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
Departments of Rheumatology, GKT School of Medicine, King's College London and Guy's Hospital, London SE1 9RT, UK and
1 Department of Immunology, GKT School of Medicine, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK
 |
Abstract
|
---|
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 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 (Vß 7.1 and Vß 12); the Vß element of the remaining clone was not recognized by the panel of antibodies used. All three clones produced interleukin 10 (IL-10) (80380 pg/ml) and two of them produced IL-4 (1080 pg/ml) and IL-5 (>5000 pg/ml). One clone produced both IL-10 and interferon
(>5000 pg/ml). 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.
 |
Introduction
|
---|
We have shown previously that antibodies to immunoglobulin binding protein (BiP) can be found in the serum of RA patients and in the serum of mice with collagen- or pristane-induced arthritis [1]. T-cell proliferative responses to BiP were also identified in the synovial compartment of patients with RA but not in that of patients who have other inflammatory joint diseases [1]. These data have since been confirmed by other groups [2].
The immunoglobulin-binding protein BiP, also known as the glucose-regulated protein 78 (GRP78), is a constitutively expressed protein associated with the lumen of the endoplasmic reticulum [3]. It is a broad-specificity molecular chaperone that transiently binds newly synthesized polypeptide chains and thereby assists in their correct folding and post-transcriptional modification. BiP is also responsible for the transfer of aberrant protein to the proteosome for degradation. BiP is expressed at high levels in the cell but is increased in situations that lead to accumulation of unfolded protein in the cell (the unfolded protein response) [3, 4] and cellular stresses, such as anoxia and glucose starvation [5]. These conditions are often found in the inflamed joint [6].
BiP is a member of the heat shock protein (HSP) 70 family of proteins [7]. In common with other members of the family, BiP has an ATP-binding domain at the N-terminus and a C-terminal peptide binding site [8]. The HSP70 family of proteins have been implicated in the pathogenesis of both experimental and human arthritis. Elevated levels of antibodies to HSP70 have been reported in RA [9] and expression of the protein has been shown to be enhanced in RA synovium [10]. In animal models of arthritis, nasal immunization with HSP70 has been reported to prevent inflammation [11] and T cells recognizing this molecule can protect against the arthritis [12]. In both reports, the T cells recognizing HSP70 were shown to produce interleukin (IL) 10. This may be an inappropriate, although beneficial, response to this protein because HSP70 is associated with the production of proinflammatory cytokines [13, 14].
HSP70 has been shown to specifically stimulate CD8 cells [15]. Furthermore, this stimulation has been shown to occur in the absence of peptide association with HSP70, suggesting that the HSP molecule itself is capable of eliciting a CD8 response [13]. We therefore decided to clone T cells from individuals whose T cells proliferated in response to BiP and to investigate their function. In this paper we show that cloned BiP-responsive T cells have the cytokine profile of regulatory cells. We propose that decreased numbers or deficient function of these regulatory T cells may contribute to the pathogenesis of RA through a lack of immune down-regulation.
 |
Materials and methods
|
---|
Purification of antigens
BiP and the control antigen, ß-galactosidase, were purified from an Escherichia coli expression system as described previously [1]. Briefly, the gene for BiP and the gene for the control protein, ß-galactosidase, were transfected into kanamycin-resistant E. coli. The construct contained a 6x histidine tag and was purified on nickel columns (Pharmacia, Amersham, UK).
Mononuclear cell purification
Mononuclear cells were isolated from peripheral blood as described previously [1]. Cells were resuspended in tissue culture medium (TCM) [RPMI 1640 supplemented with L-glutamine, penicillin, streptomycin and 10% heat-inactivated human serum (Life Technologies, Paisley, UK)] and plated out at 1x105 cells per well with or without antigen. Proliferation was determined by 3H-thymidine incorporation after 6 days [1].
Cloning of specific T cells
Mononuclear cells were plated at 1x106 cells/ml in 2 ml culture wells in the presence of 0.25 µM BiP (20 µg/ml) in TCM. Cells were cultured at 37°C in 5% CO2. After 7 days, Lymphocult-T (LC-T; Biotest, Dreieich, Germany) was added to the cultures (40 µl/ml) as a source of IL-2 (IL-2). After a further 7 days the cells were plated at 1 cell per well into 96U plates with 1x104
-irradiated autologous feeder cells and 0.25 µM BiP. LC-T was added 1 week later. The cells were then expanded using 1x104 irradiated allogeneic peripheral blood mononuclear cells (PBMC), LC-T and 2 µg/ml phytohaemagglutinin (PHA; Sigma, Poole, UK). After 1 week, LC-T was added to the wells and, after a further week, 1x104 irradiated feeder cells, LC-T and PHA were added again. The cells were expanded until sufficient cell numbers were obtained for further study.
Clone proliferation assays
Cloned cells (1x104) were incubated for 3 days with 1x105 irradiated autologous (or allogeneic) feeder cells in the presence or absence of BiP (0.25 µM, or as described), ß-galactosidase (0.15 µM) or PHA (2 µg/ml). The cells were incubated for the last 18 h with 3H-thymidine (0.2 µCi) (Amersham, Amersham, UK) before harvesting and counting. Proliferation was expressed as counts per minute (c.p.m.) or stimulation index (SI).
Flow cytometric analysis
Phenotypic analysis was carried out on responding clones using antibodies to the T-cell markers CD3, CD4 and CD8. Cells were washed in FACS (fluorescence-activated cell sorter) buffer [phosphate-buffered saline containing 1% bovine serum albumin (BDH) and 0.05% sodium azide (Sigma)] and incubated with 4 µl of antibody for 40 min on ice. Three-colour analysis was performed using a FACScan (Becton Dickinson, Oxford, UK) flow cytometer and Cellquest software (Becton Dickinson). Cells were permeabilized using Fix and Perm (Caltag, Burlingane, CA, USA) according to the manufacturer's instructions, for intracytoplasmic staining for CTLA-4. Isotype control antibodies were used throughout (all directly conjugated antibodies from Becton Dickinson).
T-cell receptor usage was determined using a panel of both fluorescein isothiocyanate (FITC)-conjugated and non-conjugated antibodies (Serotec, Oxford, UK). For conjugated antibodies, 1x104 cells were stained as above. For non-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 antibody (Becton Dickinson). Cells were run on a FACScan flow cytometer with a 488 nm laser and the results analysed using Cellquest and WinMDI software.
Cytokine determination
Supernatants were removed from cultures at various times after the last round of stimulation. Supernatants from cultures containing only irradiated feeders, LC-T and PHA were used as controls. The amount of IL-4, IL-10, IFN-
and tumour necrosis factor (TNF)-
were determined by enzyme-linked immunosorbent assay (ELISA; Pharmingen, Oxford, UK) according to the manufacturer's instructions. IL-5 concentration was determined using cytometric bead array analysis (Becton Dickinson) according to the manufacturer's instructions.
 |
Results
|
---|
During our investigation into the proliferative response of T cells from patients with RA to BiP, peripheral blood T cells from occasional healthy individuals also proliferated in response to BiP (Fig. 1
). PBMC from this individual, FC, were used to clone out BiP-responding cells and are reported here.

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1. Proliferative responses of PBMC from six normal 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.
|
|
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 incorporation in the presence of antigen double that of medium alone (i.e. a stimulation index >2.0) (Fig. 2a
). This proliferation, although small, was consistent and present when retested 4 weeks later (data not shown). The response to PHA, used as a positive control and to confirm the proliferative ability of the clones under investigation, was of the same magnitude on both occasions (SI 53724). The clones responded in a dose-dependent manner to BiP (a representative clone is shown in Fig. 2b
). There was no proliferation in response to the control antigens ß-galactosidase (Fig. 2b
) and tuberculin PPD (data not shown). The proliferation of the BiP-responsive clones FC2B5 and FC3E3 was abrogated when the clones were incubated with BiP in the presence of allogeneic presenting cells (data not shown), suggesting MHC restriction.

View larger version (16K):
[in this window]
[in a new window]
|
FIG. 2. Proliferative response of clones generated to BiP. (a) Proliferation of clones stimulated by irradiated autologous feeder cells and BiP. (b) Responses of a representative clone to BiP and the control antigen ß-galactosidase (generated in the same expression system as BiP).Values are mean and S.D. c.p.m.
|
|
Phenotyping of specific clones
Of the six BiP-specific clones isolated, five were CD8-positive and one was CD4-positive (for representative FACS profiles see Fig. 3
).

View larger version (21K):
[in this window]
[in a new window]
|
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 FC2E4 all express CD8 and no CD4. Panel (b) shows that the T-cell receptor ß-chain usage (TCRVß) of FC2B5 is Vß 12 (x-axis). Panel (e) shows that the TCRVß usage of FC2E4 is Vß 7.1 (x-axis). No antibody in the panel used stained clone FC3E3.
|
|
Vß staining of specific clones
There was no common usage of Vß elements between the clones. Figure 3
shows the FACS profiles of clones FC2B5 3E3 and 2E4; panels (a), (c) and (d) show CD4 (x-axis) and CD8 (y-axis) double staining (CD4-FITC and CD8-PE). Panels (b) and (e) show staining with antibodies to the Vß element of the T-cell receptor. Of the CD8-positive clones, one (3E3) did not have its Vß usage determined with the antibodies used, clone 2B3 expressed Vß5.1, clone 2B5 expressed Vß12, clone 2E4 expressed Vß7.1 and 3E6 expressed Vß8.
Additional phenotypic studies
Two rapidly growing CD8-positive clones were further phenotyped to examine the expression of costimulatory molecules and activation markers. Figure 4
shows expression of the CD25, 28, 56, 57, 80, 86 and CTLA-4. As expected, both clones expressed the
chain of the IL-2 receptor (CD25). The costimulation molecule CD28 was expressed on both the clones, as were the ligands for CD28, CD80 and CD86. The expression of the costimulatory molecule CTLA-4, thought to be an essential signal for some regulatory T cells [16], was expressed cytoplasmically in one of the two clones examined, but not on the surface of the cell. Interestingly, the single CD4-positive, BiP-responsive clone (FC1D5) expressed surface CLTA-4 constitutively (data not shown). None of the clones expressed the NK marker CD57 but low-level expression of CD56 was seen on both clones.

View larger version (27K):
[in this window]
[in a new window]
|
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.
|
|
Determination of cytokine production of T-cell clones
Supernatants were removed from cultures 72 h after stimulation with PHA and LC-T. Figure 5
shows IL-10 production from all clones isolated. Interestingly, the clone showing the least IL-10 production proliferated most strongly. The cytokine levels were compared with the results obtained by the feeder mix (irradiated allogeneic cells with LC-T and PHA) alone. Three strongly growing clones were tested for a number of other cytokines. IL-10, IL-5 and IL-4 production was seen in the CD8-positive clone FC3E3 and IL-10 in the CD8 clone FC2E4 (Table 1
). Clone FC2B5 produced low levels of IL-10, suggesting that the IL-10 production was not simply due to the cloning procedure, and high levels of IFN-
, 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-
(data not shown). Cytokine production by antigen stimulation of the clones was difficult to measure due to the production of IL-10 by the feeder cells when cultured with BiP (data not shown).

View larger version (8K):
[in this window]
[in a new window]
|
FIG. 5. IL-10 production of BiP-responsive clones. Levels of cytokine were determined 72 h after stimulation with PHA, LC-T and irradiated allogeneic feeder cells. Values given are above that seen with feeder mix alone.
|
|
Cytokine production by T-cell lines grown from peripheral blood and synovial fluid of an RA patient
T-cell lines were grown from RA patient K in the presence of BiP. The lines were generated using the same initial protocol used for the clones before the limiting dilution stage. On examining the cytokine profiles of three lines grown from the peripheral blood (PB) and three from the synovial fluid (SF), two of three lines generated from PB produced IL-10, whereas none of the three lines generated from the SF did (Table 2
). The six lines generated from the RA patient were all predominantly CD3+CD4+ cells, in contrast to the CD8 T cells from the normal individual.
BiP-induced IL-10 production by RA patients
BiP-induced IL-10 production was measured in PB and SF mononuclear cell populations isolated from nine patients with RA. All samples were received from the rheumatology clinic at Guy's hospital. Cells were set up at 1x106 cells per well and stimulated with 0.25 µM BiP. Supernatants were taken 72 h after stimulation and the cytokine profile was determined by ELISA. As seen in Fig. 6
, no differences were seen in the amount of IL-10 produced by cells isolated from the PB and SF from RA patients. Furthermore, there was no difference seen between the RA patients and six normal individuals (data not shown).

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 6. BiP-induced IL-10 production in paired PB and SF from RA patients. There were no differences in the levels of BiP-induced IL-10 produced by cells isolated from the PB and SF of nine RA patients.
|
|
 |
Discussion
|
---|
The aim of this study was to investigate the T-cell response to BiP in the PB of normal, healthy individuals in order to determine the nature of the cells activated by BiP and, in particular, whether they had any of the emergent characteristics of regulatory T cells. The data in this report suggest that BiP can indeed stimulate a population of CD8 T cells from normal individuals that have the cytokine profile of regulatory cells because they produce the anti-inflammatory cytokine IL-10 and, in some cases, IL-4 and IL-5. In common with other reported human regulatory T cells, they have a low but consistent proliferative capacity [17].
The role of regulatory T cells, both CD4-negative and CD8-positive, in health and disease is becoming more understood. Whereas CD4-positive regulatory T cells have been investigated extensively (for review see [18]), CD8-positive regulatory cells have proved more difficult to isolate and study. These cells, however, have been described in humans and shown to be capable of high IL-10 production [19]. In this report, IL-10-producing CD8 cells specific for influenza matrix peptide were isolated from two volunteers who had been injected with immature dendritic cells pulsed with this antigen. The induction of regulatory T cells by immature dendritic cells has been reported by a number of groups (for review see [20]). The ability of CD8 T cells to produce regulatory cytokines is a relatively new discovery in the immune arsenal, but this cell type has been implicated in autoimmunity. Experimental allergic encephalomyelitis (EAE) can be prevented by regulatory T cells of both the CD8 [21, 22] and the CD4 [23, 24] type. In the EAE model, regulatory CD8 cells have been shown to be present in naive mice, their numbers increasing with age. On exposure to myelin basic protein, this population shows a restricted clonotype, suggesting expansion of a specific regulatory population, which can affect the outcome of the disease. Interestingly, the cytotoxic potential of these cells does not correlate with their proliferative ability [21]. Jiang et al. [22] have suggested that CD8-positive regulatory cells, when induced by T-cell vaccination (with disease-causing CD4 cells), are cytotoxic for the CD4 cell and that this response is abrogated in ß2 microglobulin-deficient mice [22]. CD8 cells have also been shown to protect against oil-induced arthritis in the rat [25].
BiP stimulates a population of CD8 T cells. Of the BiP-responsive clones isolated, five out of six were CD8-positive; moreover, these clones produced a cytokine profile similar to that of regulatory CD4 cells, in that IL-10 and IL-4 were predominant. The role of CD8 T cells in RA has been the subject of some investigation and it has been shown that CD8-positive cells isolated from the SF can have a restricted clonotype, suggesting expansions of discrete CD8 populations [26, 27]. The same reports also describe the expansion of 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 the effects of CD8 cells in an animal model of arthritis (collagen-induced arthritis in HLA-DQ8 transgenic mice) has suggested that these cells may play a role in regulating the disease. In this model, which mirrors RA in that rheumatoid factor is produced, depletion of the CD8 cells results in severe disease and an increase in the proinflammatory cytokines IFN-
and TNF-
[28]. The absence of CD8 cells also results in the production of anti-nuclear antibodies in the mice.
Of the two CD8-positive BiP-responsive clones examined for the expression of CTLA-4, one constitutively expressed this molecule cytoplasmically. This molecule is transiently expressed on activated CD4 and CD8 cells, although its expression on CD8 cells has been reported to be associated with early activation [29]. The expression of this molecule on CD8 clones, which are long-term activated cells, may represent a different role. Regulatory T cells of the CD4 phenotype have been shown to express CTLA-4 constitutively, and blockade of this molecule abrogates the suppression of antigenic and polyclonal stimulation of other T cells [30, 16]. A synergistic effect of CTLA-4 blockade and depletion of CD25-positive regulatory T cells has been reported [31], suggesting two pathways of immune control.
T-cell lines generated by the incubation of SF mononuclear cells of an RA patient with BiP showed no IL-10 production, in contrast to the PB cells of the same patient. Lines generated from this patient were all CD4-positive, suggesting that the normal CD8 response to BiP may be replaced by a CD4 response in RA patients. This may be due to differential processing of the antigen in RA patients, or the in vitro observation may occur because of the relatively slow growth of the CD8-positive, IL-10-producing subset, allowing CD4 cells to outgrow them. In mixed mononuclear cell populations, however, IL-10 is produced at the same level in PB and SF cell populations, suggesting that IL-10-producing cells responsive to BiP are present in the synovial compartment.
The proportion of CD8 cells is often increased in the SF of patients with RA; furthermore, a recent report has identified a population of IL-10-producing Tc2 cells that is elevated in the SF of RA patients when compared with the PB [32]. The authors of this paper suggest that the increase in Tc2 cells may be part of an insufficient effort to reduce the inflammation in the joint. This information, together with the demonstration that members of the HSP70 family are overexpressed in the synovial joint [10] and that the conditions exist for BiP overproduction [6], could lead to the conclusion that the interaction between BiP and CD8 cells may be occurring within this compartment. Our data suggest that BiP is an antigen that may specifically stimulate CD8 cells with the ability to produce large amounts of IL-10. This IL-10 production may be part of a normal mechanism to down-modulate an immune response. The increased reactivity seen in response to BiP in the synovial compartment of RA may be an attempt by the host to reduce the inflammation present. Although, as described above, HSPs can cause the production of proinflammatory cytokines, such as TNF-
and IL-1, there is evidence that some HSPs can lead to the production of anti-inflammatory cytokines. De et al. [33] showed that the low-molecular weight HSP27 can cause the production of large amounts of IL-10, with very little TNF-
, from monocytes. The ease of isolation of BiP-responsive CD8 T cells in this study may be considered surprising, but other members of the HSP70 family have been shown to stimulate this cell type preferentially [13, 15]. In conclusion, the finding of human BiP-responding T cells in the peripheral blood of normal individuals suggests that BiP-responsive T cells may form part of a regulatory network that has a role in the control of ongoing immune responses.
 |
Acknowledgments
|
---|
This work was supported by Arthritis Research Campaign project grants P0559 and B0677.
 |
Notes
|
---|
Correspondence to: M. Bodman-Smith, Guy's Hospital, London, SE1 9RT, UK. E-mail: mark.bodman-smith{at}kcl.ac.uk 
 |
References
|
---|
- Corrigall VM, Bodman-Smith MD, Fife MS et al. The human endoplasmic reticulum molecular chaperone BiP is an autoantigen for rheumatoid arthritis and prevents the induction of experimental arthritis. J Immunol 2001;166:14928.[Abstract/Free Full Text]
- Blass S, Union A, Raymackers J et al. The stress protein BiP is overexpressed and is a major B and T cell target in rheumatoid arthritis. Arthritis Rheum 2001;44:76171.[CrossRef][ISI][Medline]
- Gething MJ. Role and regulation of the ER chaperone BiP. Semin Cell Dev Biol 1999;10:46572.[CrossRef][ISI][Medline]
- McMillan DR, Gething MJ, Sambrook J. The cellular response to unfolded proteins. intercompartmental signaling. Curr Opin Biotechnol 1994;5:5405.[Medline]
- Cai B, Tomida A, Mikami K, Nagata K, Tsuruo T. Down-regulation of epidermal growth factor receptor-signaling pathway by binding of GRP78/BiP to the receptor under glucose-starved stress conditions. J Cell Physiol 1998;177:2828.[CrossRef][ISI][Medline]
- Maddison PJ, Isenberg DA, Woo P, Glass DN. Oxford textbook of rheumatology. Oxford: Oxford University Press, 2001.
- Munro S, HR Pelham. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 1986;46:291300.[ISI][Medline]
- McKay DB. Structure and mechanism of 70-kDa heat-shock-related proteins. Adv Protein Chem 1993;44:6798.[ISI][Medline]
- Hayem G, De Bandt M, Palazzo E et al. Anti-heat shock protein 70 kDa and 90 kDa antibodies in serum of patients with rheumatoid arthritis. Ann Rheum Dis 1999;58:2916.[Abstract/Free Full Text]
- Schett G, Redlich K, Xu Q et al. Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Differential regulation of hsp70 expression and hsf1 activation in synovial fibroblasts by proinflammatory cytokines, shear stress, and antiinflammatory drugs. J Clin Invest 1998;102:30211.[Abstract/Free Full Text]
- Wendling U, Paul L, van der ZR, Prakken B, Singh M, van Eden W. A conserved mycobacterial heat shock protein (hsp) 70 sequence prevents adjuvant arthritis upon nasal administration and induces IL-10-producing T cells that cross-react with the mammalian self-hsp70 homologue. J Immunol 2000;164:27117.[Abstract/Free Full Text]
- Tanaka S, Kimura Y, Mitani A et al. Activation of T cells recognizing an epitope of heat-shock protein 70 can protect against rat adjuvant arthritis. J Immunol 1999;163:55605.[Abstract/Free Full Text]
- Breloer M, Fleischer B, von Bonin A. In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins. J Immunol 1999;162:31417.[Abstract/Free Full Text]
- Asea A, Kraeft SK, Kurt-Jones EA et al. HSP70 stimulates cytokine production through a CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000;6:43542.[CrossRef][ISI][Medline]
- Blachere NE, Li Z, Chandawarkar RY et al. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med 1997;186:131522.[Abstract/Free Full Text]
- Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med 2000;192:295302.[Abstract/Free Full Text]
- Groux H, O'Garra A, Bigler M et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997;389:73742.[CrossRef][ISI][Medline]
- Shevach EM. Regulatory T cells in autoimmmunity. Annu Rev Immunol 2000;18:42349.[CrossRef][ISI][Medline]
- Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 2001;193:2338.[Abstract/Free Full Text]
- Roncarolo MG, Levings MK, Traversari C. Differentiation of T regulatory cells by immature dendritic cells. J Exp Med 2001;193:F5F9.[ISI][Medline]
- Sun D, Whitaker JN, Wilson DB. Regulatory T cells in experimental allergic encephalomyelitis. I. Frequency and specificity analysis in normal and immune rats of a T cell subset that inhibits disease. Int Immunol 1999;11:30715.[Abstract/Free Full Text]
- Jiang H, Kashleva H, Xu LX et al. T cell vaccination induces T cell receptor Vbeta-specific Qa-1-restricted regulatory CD8(+) T cells. Proc Natl Acad Sci USA 1998;95:45337.[Abstract/Free Full Text]
- Olivares-Villagomez D, Wensky AK, Wang Y, Lafaille JJ. Repertoire requirements of CD4+ T cells that prevent spontaneous autoimmune encephalomyelitis. J Immunol 2000;164:5499507.[Abstract/Free Full Text]
- Van de Keere F, Tonegawa S. CD4(+) T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice. J Exp Med 1998;188:187582.[Abstract/Free Full Text]
- Jansson AM, Lorentzen JC, Bucht A. CD8+ cells suppress oil-induced arthritis. Clin Exp Immunol 2000;120:5326.[CrossRef][ISI][Medline]
- Gonzalez-Quintial RR, Baccala R, Pope RM, Theofilopoulos AN. Identification of clonally expanded T cells in rheumatoid arthritis using a sequence enrichment nuclease assay. J Clin Invest 1996;97:133543.[Abstract/Free Full Text]
- Fitzgerald JE, Ricalton NS, Meyer AC et al. Analysis of clonal CD8+ T cell expansions in normal individuals and patients with rheumatoid arthritis. J Immunol 1995;154:353847.[Abstract/Free Full Text]
- Taneja V, Taneja N, Painsansinsup T et al. CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: Implications for rheumatoid arthritis. J Immunol 2002;168:586775.[Abstract/Free Full Text]
- Slifka MK, Whitton JL. Activated and memory CD8+ T cells can be distinguished by their cytokine profiles and phenotypic markers. J Immunol 2000;164:20816.[Abstract/Free Full Text]
- Takahashi T, Tagami T, Yamazaki S et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 2000;192:30310.[Abstract/Free Full Text]
- Sutmuller RP, van Duivenvoorde LM, van Elsas A et al. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 2001;194:82332.[Abstract/Free Full Text]
- Berner B, Akca D, Jung T, Muller GA, Reuss-Borst MA. Analysis of Th1 and Th2 cytokines expressing CD4+ and CD8+ T cells in rheumatoid arthritis by flow cytometry. J Rheumatol 2000;27:112835.[ISI][Medline]
- De AK, Kodys KM, Yeh BS, Miller-Graziano C. Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus. J Immunol 2000;165:39518.[Abstract/Free Full Text]
Submitted 6 August 2002;
Accepted 15 November 2002