BIP, A PUTATIVE AUTOANTIGEN IN RHEUMATOID ARTHRITIS, STIMULATES IL-10-PRODUCING CD8- POSITIVE T CELLS FROM NORMAL INDIVIDUALS
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Rheumatology 2003;42:637–644
doi:10.1093/rheumatology/keg204, available online at www.rheumatology.oupjournals.org
Advance Access publication 28 February 2003
BiP, a putative autoantigen in rheumatoid
arthritis, stimulates IL-10-producing CD8-
positive T cells from normal individuals
M. D. Bodman-Smith, V. M. Corrigall, D. M. Kemeny1 and
G. S. Panayi
Objectives. We have reported that synovial fluid T cells from patients with
rheumatoid arthritis (RA) proliferate in response to the endoplasmic reticulum
molecular chaperone immunoglobulin binding protein (BiP). The aim of the present
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work was to clone and define T cells responding to this protein.
Methods. T-cell clones were generated from the peripheral blood of an individual
known to respond to BiP by limiting dilution of BiP-stimulated peripheral blood
mononuclear cells. T-cell receptor usage of BiP-responsive clones was determined
by monoclonal antibody staining followed by flow cytometric analysis. Cytokine
production by the BiP-responsive clones was determined by analysis of post-
stimulation supernatants by ELISA. Additional phenotyping was performed by
flow cytometry.
Results. Of 49 clones isolated, six were shown to proliferate in response to BiP.
Proliferation was low but consistent. One clone expressed CD4 and five were CD8-
positive. Three clones, all CD8+, grew strongly and were investigated further.
T-cell receptor usage was determined in two clones (Vb 7.1 and Vb 12); the Vb
element of the remaining clone was not recognized by the panel of antibodies used.
All three clones produced interleukin 10 (IL-10) (80–380 pguml) and two of them
produced IL-4 (10–80 pguml) and IL-5 (>5000 pguml). One clone produced both
IL-10 and interferon c (>5000 pguml). Additional phenotyping of these clones
showed them to express CD25, CD28, CD80 and 86 but not CD56 or 57. One
clone constitutively expressed CTLA-4 cytoplasmically.
Conclusions. This study demonstrates that a population of CD8+ T cells with the
cytokine profile of Tc2 cells can be stimulated by the chaperone BiP. These cells
may perform a regulatory role in the normal response to inflammation. The
increase in response to this antigen in the synovial joint in RA may indicate an
attempt to regulate the ongoing inflammation.
KEY WORDS: Heat shock protein, CD8, IL-10.
We have shown previously that antibodies to immuno- who have other inflammatory joint diseases w1x. These
globulin binding protein (BiP) can be found in the serum data have since been confirmed by other groups w2x.
of RA patients and in the serum of mice with collagen- The immunoglobulin-binding protein BiP, also known
or pristane-induced arthritis w1x. T-cell proliferative as the glucose-regulated protein 78 (GRP78), is a con-
responses to BiP were also identified in the synovial com- stitutively expressed protein associated with the lumen of
partment of patients with RA but not in that of patients the endoplasmic reticulum w3x. It is a broad-specificity
Departments of Rheumatology, GKT School of Medicine, King’s College London and Guy’s Hospital, London SE1 9RT, UK and 1Department of
Immunology, GKT School of Medicine, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
Received 6 August 2002; revised version accepted 15 November 2002.
Correspondence to: M. Bodman-Smith, Guy’s Hospital, London, SE1 9RT, UK. E-mail: mark.bodman-smith@kcl.ac.uk
637
Rheumatology 42 ß British Society for Rheumatology 2003; all rights reserved638 M. D. Bodman-Smith et al.
molecular chaperone that transiently binds newly syn- Cloning of specific T cells
thesized polypeptide chains and thereby assists in their Mononuclear cells were plated at 1 3 106 cellsuml in 2 ml culture
correct folding and post-transcriptional modification. wells in the presence of 0.25 mM BiP (20 mguml) in TCM. Cells
BiP is also responsible for the transfer of aberrant pro- were cultured at 378C in 5% CO2. After 7 days, Lymphocult-T
tein to the proteosome for degradation. BiP is expressed (LC-T; Biotest, Dreieich, Germany) was added to the cultures
at high levels in the cell but is increased in situations that (40 mluml) as a source of IL-2 (IL-2). After a further 7 days
lead to accumulation of unfolded protein in the cell the cells were plated at 1 cell per well into 96U plates with
(the unfolded protein response) w3, 4x and cellular stresses, 1 3 104 c-irradiated autologous feeder cells and 0.25 mM BiP.
LC-T was added 1 week later. The cells were then expanded
such as anoxia and glucose starvation w5x. These
using 1 3 104 irradiated allogeneic peripheral blood mono-
conditions are often found in the inflamed joint w6x. nuclear cells (PBMC), LC-T and 2 mguml phytohaemagglutinin
BiP is a member of the heat shock protein (HSP) 70 (PHA; Sigma, Poole, UK). After 1 week, LC-T was added
family of proteins w7x. In common with other members to the wells and, after a further week, 1 3 104 irradiated feeder
of the family, BiP has an ATP-binding domain at the cells, LC-T and PHA were added again. The cells were expanded
N-terminus and a C-terminal peptide binding site w8x. until sufficient cell numbers were obtained for further study.
The HSP70 family of proteins have been implicated
in the pathogenesis of both experimental and human
Clone proliferation assays
arthritis. Elevated levels of antibodies to HSP70 have Cloned cells (1 3 104) were incubated for 3 days with 1 3 105
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been reported in RA w9x and expression of the protein irradiated autologous (or allogeneic) feeder cells in the presence
or absence of BiP (0.25 mM, or as described), b-galactosidase
has been shown to be enhanced in RA synovium w10x. In
(0.15 mM) or PHA (2 mguml). The cells were incubated for the
animal models of arthritis, nasal immunization with last 18 h with 3H-thymidine (0.2 mCi) (Amersham, Amersham,
HSP70 has been reported to prevent inflammation w11x UK) before harvesting and counting. Proliferation was
and T cells recognizing this molecule can protect against expressed as counts per minute (c.p.m.) or stimulation index (SI).
the arthritis w12x. In both reports, the T cells recognizing
HSP70 were shown to produce interleukin (IL) 10. This Flow cytometric analysis
may be an inappropriate, although beneficial, response Phenotypic analysis was carried out on responding clones using
to this protein because HSP70 is associated with the antibodies to the T-cell markers CD3, CD4 and CD8. Cells
production of proinflammatory cytokines w13, 14x. were washed in FACS (fluorescence-activated cell sorter) buffer
HSP70 has been shown to specifically stimulate CD8 wphosphate-buffered saline containing 1% bovine serum albu-
min (BDH) and 0.05% sodium azide (Sigma)x and incubated
cells w15x. Furthermore, this stimulation has been shown
with 4 ml of antibody for 40 min on ice. Three-colour analysis
to occur in the absence of peptide association with was performed using a FACScan (Becton Dickinson, Oxford,
HSP70, suggesting that the HSP molecule itself is UK) flow cytometer and Cellquest software (Becton Dickinson).
capable of eliciting a CD8 response w13x. We therefore Cells were permeabilized using Fix and Perm (Caltag, Burlingane,
decided to clone T cells from individuals whose T cells CA, USA) according to the manufacturer’s instructions, for
proliferated in response to BiP and to investigate their intracytoplasmic staining for CTLA-4. Isotype control anti-
function. In this paper we show that cloned BiP- bodies were used throughout (all directly conjugated antibodies
responsive T cells have the cytokine profile of regulatory from Becton Dickinson).
cells. We propose that decreased numbers or deficient T-cell receptor usage was determined using a panel of
function of these regulatory T cells may contribute to both fluorescein isothiocyanate (FITC)-conjugated and non-
conjugated antibodies (Serotec, Oxford, UK). For conjugated
the pathogenesis of RA through a lack of immune
antibodies, 1 3 10 4 cells were stained as above. For non-
down-regulation. conjugated antibodies, cells were incubated for 40 min on ice
with the primary antibody, washed twice in FACS buffer, then
incubated for 40 min with a FITC-conjugated goat anti-mouse
Materials and methods antibody (Becton Dickinson). Cells were run on a FACScan
flow cytometer with a 488 nm laser and the results analysed
Purification of antigens using Cellquest and WinMDI software.
BiP and the control antigen, b-galactosidase, were purified Cytokine determination
from an Escherichia coli expression system as described pre-
Supernatants were removed from cultures at various times after
viously w1x. Briefly, the gene for BiP and the gene for the control
the last round of stimulation. Supernatants from cultures
protein, b-galactosidase, were transfected into kanamycin-
containing only irradiated feeders, LC-T and PHA were
resistant E. coli. The construct contained a 6 3 histidine tag
used as controls. The amount of IL-4, IL-10, IFN-c and tumour
and was purified on nickel columns (Pharmacia, Amersham,
necrosis factor (TNF)-a were determined by enzyme-linked
UK).
immunosorbent assay (ELISA; Pharmingen, Oxford, UK)
Mononuclear cell purification according to the manufacturer’s instructions. IL-5 concentra-
tion was determined using cytometric bead array analysis
Mononuclear cells were isolated from peripheral blood as
(Becton Dickinson) according to the manufacturer’s instructions.
described previously w1x. Cells were resuspended in tissue
culture medium (TCM) wRPMI 1640 supplemented with
L-glutamine, penicillin, streptomycin and 10% heat-inactivated Results
human serum (Life Technologies, Paisley, UK)x and plated out
at 1 3 105 cells per well with or without antigen. Proliferation During our investigation into the proliferative response
was determined by 3H-thymidine incorporation after 6 days w1x. of T cells from patients with RA to BiP, peripheral bloodBiP stimulates IL-10-producing CD8 T cells 639
T cells from occasional healthy individuals also prolif-
erated in response to BiP (Fig. 1). PBMC from this
individual, FC, were used to clone out BiP-responding
cells and are reported here.
Growth of T-cell clones
Of 420 wells seeded, 49 showed growth and were
expanded. Of the clones isolated, six had proliferative
responses to BiP, as measured by 3H-thymidine incor-
poration in the presence of antigen double that of
medium alone (i.e. a stimulation index >2.0) (Fig. 2a).
FIG. 1. Proliferative responses of PBMC from six normal
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individuals (indicated by their initials) to the human chaperone
protein BiP. Mean and S.D. c.p.m. (triplicate wells) for different
concentrations of concentrations of BiP.
FIG. 3. Flow cytometric analysis of clones FC2B5, 3E3 and
2E4. Panels (a), (c) and (d) show CD4 (x-axis) and CD8
(y-axis) staining of the three clones. FC2B5, FC3E3 and
FIG. 2. Proliferative response of clones generated to BiP. (a) FC2E4 all express CD8 and no CD4. Panel (b) shows that the
Proliferation of clones stimulated by irradiated autologous T-cell receptor b-chain usage (TCRVb) of FC2B5 is Vb 12
feeder cells and BiP. (b) Responses of a representative clone to (x-axis). Panel (e) shows that the TCRVb usage of FC2E4 is
BiP and the control antigen b-galactosidase (generated in the Vb 7.1 (x-axis). No antibody in the panel used stained clone
same expression system as BiP).Values are mean and S.D. c.p.m. FC3E3.640 M. D. Bodman-Smith et al.
This proliferation, although small, was consistent and FC2B5 3E3 and 2E4; panels (a), (c) and (d) show CD4
present when retested 4 weeks later (data not shown). (x-axis) and CD8 (y-axis) double staining (CD4-FITC
The response to PHA, used as a positive control and to and CD8-PE). Panels (b) and (e) show staining with
confirm the proliferative ability of the clones under antibodies to the Vb element of the T-cell receptor. Of
investigation, was of the same magnitude on both the CD8-positive clones, one (3E3) did not have its Vb
occasions (SI 53–724). The clones responded in a dose- usage determined with the antibodies used, clone 2B3
dependent manner to BiP (a representative clone is expressed Vb5.1, clone 2B5 expressed Vb12, clone 2E4
shown in Fig. 2b). There was no proliferation in expressed Vb7.1 and 3E6 expressed Vb8.
response to the control antigens b-galactosidase
(Fig. 2b) and tuberculin PPD (data not shown). The
Additional phenotypic studies
proliferation of the BiP-responsive clones FC2B5 and Two rapidly growing CD8-positive clones were further
FC3E3 was abrogated when the clones were incubated phenotyped to examine the expression of costimulatory
with BiP in the presence of allogeneic presenting cells molecules and activation markers. Figure 4 shows expre-
(data not shown), suggesting MHC restriction. ssion of the CD25, 28, 56, 57, 80, 86 and CTLA-4. As
expected, both clones expressed the a chain of the IL-2
Phenotyping of specific clones receptor (CD25). The costimulation molecule CD28 was
Of the six BiP-specific clones isolated, five were CD8- expressed on both the clones, as were the ligands for
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positive and one was CD4-positive (for representative CD28, CD80 and CD86. The expression of the costi-
FACS profiles see Fig. 3). mulatory molecule CTLA-4, thought to be an essential
signal for some regulatory T cells w16x, was expressed
Vb staining of specific clones cytoplasmically in one of the two clones examined, but
There was no common usage of Vb elements between the not on the surface of the cell. Interestingly, the single CD4-
clones. Figure 3 shows the FACS profiles of clones positive, BiP-responsive clone (FC1D5) expressed surface
FIG. 4. Phenotypic analysis of BiP-responsive CD8 clones FC2B5 and FC3E3 by flow cytometry. In each case the isotype control is
shown by the thin line and the test antibody by the bold line. Both clones expressed CD25 and low levels of the T-cell costimulatory
molecule CD28. CD56 was expressed at very low levels and there was no expression of CD57. The ligands for CD28, CD80 and
CD86 were both expressed in both clones. The ‘negative’ stimulatory molecule CTLA-4 was not expressed on the surface of either
clone. However, constitutive cytoplasmic expression was seen in FC3E3.BiP stimulates IL-10-producing CD8 T cells 641
TABLE 2. Cytokine production (pguml) of T-cell lines generated against
BiP from PB and SF of patients with RA
Clone IL-4 IL-10 IFN-c
KPB2E4 20 0 410
KPB2C4 20 100 3830
KPB2C5 390 120 0
KSF3D4 130 0 0
KSF1B4 20 0 0
KSF3A5 30 0 1500
difficult to measure due to the production of IL-10 by
the feeder cells when cultured with BiP (data not shown).
Cytokine production by T-cell lines grown from
peripheral blood and synovial fluid of an RA patient
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FIG. 5. IL-10 production of BiP-responsive clones. Levels of T-cell lines were grown from RA patient K in the
cytokine were determined 72 h after stimulation with PHA, presence of BiP. The lines were generated using the same
LC-T and irradiated allogeneic feeder cells. Values given are initial protocol used for the clones before the limiting
above that seen with feeder mix alone. dilution stage. On examining the cytokine profiles of
three lines grown from the peripheral blood (PB) and
CLTA-4 constitutively (data not shown). None of the three from the synovial fluid (SF), two of three lines
clones expressed the NK marker CD57 but low-level generated from PB produced IL-10, whereas none of the
expression of CD56 was seen on both clones. three lines generated from the SF did (Table 2). The six
lines generated from the RA patient were all predomi-
Determination of cytokine production of T-cell nantly CD3+CD4+ cells, in contrast to the CD8 T cells
clones from the normal individual.
Supernatants were removed from cultures 72 h after
stimulation with PHA and LC-T. Figure 5 shows IL-10 BiP-induced IL-10 production by RA patients
production from all clones isolated. Interestingly, the BiP-induced IL-10 production was measured in PB and
clone showing the least IL-10 production proliferated SF mononuclear cell populations isolated from nine
most strongly. The cytokine levels were compared with patients with RA. All samples were received from the
the results obtained by the feeder mix (irradiated allo- rheumatology clinic at Guy’s hospital. Cells were set up
geneic cells with LC-T and PHA) alone. Three strongly at 1 3 106 cells per well and stimulated with 0.25 mM BiP.
growing clones were tested for a number of other cytokines. Supernatants were taken 72 h after stimulation and the
IL-10, IL-5 and IL-4 production was seen in the CD8- cytokine profile was determined by ELISA. As seen in
positive clone FC3E3 and IL-10 in the CD8 clone Fig. 6, no differences were seen in the amount of IL-10
FC2E4 (Table 1). Clone FC2B5 produced low levels of produced by cells isolated from the PB and SF from RA
IL-10, suggesting that the IL-10 production was not patients. Furthermore, there was no difference seen
simply due to the cloning procedure, and high levels of
IFN-c, IL-4 and IL-5. Interestingly, the single CD4-
positive clone isolated from this individual showed lower
IL-10, low IL-4 and no IL-5 production, but produced
elevated levels of TNF-a (data not shown). Cytokine
production by antigen stimulation of the clones was
TABLE 1. Cytokine production profile of BiP-responsive clones
Clone IL-4 IL-10 TNF-a IFN-c IL-5
FC2B5 80 10 0 5110 >5000
FC2E4 0 80 0 0 n.d.
FC3E3 10 380 40 10 >5000
Culture supernatants were taken 72 h after stimulation and cytokine
levels determined by capture ELISA (except IL-5, determined by
cytometric bead array) and expressed as pguml. In each case the
cytokine level given indicates the amount of cytokine seen in the clone FIG. 6. BiP-induced IL-10 production in paired PB and SF
cultures above the level determined for the feeder cells alone (10, 20, 20, from RA patients. There were no differences in the levels of
170 and 90 pguml respectively). BiP-induced IL-10 produced by cells isolated from the PB and
n.d., not determined. SF of nine RA patients.642 M. D. Bodman-Smith et al.
between the RA patients and six normal individuals w26, 27x. The same reports also describe the expansion of
(data not shown). CD4 clonotypes but only in the PB of the patients,
suggesting that the CD8 cells may be of relevance to the
inflammation in the joint. Furthermore, investigation of
Discussion the effects of CD8 cells in an animal model of arthritis
(collagen-induced arthritis in HLA-DQ8 transgenic
The aim of this study was to investigate the T-cell mice) has suggested that these cells may play a role in
response to BiP in the PB of normal, healthy individuals regulating the disease. In this model, which mirrors RA
in order to determine the nature of the cells activated by in that rheumatoid factor is produced, depletion of the
BiP and, in particular, whether they had any of the CD8 cells results in severe disease and an increase in the
emergent characteristics of regulatory T cells. The data proinflammatory cytokines IFN-c and TNF-a w28x. The
in this report suggest that BiP can indeed stimulate a absence of CD8 cells also results in the production of
population of CD8 T cells from normal individuals that anti-nuclear antibodies in the mice.
have the cytokine profile of regulatory cells because they Of the two CD8-positive BiP-responsive clones
produce the anti-inflammatory cytokine IL-10 and, in examined for the expression of CTLA-4, one constitu-
some cases, IL-4 and IL-5. In common with other tively expressed this molecule cytoplasmically. This
reported human regulatory T cells, they have a low but molecule is transiently expressed on activated CD4 and
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consistent proliferative capacity w17x. CD8 cells, although its expression on CD8 cells has been
The role of regulatory T cells, both CD4-negative and reported to be associated with early activation w29x. The
CD8-positive, in health and disease is becoming more expression of this molecule on CD8 clones, which are
understood. Whereas CD4-positive regulatory T cells long-term activated cells, may represent a different role.
have been investigated extensively (for review see w18x), Regulatory T cells of the CD4 phenotype have been
CD8-positive regulatory cells have proved more difficult shown to express CTLA-4 constitutively, and blockade
to isolate and study. These cells, however, have been of this molecule abrogates the suppression of antigenic
described in humans and shown to be capable of high and polyclonal stimulation of other T cells w30, 16x. A
IL-10 production w19x. In this report, IL-10-producing synergistic effect of CTLA-4 blockade and depletion of
CD8 cells specific for influenza matrix peptide were CD25-positive regulatory T cells has been reported w31x,
isolated from two volunteers who had been injected with suggesting two pathways of immune control.
immature dendritic cells pulsed with this antigen. The T-cell lines generated by the incubation of SF mono-
induction of regulatory T cells by immature dendritic nuclear cells of an RA patient with BiP showed no IL-10
cells has been reported by a number of groups (for production, in contrast to the PB cells of the same
review see w20x). The ability of CD8 T cells to produce patient. Lines generated from this patient were all CD4-
regulatory cytokines is a relatively new discovery in the positive, suggesting that the ‘normal’ CD8 response to
immune arsenal, but this cell type has been implicated in BiP may be replaced by a CD4 response in RA patients.
autoimmunity. Experimental allergic encephalomyelitis This may be due to differential processing of the antigen
(EAE) can be prevented by regulatory T cells of both in RA patients, or the in vitro observation may occur
the CD8 w21, 22x and the CD4 w23, 24x type. In the EAE because of the relatively slow growth of the CD8-
model, regulatory CD8 cells have been shown to be positive, IL-10-producing subset, allowing CD4 cells to
present in naive mice, their numbers increasing with age. outgrow them. In mixed mononuclear cell populations,
On exposure to myelin basic protein, this population however, IL-10 is produced at the same level in PB and
shows a restricted clonotype, suggesting expansion of a SF cell populations, suggesting that IL-10-producing cells
specific regulatory population, which can affect the responsive to BiP are present in the synovial compartment.
outcome of the disease. Interestingly, the cytotoxic The proportion of CD8 cells is often increased in the
potential of these cells does not correlate with their SF of patients with RA; furthermore, a recent report has
proliferative ability w21x. Jiang et al. w22x have suggested identified a population of IL-10-producing Tc2 cells that
that CD8-positive regulatory cells, when induced by is elevated in the SF of RA patients when compared with
T-cell vaccination (with disease-causing CD4 cells), are the PB w32x. The authors of this paper suggest that the
cytotoxic for the CD4 cell and that this response is increase in Tc2 cells may be part of an insufficient effort
abrogated in b2 microglobulin-deficient mice w22x. CD8 to reduce the inflammation in the joint. This informa-
cells have also been shown to protect against oil-induced tion, together with the demonstration that members of
arthritis in the rat w25x. the HSP70 family are overexpressed in the synovial joint
BiP stimulates a population of CD8 T cells. Of the w10x and that the conditions exist for BiP overproduction
BiP-responsive clones isolated, five out of six were w6x, could lead to the conclusion that the interaction
CD8-positive; moreover, these clones produced a cyto- between BiP and CD8 cells may be occurring within this
kine profile similar to that of regulatory CD4 cells, in compartment. Our data suggest that BiP is an antigen
that IL-10 and IL-4 were predominant. The role of that may specifically stimulate CD8 cells with the ability
CD8 T cells in RA has been the subject of some to produce large amounts of IL-10. This IL-10 produc-
investigation and it has been shown that CD8-positive tion may be part of a normal mechanism to down-
cells isolated from the SF can have a restricted clono- modulate an immune response. The increased reactivity
type, suggesting expansions of discrete CD8 populations seen in response to BiP in the synovial compartment ofBiP stimulates IL-10-producing CD8 T cells 643
RA may be an attempt by the host to reduce the cytokines, shear stress, and antiinflammatory drugs. J Clin
inflammation present. Although, as described above, Invest 1998;102:302–11.
HSPs can cause the production of proinflammatory 11. Wendling U, Paul L, van der ZR, Prakken B, Singh M,
cytokines, such as TNF-a and IL-1, there is evidence van Eden W. A conserved mycobacterial heat shock
protein (hsp) 70 sequence prevents adjuvant arthritis
that some HSPs can lead to the production of anti- upon nasal administration and induces IL-10-producing
inflammatory cytokines. De et al. w33x showed that the T cells that cross-react with the mammalian self-hsp70
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