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and Are Protective in Adjuvant Arthritis1
*
Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands; and
Department of Immunobiology, Biomedical Primate Research Center, Rijswijk, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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.
Enhanced autoproliferation and expression of the cell surface molecule
B7.2 by these T cells were observed in response to syngeneic
heat-shocked APC, which indicated that the autoproliferation and
expression of B7.2 resulted from the recognition of endogenously
expressed and processed hsp. Despite their strong autoreactivity, upon
transfer such T cells were found to induce a significant disease
reduction in adjuvant arthritis. In contrast, T cells both primed and
restimulated with peptide M256–270 became unresponsive toward
syngeneic APC as well as toward the conserved core peptide M256–265,
and they were devoid of protective capacity. This study demonstrates
that the loss of self-tolerance toward hsp60 does not necessarily lead
to autoimmune disease, but that hsp60-specific self-reactive and
autoproliferative T cells may mediate T cell regulation in
arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many laboratories have reported cross-reactive T cell recognition of Mt hsp60 and self hsp60, most likely due to the high degree of homology between Mt hsp60 and its mammalian homologue (9, 10, 11, 12). This strong sequence conservation is not restricted to hsp; however, hsp are unique in their up-regulated expression in response to various stress stimuli (13), and elevated expression of hsp60 has been found in inflamed synovia of arthritis patients and arthritic rats (14, 15). Thus, bacterial hsp-primed T cells, cross-reactive with self hsp60, might play an important role in the regulation of pathogenic autoimmune responses.
We investigated whether the protective effect of M256–270-reactive T
cells in AA was mediated through cross-reactivity with self and whether
it was associated with the production of regulatory cytokines. The
exact requirements for generating such T cells were studied, and we
provide data indicative of a protective mechanism of these T cells in
AA. We show now the importance of cross-reactivity with self, as T
cells reactive to the highly conserved peptide M256–265, which were
able to confer protection, were strongly autoproliferative, induced
IL-4/IL-10 and IFN-, and expressed B7.2. On the other hand, T cells
that were restricted to nonconserved parts of peptide M256–270 and
that were unable to confer protection did not autoproliferate, had a
different cytokine profile, and did not express B7.2.
Thus, we provide evidence that the induction of IL-4/IL-10- and
IFN--secreting and/or selectively B7.2-expressing, autoproliferative
regulatory T cells specific for self-hsp is crucial to the observed
protection in AA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male inbred Lewis rats (RT1l MHC haplotype) were obtained from the University of Limburg (Maastricht, The Netherlands), and male Fisher F344 rats were obtained from Harlan/Olac (Horst, The Netherlands). The animals were housed under conventional conditions. Rats were 7–9 wk old at the start of each experiment.
Ags and adjuvants
Heat-killed Mycobacterium tuberculosis (Mt) strain H37Ra and IFA were obtained from Difco (Detroit, MI). Dimethyldioctadecyl ammonium bromide (Eastman Kodak, Rochester, NY) was used as adjuvant (16). It was prepared as a 20 mg/ml suspension in PBS, sonicated, and heated to produce a gel, which was mixed 1/1 with 2 mg/ml peptide solution before immunization. Mt hsp60 peptides M256–270 (ALSTLVVNKIRGTFK), M256–265 (ALSTLVVNKI), and M211–225 (AVLEDPYILLVSSKV) and rat hsp60 peptides R256–270 (ALSTLVLNRLKVGLQ) and R256–265 (ALSTLVLNRL) were made by solid phase peptide synthesis (17) and contained an amide group at the C-terminus. Peptides were checked by reverse phase HPLC and fast atom bombardment-mass spectrometry.
Generation of peptide-specific T cell lines
Rats were lightly anesthetized using ether and immunized with 50 µg of synthetic peptide in 50 µl of PBS/dimethyldioctadecyl ammonium bromide in each hind footpad (i.e., 100 µg/rat). Ten days later, draining popliteal lymph nodes were removed, disaggregated, washed twice, and used as a source of primed lymph node cells (PLNC). T cell lines were generated by culturing PLNC at 5 x 106/ml in culture medium (IMDM, Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 5 x 10-5 M 2-ME), and 2% naive inactivated rat serum (NRS) in the presence of 10 µg/ml peptide. After 3 days, viable cells were harvested using a Ficoll-Isopaque gradient and expanded for 4 days in culture medium supplemented with 10% EL-4 supernatant and 10% FCS. Seven days after initial stimulation, 4 x 105 T cells/ml were restimulated with 1 x 106/ml irradiated (3000 rad) syngeneic splenocytes as a source of APC and with 10 µg/ml peptide in culture medium supplemented with 2% NRS. Lines were maintained in this 7-day restimulation cycle. The M52ext T cell line was generated by immunization and restimulation with peptide M256–270, the M52core T cell line by immunization with peptide M256–270 and restimulation with the core-peptide M256–265, and the T cell line M43 by immunization and restimulation with peptide M211–225 (6).
T cell proliferation assay
T cell lines were cultured in triplicate in 200-µl flat-bottom microtiter wells (Costar, Cambridge, MA) at 2 x 104 T cells/well with or without Ag and 2 x 105 irradiated syngeneic splenocytes/well as APC in culture medium supplemented with 1% NRS. Responses to a dose range of individual peptides were tested; Con A (2.5 µg/ml) and human rIL-2 (10 U/ml; PharMingen, San Diego, CA) were used as positive controls, and wells without Ag or mitogen were used as negative controls. In some experiments heat-shocked or normally treated splenocytes (or adherent splenocytes; see below) without added peptide were used as APC. Isolation and heat shock treatment were performed as described below. If adherent APC were used, 2 x 104 T cells/well were cultured in triplicate in 200-µl flat-bottom microtiter wells with 1 x 105 irradiated adherent APC/well in culture medium supplemented with 1% NRS.
Cultures were incubated for 96 h at 37°C in 5% CO2 and pulsed with [3H]thymidine (0.4 µCi/well; sp. act., 1 Ci/mmol; Amersham, Arlington Heights, IL) for the final 16–18 h. [3H]thymidine incorporation was measured using a liquid scintillation beta counter (Wallac Oy, Turku, Finland). Results are expressed as the mean counts per minute ± SD of triplicate wells.
Heat shock treatment of APC (splenocytes)
Rat splenocytes were isolated, reconstituted to a concentration of 1 x 107/ml in culture medium supplemented with 1% NRS, and, under a 5% CO2 atmosphere, heat shocked for 30 min at 43°C or left untreated at 37°C. After a recovery period of 4 h at 37°C, the cells were irradiated, washed, and used in a proliferation assay as heat-shocked (43°C) or normally treated (37°C) APC, as described above. Under these conditions heat-shocked splenocytes were found to have up-regulated the expression of hsp60 when analyzed by Western blotting.
Isolation and heat shock treatment of adherent APC
Splenocytes (1 x 107/ml), obtained from Lewis or Fisher rats, in culture medium supplemented with 10% FCS were allowed to adhere for 1 h at 37°C under a 5% CO2 atmosphere in a 24-well plate (Costar, Cambridge, MA). Nonadherent cells were removed by washing extensively with warm culture medium. Adherent cells were allowed to recover for 15 min under a 5% CO2 atmosphere in culture medium supplemented with 1% NRS before heat stress treatment. Adherent cells were heat shocked for 30 min at 43°C or were left untreated at 37°C and allowed to recover for 4 h at 37°C in 5% CO2. After washing the adherent cells once, 4 x 105/ml T cells in culture medium supplemented with 1% NRS were added 7 days after the last restimulation to the 24-well plate containing adherent APC. Thus, incubated T cells were analyzed by flow cytometry after 20–24 h. If used in proliferation assays, adherent cells were first irradiated (3000 rad) and subsequently used as described above.
Blocking with anti-MHC class II mAbs
Anti-MHC class II mAb were added in proliferation assays to determine the MHC restriction of the autoproliferation. OX6 (anti-RT1.Bl) and OX17 (anti-RT1.Dl), both mouse IgG1, were added at 5 and 20 µg/ml.
Flow cytometry
Expression of the molecule B7.2 on the surface of peptide-specific T cell lines was analyzed after 20–24 h of incubation using the following protocol. APC were isolated and treated as described above for isolation and heat shock treatment of adherent APC. T cell lines (4 x 105/ml in culture medium supplemented with 1% NRS) were added 7 days after the last restimulation to normally treated or heat-shocked APC obtained as described above. After 20–24 h of incubation, viable cells were collected by Ficoll-Isopaque gradient centrifugation, washed twice with culture medium, counted, and reconstituted to 4 x 105 cells/ml. Cells were washed once with FACS buffer (PBS containing 1% BSA, 0.1% NaN3, and 4% NRS) and incubated for 30 min on ice in FACS buffer-diluted (1/2) mouse anti-rat B7.2 mAb (anti-CD86, 24F, IgG1 isotype). Cells were washed twice and incubated for 30 min with FACS buffer-diluted (1/300) PE-conjugated goat anti-mouse total IgG Ab (PharMingen). Cells were washed twice and resuspended in FACS buffer without NRS. Residual dead cells were excluded based on the forward/side scatter by raising the threshold on forward scatter, and the PE fluorescence of the T cell population was measured in the fluorescent light 2 channel of a FACScan (Becton Dickinson, Mountain View, CA) after gating on the fluorescent light 1-negative population when incubated with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester-labeled APC (20). Data were analyzed using CellQuest software (Becton Dickinson). The isotype control mAb was UD15 (IgG1, anti-choramphenicol) (18). The 24F hybridoma was a gift from Dr. H. Yagita (Juntendo University School of Medicine, Tokyo, Japan).
Cytokine PCR analysis
For detection of cytokine mRNA levels, RT-PCR analysis was
performed. T cells were stimulated for 48 h with APC only, as
described above for the proliferation assay. Total RNA was isolated
from cell pellets by extraction with RNAzol (Life Technologies). The
isolated RNA and 0.5 µg oligo(dT)12–18 were
heated at 55°C for 10 min and cooled to room temperature.
First-strand synthesis was performed by incubating 1 µg of RNA in a
reaction mixture (total volume, 20 µl) containing 5 mM Tris-HCl, pH
8.3, at 42°C, 50 mM KCl, 10 mM MgCl2, 2 mM DTT,
25 U reverse transcriptase, and 1 mM each of dATP, dCTP, dGTP, and
dTTP. The mixture was incubated at 42°C for 1.5 h to form cDNA.
The mixture was cooled on ice and diluted with 6 vol of
H2O. cDNA (4 µl) was incubated in a mixture
(total volume, 20 µl) containing 0.1 mM dATP, dCTP, dGTP, and dTTP;
50 mM KCl; and 10 mM Tris-HCl, pH 9.0, at 25°C and 1.5 mM
MgCl2, 0.01% gelatin, 0.1% Triton X-100, 50 ng
each of the desired 5' and 3' primers (see below), and 0.625 U of Taq
DNA polymerase I (Perkin-Elmer, Norwalk, CT). The mixture was incubated
for 2 min at 94°C, followed by 35 cycles of 10 s at 94°C,
30 s at 60°C, and 30 s at 72°C, followed by 10 min at
72°C, in a PCR apparatus (model 9600, Perkin-Elmer). For detection of
IL-4 mRNA, a nested PCR was performed with first amplification for 35
cycles with full-length primers, followed by a second amplification for
25 cycles with nested primers. PCR products were visualized on a 2%
agarose gel stained with ethidium bromide. The following rat-specific
primer pairs were used: G3PDH (452-bp fragment), 5'-ACC ACA GTC CAT GCC
ATC AC and 3'-TCC ACC ACC CTG TTG CTG TA; IL-4, first PCR round (460-bp
fragment, including XbaI/BamHI restriction sites
as indicated in lowercase), 5'-CCg gat ccA TGG GTC TCA GCC CCC ACC T
and 3'-GCt cta gaT TAG GAC ATG GAA GTG CAG GAC T; for nested PCR
(238-bp fragment), 5'-ATG CAC CGA GAT GTT TGT ACC and 3'-TTT CAG TGT
TCT GAG CGT GGA; IL-10 (127-bp fragment), 5'-TGC CAA GCC TTG TCA GAA
ATG ATC AAG and 3'-GTA TCC AGA GGG TCT TCA GCT TCT CTC; and IFN-
(419-bp fragment), 5'-CCC TCT CTG GCT GTT ACT GC and 3'-CTC CTT TTC CGC
TTC CTT AG. The appropriate length of the PCR products was confirmed
using 
Pst as the m.w. marker. Note that for detection of IL-4 mRNA,
a nested PCR was required.
Modulation of arthritis with peptide-specific T cell lines
The T cell lines M52ext and M52core were tested for their protective capacity by i.v. administration on the day of arthritis induction. T cell lines were restimulated in vitro with irradiated syngeneic splenocytes and 10 µg/ml specific peptide (M256–270 for the M52ext T cell line and M256–265 for the M52core T cell line). Three days later viable cells were isolated by Ficoll-Isopaque gradient centrifugation, washed twice in PBS, and suspended to a concentration of 2.5 x 107/ml in PBS. Immediately before induction of AA, T cells (5 x 106/200 µl) were injected i.v. in the tail vein (n = 4/group). Control rats received 200 µl of PBS (n = 4).
Induction and clinical assessment of AA
AA was induced by a single intradermal injection in the base of the tail with 100 µl of 5 mg/ml Mt in IFA (0.5 mg/rat). Rats were examined for clinical signs of arthritis in a blind set-up. The severity of the disease was assessed by scoring each paw from zero to four based on the degree of swelling, erythema, and deformity of the joints; thus, the maximum possible score was 16 (3). Simultaneously, the weights of the animals were measured.
Statistical analysis
Significant differences between the arthritis scores of the
experimental groups were determined by the two-tailed Mann-Whitney
U test. Differences were taken to be significant at
p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously we showed that short term M256–270-specific T cell
lines were protective against AA and avridine-induced arthritis. These
T cells also showed proliferative responses to the conserved core
sequence M256–265 and to rat hsp60 (6). In the present
study we determined the exact requirements of this autoreactivity for
protection. Therefore, we generated two different M256–270-specific T
cell lines by priming with peptide M256–270 and subsequent
restimulation either with M256–270 (e.g., T cell line
M52ext) or with the conserved core peptide
M256–265 (e.g., T cell line M52core). Repetitive
restimulation with the conserved core generated T cells (e.g., the
M52core line) that were highly proliferative in
response to naive, irradiated syngeneic APC in the absence of
exogenously added Ag (Fig. 1
). The
M52ext T cell line, restimulated with the
full-length peptide, M256–270, lost its ability to proliferate upon
culture with naive APC. Between different experiments, however,
variations in the magnitude of the autoproliferation of the
M52core T cell line were observed, probably due
to a variable degree of manipulation-induced stress in the APC. In the
infrequent experiments in which a low level of autoproliferation was
found, weak responses of the M52core line toward
the rat hsp60 homologue peptides, R256–270 and R256–265 (stimulation
index, 1.5–2.5), were observed. The T cell line
M52ext hardly proliferated upon stimulation with
the conserved core peptide M256–265, whereas the
M52core T cell line proliferated almost equally
in response to peptides M256–270 and M256–265 (Fig. 2). Stimulation with peptide M256–270
evoked comparable proliferative responses in both T cell lines, which
implies that the nonconserved C-terminal part of peptide M256–270
forms the dominant specificity of the M52ext T
cell line. This appeared to correlate with the inability of this line
and similarly obtained lines to recognize the self-epitope on
APC.
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To determine the MHC class II restriction of the autoproliferative
response of the M52core T cell line upon
stimulation with naive APC, specific Abs against
RT1.Bl (OX6) or RT1.Dl
(OX17) were added simultaneously with APC. The response upon
recognition of the self-epitope was RT1.Bl
restricted, as the autoproliferative response was largely blocked in
the presence of a low concentration (5 µg/ml) and was almost
completely blocked by a higher (20 µg/ml) concentration of OX6.
Addition of OX17 Ab (anti-rat RT1.Dl) had no
effect (Fig. 3; one of three
experiments).
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To obtain further evidence that the autoproliferative T cell line
M52core recognized the self-hsp epitope on naive
APC, we tested whether heat shock-induced up-regulation of self-hsp60
could enhance the autoproliferative responses. Therefore, we monitored
the (auto)proliferative responses of T cell lines
M52core, M52ext and of M43
(a T cell line generated against nonconserved peptide M211–225 of
mycobacterial hsp60) toward otherwise naive APC kept at 37°C or
exposed to 43°C. In one experiment with a relatively low level of
autoproliferation, the proliferative response of the
M52core T cell line was clearly enhanced upon
stimulation with heatshocked APC (43°C) compared with the effect of
normally treated APC (37°C; Fig. 4A). The T cell line
M52ext showed a minor elevated response, which is
in agreement with the lack of autoproliferation and with the relatively
reduced proliferation upon stimulation with the conserved core
M256–265. The control T cell line M43 did not proliferate upon
stimulation with normally treated or heat-shocked APC. In three other
experiments we were unable to see enhanced autoproliferation toward
heat-shocked splenocytes by the M52core T cell
line (data not shown). However, in these experiments the
autoproliferation toward normally treated (37°C) cells was already
very high, thereby probably masking any effect of heat-shocked APC. In
a similarly performed experiment using adherent APC obtained from
the MHC class II-identical Fisher rat, autoproliferative responses of
the M52core T cell line toward normally treated
APC were low. Also in this case heat-shocked APC induced highly
enhanced autoproliferative responses, but not when T cell lines
M52ext or M43 were analyzed (Fig. 4B).
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Recognition of self-epitope on APC induces IL-4, IL-10, and
IFN-
Next we determined whether there were any differences between the
cytokines produced by the highly autoproliferative
M52core T cell line and the
M52ext T cell line, which had lost its
autoreactivity upon repetitive restimulation with peptide M256–270.
Cytokine mRNA levels of T cell line M52core and T
cell line M52ext were determined after 48 h
of stimulation by naive syngeneic APC. We observed that the T cell line
M52core produced more IL-4, IL-10, and IFN-
than the M52ext T cell line upon stimulation with
irradiated naive APC. In the experiment shown (Fig. 5), T cell lines were used after four
cycles of restimulation in vitro with peptide M256–265 or peptide
M256–270, respectively. G3PDH levels are shown as a control for the
amount of cDNA. To determine whether this differential cytokine
production was related to the autoreactivity displayed by the T cell
lines, we followed over time the cytokine mRNA levels in the
M52core and M52ext T cell
lines after successive restimulation cycles. Upon successive
restimulation with peptide M256–270, the observed loss of
autoreactivity of the M52ext T cell line (Fig. 1
)
was paralleled by an increasingly reduced production of IL-4, IL-10,
and IFN- (data not shown). Therefore, recognition of the
self-epitope on naive APC appeared to be essential for production of
IL-4, IL-10, and IFN-.
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We reported previously that M256–270-specific T cells express
B7.2 upon recognition of the self-hsp60 peptide R256–265
(20). In the present study we investigated whether the
natural self-epitope was also able to up-regulate B7.2 expression on
M256–270-specific T cells. For this experiment we used the
M52core T cell line, which showed enhanced
autoproliferative responses toward heat-shocked naive syngeneic APC,
and the M52ext T cell line, which did not
recognize the self-epitope on naive APC (Fig. 4). B7.2 expression by
both T cell lines was analyzed after 20–24 h of incubation in the
presence of normally treated or heat-shocked adherent APC. Parallel to
its inability to recognize the self-epitope on naive APC, the
M52ext T cell line did not express B7.2 upon
incubation with heat-shocked APC (Fig. 6, upper part). Also, the M211–225-specific T cell line did
not express B7.2 after incubation with normally treated or heat-shocked
APC (data not shown). Parallel to an increase in autoproliferation, the
M52core T cell line increased its B7.2 expression
when stimulated with heat-shocked APC compared with normally treated
APC (Fig. 6, lower part). This up-regulated surface
expression of B7.2 was induced after 20–24 h of culture in the
presence of APC, as B7.2 expression was not detectable on these T cells
when analyzed immediately after addition to APC (time zero; data not
shown). Thus, upon recognition of the natural self-epitope,
M52core T cells expressed the same phenotype as
described previously for M256–270-specific T cells upon stimulation
with the rat peptide R256–265 (19) by up-regulating
B7.2.
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We proposed previously that only conserved epitopes of
mycobacterial hsp were able to induce protection against arthritis,
most likely because of their ability to induce cross-reactivity with
the self-epitope (6, 7). In the present study we have
characterized two T cell lines, both of which were generated against
the mycobacterial hsp60 peptide M256–270, but which demonstrated
differences not only in their ability to recognize the most conserved
core of this peptide, but also in their autoproliferative responses
toward naive APC. Therefore, we investigated whether the presence or
the absence of autoproliferation displayed by these two T cell lines
would influence their protective capacity. Thus, we transferred the
activated T cell lines (after nine restimulations in vitro) into rats
on the day of AA induction. The highly autoproliferative T cell line
M52core reduced the severity of the disease
significantly and tended to delay the onset (Fig. 7). Incidence rates on day 12 were 100,
50, and 25% for the PBS, M52ext, and
M52core groups, respectively. On day 13 incidence
rates were 100, 100, and 50%, respectively. The T cell line
M52ext, which was not autoreactive, had a minor,
but not significant, effect on disease development compared with that
in PBS-treated animals. A similar pattern was seen when the loss of
body weight was compared among the different groups; animals that had
received the M52core T cell line showed
significantly reduced weight loss compared with the control and
M52ext groups (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the anti-inflammatory cytokines IL-4 and IL-10. Despite
their strong autoreactivity to naive splenic APC, upon transfer such T
cells were found to induce a significant reduction of AA.
The M52core T cell line recognized the naive
APC-derived self-epitope in the context of MHC class II, as its
response was blocked with anti-RT1.Bl mAb
OX6. In different experiments variations in the levels of
autoproliferation were observed, probably caused by varying endogenous
hsp levels due to the variations in manipulation stress induced in the
APC during their isolation (e.g., adherence to plastic surfaces may
up-regulate hsp expression (20)). In the incidental
situation of relatively low autoreactivity, we found that exposure of
APC to heat shock strongly enhanced the autoproliferative responses of
T cell line M52core, indicating that these
responses resulted from the recognition of endogenously expressed and
processed hsp. The enhanced autoproliferation in response to
heat-shocked APC (Lewis rat splenocytes) was clearly observed in one of
four experiments. The experiment requires T cells that have become
peptide specific, and therefore, the experiments could only be
conducted after four or more peptide restimulations in vitro. However,
successive restimulation cycles made the M52core
T cells become more autoproliferative in response to normal APC (Fig. 1), and thus, enhancement of autoproliferation with heat-shocked APC
became masked. However, a similar experiment was performed with
adherent APC derived from Fisher rats (that have the same MHC class II
phenotype as Lewis rats). In this case autoproliferative responses
(toward normally treated APC) of M52core T cells
were relatively low, but again became highly enhanced upon exposure to
heat-shocked APC.
Attempts to define the endogenous hsp60 epitope more precisely were
only partly successful. Weak responses of the
M52core T cell line to synthetic rat homologue
peptides R256–270 and R256–265 were only found in the infrequent
situation of relatively low levels of autoproliferation. Most likely,
the otherwise already high levels of autoproliferation induced by naive
APC prevented the detection of responses induced by the rat homologue
peptides. Alternatively, the natural hsp epitope may differ in length
from the synthetic rat hsp60 peptides we tested. Also interesting in
this respect are two recent reports on CD4+ T
cell clones specific for the MHC class II
E
52–68 peptide. These T cells discriminated
between exogenously added peptide and endogenously processed
peptide even when bound to the same MHC I-Ab
molecule (21, 22). Thus, in our case also the endogenously
processed self hsp60 peptide and the corresponding exogenously added
peptide may form two distinct TCR ligands for the
M52core T cell line. Clearly, the presence or the
absence of an autoproliferative response by the two T cell lines was
related to the recognition of a different epitope, as the
M52ext T cell line proliferated in response to
peptide M256–270 and lost its recognition of peptide M256–265. In
contrast, the M52core T cell line recognized
peptides M256–270 and M256–265 equally well. We have shown previously
that both peptides have similar MHC class II binding affinities
(19). This excludes that the effects induced by peptide
restimulation are due to a difference in MHC class II binding.
Besides a difference in specificity between the T cell lines, we found
that the autoproliferative M52core T cell line
produced IL-4/IL-10 and IFN- upon recognition of the self-Ag on APC.
Although the simultaneous production of cytokines characteristic of Th1
cells (IFN-) together with those characteristic of Th2 cells
(IL-4/IL-10) is uncommon, similar observations in the Lewis rat have
been made with autoantigen-specific T cells; e.g., it was shown that T
cell clones specific for the myelin basic protein autoantigen produced
cytokines of both subsets at the same time (23). In the
latter study the relative balance between cytokines of both subsets
produced by the autoreactive T cells was found to depend on the
activation conditions. Another option is that the
M52core T cell line could be an example of T
cells that display a Tr1-like phenotype (simultaneously producing
IFN- and IL-10) and that have a regulatory role in vivo
(24). On the other hand, it is quite well possible that
the uncloned M52core T cell line contains
subpopulations (e.g., Th1-like and Th2-like), each expressing different
cytokines. Detailed characterization of the
M52core T cells combined with intracellular FACS
staining experiments will be needed to clarify this further.
Cytokine production was related to autoproliferation, since a decreased
production was accompanied by a decreased ability to recognize the
APC-presented self-epitope, as seen with the
M52ext T cell line. Only the autoproliferative T
cell line, inducing higher levels of IL-4/IL-10 and IFN-, was able
to protect against AA. Thus, protection correlated with the ability of
these T cells to recognize the autoantigen as present on stressed APC
and possibly on T cells (25) as well as with the
subsequent production of IL-4/IL-10 and IFN-. In the nonobese
diabetic mouse the induction of T cells that are autoproliferative to
endogenously processed and presented self-Ags by naive APC has been
suggested as a basis of nonobese diabetic autoimmune disease in vivo
(26). As in that study we demonstrate here a loss of
self-tolerance in vitro. However, when the autoproliferative T cell
line was tested in vivo, we observed a reduction of autoimmune disease,
indicating that autoproliferative responses toward stress-inducible
proteins may function differently.
The development of autoproliferative, hsp-specific T cells that induce regulatory cytokines is possibly driven by their ability to recognize Ag (every cell constitutively expresses hsp) in the periphery in the absence of signal 2 (18, 27, 28, 29). Active regulatory mechanisms such as immune deviation (30, 31) (a switch from a Th1 to a Th2 response or the development of IL-10/TGF-ß-producing cells) and the induction of anergy (32, 33, 34) have been described for the maintenance of peripheral tolerance, and this could explain our findings that eliciting responses to self-hsp60/70 induces T cells with a Th2 phenotype or a Tr1 phenotype induced by IL-10.
Moreover, apoptotic cells have been shown to produce IL-10 and to induce immune deviation of the phagocytosing APC (35), and several stress conditions have been shown to induce IL-10 expression (36). Together with the finding that apoptotic cells (37) and stressed cells (13) up-regulate the expression of hsp, this could indicate that upon uptake of apoptotic or stressed cells by macrophages and immature dendritic cells or during recognition of self-hsp on stressed cells, self-hsp reactive T cells become primed in an IL-10-inducing environment. Also the priming of self-hsp60 cross-reactive T cells in the gut by bacterial hsp60, where IL-10 (and TGF-ß)-biased cells are generated due to the local cytokine environment (38), may favor the development of IL-4- and IL-10-producing, regulatory, autoproliferative, hsp-specific T cells. Several reports support the regulatory effect of IL-4 and IL-10 in experimental models of arthritis as well as in other autoimmune models (40, 41, 42, 43, 44). Furthermore, we found recently that protection by preimmunization with Mt hsp70 was related to the priming of autoreactive, IL-4- and IL-10-producing T cells (7).
These autoproliferative T cells may then down-modulate the pathogenic T cells (MHC class II positive in the rat) either directly through recognition of self-hsp60 on the pathogenic T cells (19, 44), thereby inducing anergy in the latter T cells (46, 47), or indirectly through bystander suppression. In a previous study we reported that M256–270-specific T cells preferentially express B7.2 upon recognition of the rat hsp60 peptide R256–265 (19). In the present study we now also found expression of B7.2 on the M52core T cells upon exposure to heat-shocked APC, but, again, not on control T cell lines. There are several reports that suggest a regulatory role of B7.2 expression on T cells. First, B7.2 expressed on T cells was described to bind preferentially to CTLA-4 (47). Second, this interaction was responsible for the down-modulation of T cell responses in vitro (48). Third, B7.2-expressing T cell tumors were reported to suppress the induction of anti-tumor immunity in vivo (47), and recently the same group showed that IL-4- and/or IL-10-producing CD4+ T cells played a role in this suppression (49). Given this indirect evidence for an association of B7.2 and T cell regulation, the expression of B7.2 upon exposure to stressed APC may be mechanistically relevant to the modulation of AA development by the M52core T cell line. Another indirect mechanism of regulation could operate through modulation of the APC by the induced IL-4/IL-10 to generate a more Th2 type of response to the autoantigen(s) or to induce anergy (50, 51, 52, 53). Subsequent experiments, both in vitro and in vivo, to inhibit the actions of these cytokines using specific Abs will be needed to clarify their exact roles in the protection mediated by the autoproliferative, hsp-specific T cells in experimental arthritis.
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2 Address correspondence and reprint requests to Dr. Ruurd van der Zee, Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; AA, adjuvant arthritis; Mt, Mycobacterium tuberculosis; PLNC, primed lymph node cells; NRS, naive inactivated rat serum; hsp60, 60-kDa heat shock protein.
Received for publication February 17, 2000. Accepted for publication September 18, 2000.
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