid arthritis synovial T cell clones
which recognize mycobacterial65 kilodalton heat shock
protein. Int Immunol (in press)
Thole JER, van der Zee R: The 65 kD antigen: molecular
studies on a ubiquitous antigen, Molecular Biology of
the Mycobacteria. Edited by J McFadden. London,
Academic Press, 1990
O'Brien RL, Happ MP, Dallas A, Palmer E, Kubo R,
Born WK: Stimulation of a major subset of lymphocytes
expressing T cell receptor y6 by an antigen derived from
Mycobacterium tuberculosis. Cell 57567474, 1989
Janis EM, Kaufmann SHE, Schwartz RH, Pardoll DM:
Activation of y6 T cells in the primary immune response
50.
51.
52.
53.
54.
to Mycobacterium tuberculosis. Science 244:713-716,
1989
Porcelli S, Brenner MB, Greenstein JL, Balk SP, Terhorst C, Bleicher PA: Recognition of cluster of differentiation 1 antigens by human CD4-CD8- cytolytic T
lymphocytes. Nature 341:447450, 1989
Kaufmann SHE: Heat shock proteins and immune response. Immunol Today 1 1 : 129-136, 1990
Kaufmann SHE: CD8' T lymphocytes in intracellular
microbial infections. Immunol Today 9: 168-174, 1988
Steinhoff U , Kaufmann SHE: Specific lysis by CD8' T
cells of Schwann cells expressing Mycobacterium leprae
antigens. Eur J Immunol 18:969-972, 1988
Torisu M, Miyahara T , Shinohara N , Ohsato K ,
Sonozaki H: A new side effect of BCG immunotherapy:
BCG-induced arthritis in man. Cancer Immunol Immunother 5:77-83, 1978
Документ
Категория
Без категории
Просмотров
3
Размер файла
1 053 Кб
Теги
эspecific, inflammation, heat, protein, mycobacterium, cytotoxic, shock, synovial, human, lymphocytes, rheumatoid
1/--страниц
Пожаловаться на содержимое документа