Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.     

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.     

Diversity in antigen recognition by Mycobacterium tuberculosis-reactive T cell clones from the synovial fluid of rheumatoid arthritis patients

In a previous study we have shown that synovial fluid mononuclear cells from many rheumatoid arthritis (RA) patients exhibit an enhanced response to M. tuberculosis antigens as compared to peripheral blood mononuclear cells. The 65-kDa heat-shock protein of M. tuberculosis was shown not to play an important role in this response, therefore other mycobacterial proteins must be involved. In this study we have investigated the possibility that synovial fluid T cells from RA patients predominantly recognize a limited number of M. tuberculosis antigens, as a result of a lesion-specific activation of only those M. tuberculosis-reactive T cells that have cross-reacted with joint-related autoantigens. From the synovial fluid of four RA patients M. tuberculosis-reactive T cell clones were isolated and analyzed for their phenotype, HLA-DR restriction and proliferation to immunoblot fractions containing sodium dodecyl sulfate-polyacrylamide gel-separated M. tuberculosis proteins of known molecular weight range. The overall M. tuberculosis immunoblot recognition pattern of the clones was strikingly heterogeneous. Within a panel of 15 clones 12 different antigenic specificities could be distinguished. In other words, we did not observe a dominant recognition of a few M. tuberculosis antigens by synovial fluid T cells. This argues against the hypothesis that the elevated synovial T cell reactivity against M. tuberculosis is a reflection of an in vivo expansion of a limited number of different types of M. tuberculosis-reactive T cells as a result of a cross-reaction with putative joint autoantigens.     

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring

The 65-kD hsp from Mycobacterium tuberculosis has been reported to induce an autopathogenic subset of T cells in at least two animal models of autoimmune disease. Reports of increased expression of human hsp60 in the inflamed synovial tissue of rheumatoid arthritis (RA) patients, increased proliferation of RA synovial fluid T cells to mycobacterial hsp65, and increased levels of anti-mycobacterial hsp65 antibody in synovial fluid, have suggested that the highly homologous human (hu) hsp60 may be recognized as an autoantigen in RA patients. In the present study, we have examined by ELISA the serum IgG antibody levels to mycobacterial hsp65 and hu hsp60, as well as to the Escherichia coli hsp60, groEL, in patients with RA, systemic lupus erythematosus (SLE), Reiter's syndrome, active tuberculosis, and normal controls. In all these groups, the levels of anti-groEL and anti-hu hsp60 were significantly higher than the anti-mycobacterial hsp65. Anti-hu hsp60 was positively correlated with anti-groEL, but not with anti-mycobacterial hsp65. Anti-hu hsp60 was competitively inhibited by either soluble groEL or hu hsp60, but little or none by mycobacterial hsp65. Reiter's sera were found to have somewhat higher levels of anti-groEL and anti-hu hsp60 than did normal controls. We conclude that IgG anti-hu hsp60 autoantibodies arise primarily as a consequence of the humoral immune response to E. coli groEL through the recognition of cross-reactive epitopes.     

Clonal analysis of T cell infiltrates in synovial tissue of patients with rheumatoid arthritis

To investigate the possibility of the presence of disease-relevant, antigen-specific immune reactions in rheumatoid arthritis (RA), clonal diversity of the T cells in the synovial tissue was examined. T cells were directly cloned by in vitro stimulation with phytohemagglutin and interleukin 2 from both the peripheral blood and inflammatory synovial tissue. Their clonotypes were defined by analyzing rearrangement patterns of T cell receptor (TCR) beta and gamma chain genes using Southern blotting. In total, 111 clones from the synovial tissue (four patients) and 45 from the peripheral blood (one patient) were studied. Although most of the clones were unique in their TCR gene rearrangement patterns, 2 clones from the synovial tissue of one patient had identical patterns. These 2 clones were CD3+, 4-, 8+. Since phenotypic analysis of 82 clones from the synovial tissues revealed that CD8+ T cell clones were less frequent (24%) than CD4+ clones, the clonal identity observed here in 2 clones may not be negligible. Furthermore, 1 CD8+ clone from the peripheral blood of the same patient also had the same clonotype. These results may suggest selective trafficking or proliferation of CD4-, CD8+ T cells in RA synovial tissue.     

Proliferative response of synovial fluid and peripheral blood mononuclear cells to arthritogenic and non-arthritogenic microbial antigens and to the 65-kDa mycobacterial heat-shock protein

Cellular immune responses to microbial antigens have been implicated in the pathogenesis of some forms of arthritis including reactive arthritis, Reiter's syndrome, ankylosing spondylitis and rheumatoid arthritis. We investigated the proliferative T cell responses of paired peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MC) to so-called arthritogenic bacteria (Yersinia entero-colitica and Salmonella typhimurium), to control antigens, such as Candida albicans, mumps virus and purified protein derivative, to the recombinant mycobacterial 65-kDa heat-shock protein (hsp 65) and the mitogen phytohemagglutinin (PHA) in 16 patients with different inflammatory rheumatic diseases. The [³H]thymidine uptake of unstimulated cells (medium control) as well as the proliferative response to the different antigens tested was markedly increased in SFMC irrespective of the underlying rheumatic disease. In contrast, mitogenic stimulation was decreased in SFMC. The proliferative response to the hsp 65 correlated significantly with the responses to Yersinia, Salmonella and Candida. These results may reflect an enhanced function of SF antigen-presenting cells, different functional properties and subset distributions of PB and SF T cells with a preferetial accumulation of helper-inducer/memory T cells or a maintenance of an ongoing immune response by T cells cross-recognizing self epitopes such as epitopes located on the hsp 65. Thus, care should be taken in the interpretation of SF T cell responses to microbial antigens as diagnostic tools in arthritis.     

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: Selective in vivo expansion of T cells bearing defined receptors

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either Vβ5.2 or a closely related β chain, Vβ5.6.The α chains expressed by Vβ5.2⁺ and Vβ5.6⁺ clones were from different families, Vα2.4 and Vα23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either Vβ5.2 or Vβ5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of Vβ5. Conservation of the β chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing α and β chains of representative Vβ5.2⁺ and Vβ5.6⁺ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of a chain usage, since all clones expressed a member of the Vαl family, but again CDR3 sequence conservation was not apparent, β chain usage was not restricted since different clones expressed Vβ6.7, Vβ22.3 and Vβ12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical α and β TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.     

T cell receptor repertoire in rheumatoid arthritis

CD4+ T cells are a major component of the inflammatory infiltrate in rheumatoid synovitis. Within synovial lesions, clonal CD4+ T cell populations are detectable, supporting the notion of an antigen specific recognition even in the joint. In general, the clonal size of individual T cell clones is small and does not lead to a marked distortion of the synovial T cell receptor (TCR) repertoire. Comparison of TCR sequences derived from different patients has not provided evidence for common sequences. Either multiple antigens are recognized or the TCR repertoire is sufficiently plastic with a multitude of different TCR structures responding to the same antigen(s). However, within one individual, the repertoire of clonal T cell populations is restricted. Identical T cell clones can be identified in different joints and at different timepoints of the disease, emphasizing that the spectrum of antigens recognized is conserved over time and that the T cell response pattern is not subject to evolution. Characterization of antigens involved in the latter stages of the disease may thus provide critical information on disease-initiating events. Recent data have led to the new concept that the role of T cells in rheumatoid arthritis (RA) is not limited to synovial inflammation. Evidence has been provided that the premorbid TCR repertoires of RA patients and normal controls can be distinguished. The T cell repertoire in RA patients is prone to recognize certain microbial products and autoantigens. The selection of this response pattern can only partially be attributed to the disease associated HLA-DRB1 alleles. Additional factors common in RA patients but not in HLA-DR matched control individuals seem to be important in shaping the TCR repertoire. Furthermore, the repertoire of mature T cells in RA patients is characterized by oligoclonality which involves T cells in the peripheral blood compartment. Possibly, these clonal T cell populations react to widespread autoantigens, raising the possibility that RA patients have a defect in controlling peripheral tolerance and an anomaly of lymphoproliferation. In contrast to joint residing CD4+ T cells, expanded clonotypes isolated from the blood of different patients have been described to share TCR beta chain structures. How these characteristic features of the global TCR repertoire in RA patients translate into mechanisms of disease remains to be elucidated.     

Presence of human 65 kD heat shock protein (hsp) in inflamed joints and subcutaneous nodules of RA patients

Monoclonal antibodies to the human homologue of the bacterial 65 kD heat shock protein (hsp) were used to investigate the tissue distribution of endogenous hsp 65 in normal versus rheumatoid synovial tissue, in subcutaneous nodules of patients with rheumatoid arthritis (RA) and in several instances of non-rheumatoid inflammation. A strong reactivity of the anti-hsp antibody was found in the cartilage-pannus junction in rheumatoid joints and in rheumatoid nodules, but not in normal joints or in normal or inflamed kidney or liver (irreversible graft rejection, chronic glomerulonephritis or primary biliary cirrhosis). The findings provide a new hypothetical explanation for a role of T cells reactive with the 65 kD hsp in the generation of both articular and extra-articular lesions in chronic rheumatoid arthritis.     

Presence of Human 65 kD Heat Shock Protein (hsp) in Inflamed Joints and Subcutaneous Nodules of RA Patients

Monoclonal antibodies to the human homologue of the bacterial 65 kD heat shock protein (hsp) were used to investigate the tissue distribution of endogenous hsp 65 in normal versus rheumatoid synovial tissue, in subcutaneous nodules of patients with rheumatoid arthritis (RA) and in several instances of non-rheumatoid inflammation. A strong reactivity of the anti-hsp antibody was found in the cartilage-pannus junction in rheumatoid joints and in rheumatoid nodules, but not in normal joints or in normal or inflamed kidney or liver (irreversible graft rejection, chronic glomerulonephritis or primary biliary cirrhosis). The findings provide a new hypothetical explanation for a role of T cells reactive with the 65 kD hsp in the generation of both articular and extra-articular lesions in chronic rheumatoid arthritis.     

Synoviocytes Type A Bind Exogenous Antigens Recognized by Antibodies Present in Rheumatoid Arthritis

The presence of antibodies in rheumatoid arthritis (RA) patients to antigens on the synoviocyte surface has recently been reported (Scand. J. Immunol. 27, 295, 1988). Here we have further characterized these antigens and found that they are exogenous proteins acquired from the bovine serum used in the culture medium. By immunoprecipitation and ELISA studies, we have identified bovine albumin and transferrin as the antigens recognized by the RA antibodies. These specificities were found not only in the sera but also in the synovial fluid from RA patients. A comparative study with a large panel of RA sera did not show a correlation in the antibody specificities for bovine albumin, bovine transferrin, or the 65-kDa heat shock protein from Mycobacterium bovis. Similar experiments using rabbit and monkey sera as well as human synovial fluid and serum as a source of antigen did not reveal any reactivity with a highly positive RA serum. By sequence alignment, a high degree of homology between residues 142-156 from bovine albumin and residues 65-78 from human pro-collagen alpha 1 (I) was found. The capacity of the synoviocytes to bind exogenous antigens and the presence of antibodies to bovine proteins, normally present in the diet, suggest a role for these type A synoviocytes as well as a possible involvement of food antigens in the pathogenesis of RA.     

Evidence for monoclonal expansion of synovial T cells bearing V alpha 2.1/V beta 5.5 gene segments and recognizing a synthetic peptide that shares homology with a number of putative autoantigens.

A peptide of 15 amino acids derived from the cereal glycine-rich cell wall protein (GRP), sharing a significant homology with Epstein-Barr virus nuclear antigen-1 (EBNA-1), fibrillar and procollagen, stimulated synovial fluid (SF) T cells from juvenile (JRA) and adult (RA) rheumatoid arthritis patients. An overexpression of the V alpha 2 gene family was found in the SF from patients who responded significantly to the peptide. To investigate in more detail the SF T-cell responses to the GRP peptide, we established peptide-specific T-cell lines and clones from a DR8+ positive JRA patient with pauciarticular form. The T-cell clones were phenotyped as T-cell receptor (TCR)alpha beta+/CD4+ and their clonality was investigated by polymerase chain reaction (PCR) and flow cytometric analysis. TCR sequences from different clones demonstrated that the clones were identical and used the V alpha 2.1/J alpha 6 combined with V beta 5.5/J beta 2.7 gene segments. Interestingly, direct sequencing of the V alpha 2 family PCR product obtained from cDNA prepared from freshly isolated SF mononuclear cells identified the same TCR sequence as that used by the clones, suggesting the monoclonality of SF CD4+ T cells bearing V alpha 2.1/J alpha 6 gene products. The present data suggest a recruitment and expansion of a SF T-cell subpopulation, and also support the hypothesis that autoimmune diseases can be triggered by protein epitopes with crucial amino acids homologous to self-proteins.     

Immune Responses to 6 and 30-kDa Mycobacterial Antigens in Rheumatoid Patients, and Vβ Usage by Specific Synovial T-Cell Lines and Fresh T Cells

We have investigated both the humoral and the cellular immune responses of patients with juvenile rheumatoid arthritis (JRA) and rheumatoid arthritis (RA) to mycobacterial antigens. The JRA group was not Bacillus Calmette Guerin (BCG) vaccinated whilst the majority of the RA group was. As determined by immunoblotting, 79% of sera from patients with JRA reacted mainly with a 18.6-kDa protein (P18.6), whilst 70% of sera from patients with RA reacted mainly with a 30-kDa protein (P30) of BCG, M. tuberculosis and M. kansasii. In contrast, only a moderate proportion of the control sera (25% of adult and 20% of children) showed reactivity to P30, and none of the samples had significant reactivity with the P18.6 antigen. Furthermore, T-cell proliferation to the P18.6 and P30 antigens was detected in the majority of JRA and RA patients, and was nearly always higher in synovial fluid (SF) than in the peripheral blood (PB). We also investigated the usage of V beta family genes in P18.6 and P30 antigen-specific T-cell lines established from the SF of one patient with active RA. We showed that V beta 2, -4, -5, -6, -7, -14, -17, -18 and V beta 19 were over-represented compared with other known V beta families. We also noted that the proportion of V beta 14 was higher in freshly isolated SF mononuclear cells compared with the blood in this patient and in 2 out of 4 other RA patients examined. Other V beta families such as V beta 6, V beta 8, V beta 16, V beta 18 and V beta 19 were also over-represented in the SF compared with the blood in some patients. Taken together our results provide more information concerning the role of mycobacterial antigens in RA and suggest that there may be an in vivo clonal expansion of T lymphocytes in the synovium.     

Immunohistochemical localization of HTLV-I p19- and p24-related antigens in synovial joints of patients with rheumatoid arthritis.

In formalin-fixed, paraffin-embedded synovial tissues from patients with early proliferative rheumatoid arthritis (RA), immunoreactivity could be demonstrated utilizing monoclonal IgG antibodies reactive with the p19 and p24 protein of human T cell leukemia virus (HTLV-I). Additionally, surgical specimens of fresh unfixed synovial tissues from patients with RA also demonstrated immunoreactivity. At the light microscopic level, both HTLV-I antigens were detected in approximately 45% of the rheumatoid synovial tissues by the immunocolloidal gold method with silver enhancement (IGSS) and the avidin-biotin-complex technique (ABC), whereas six of eight of the frozen RA specimens stained positive by immunofluorescence. Patients whose synovial tissues were immunoreactive by immunofluorescence were seronegative to HTLV-I antigens as determined by ELISA and immunoblotting. Conversely, cases with osteoarthritis, juvenile rheumatoid arthritis, psoriatic arthritis, Dupuytren's contracture, and gangrene were shown to be nonreactive by immunohistochemistry. The results indicate that expression of antigens is related to or crossreactive with HTLV-I in synovial tissues from patients with rheumatoid arthritis.     

Heterogeneity of T cell receptor idiotypes in rheumatoid arthritis.

The nature of the T cell response in the rheumatoid synovium was investigated by using monoclonals MX9 and 42/1C1, which recognize the V beta 8 and V beta 5 T cell receptor gene families respectively. The blood and synovial T cells of ten patients with rheumatoid arthritis were compared. The majority (8/10) had different numbers of V beta 5 and V beta 8 cells in the joints from those in the blood, indicating that the T cells in the joints were not a sample of those in the blood. In three patients both V beta 5 and V beta 8 cells in the joint were augmented in number, suggesting that the T cells selectively retained in the joint were not members of a single clone, but derived from many clones. Some patients had increased levels of V beta 5 or V beta 8 alone in the joint indicating that heterogeneity existed between patients. These results are not consistent with the preferential or dominant use of a single V beta gene family in the T cells involved in the rheumatoid arthritic joints.     

Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides

Prediction, identification and analysis of T cell epitopes in protein antigens has become a central theme in fundamental and applied immunology. However, while for the characterization of linear B cell epitopes the so-called Pepscan procedure was found to be extremely effective, no such technique has so far been available for T cell studies. Recently, we described the identification and localization of a T cell epitope in a mycobacterial 65-kDa shock protein in the model of adjuvant arthritis. This was done by molecular cloning and conventional solid-phase synthesis techniques. We now show that the delineation of such a T cell epitope and its further characterization can be accomplished in a much more rapid and efficient manner by a modification of the existing Pepscan technique. We show for the first time that several hundreds of peptides, simultaneously synthesized in an automated way on activated polyethylene rods, can be easily recovered from these rods in adequate quantities, enabling a systematic analysis of T cell epitopes. Synthesis of sequentially overlapping peptides along the 65-kDa protein revealed that the adjuvant arthritis T cell clones are fully stimulated by peptides that comprise a minimal sequence of seven residues, corresponding to positions 180-186 in the sequence of the 65-kDa protein of M. bovis Bacillus Calmette Guerin (BCG). Detailed examination of the epitope by peptides containing a single amino acid substitution showed that, apart from one conservative replacement (Glu----Asp), the requirement for the native residue at all positions in peptide 180-186 was absolute for full T cell stimulation. Their indispensability was confirmed with deletion and insertion peptides. It is concluded that the occurrence of indifferent or spacer residues in a minimal stimulatory sequence, as observed by others, is not a general feature of T cell epitopes.     

Inflammation activates self hsp60-specific T cells

Injection of incomplete Freund's adjuvant (IFA) into the footpads of BALB/c mice induced an acute inflammation. Draining popliteal lymph nodes showed major histocompatibility complex (MHC) class II-restricted proliferation when challenged in vitro with recombinant Mycobacterium bovis 65-kDa heat shock protein (hsp65). αβ Tcell receptor-positive, CD4⁺, hsp65-specific T cell lines and clones were generated from these lymph nodes, and 87% of clones responded to a P galactosidase fusion protein containing residues 238–573 of human hsp60. Seventy percent of these hsp60-responsive clones also responded to a synthetic peptide corresponding to residues 412–423 of the mouse hsp60. This peptide also induced significant responses in IFA-primed lymph node cells but not in lymphoid cells from unimmunized mice. These results demonstrate that T cells specific for epitopes in self hsp60 are activated during inflammatory responses induced in the absence of exogenous bacterial hsp65. The findings of this study may provide a basis for understanding the often reported isolation of mycobacterial hsp65-responsive T cells from inflammatory sites of arthritis patients, and the protective effects of preimmunization with hsp65 in experimental models of arthritis.     

Juvenile rheumatoid arthritis patients manifest immune reactivity to the mycobacterial 65-kDa heat shock protein, to its 180-188 peptide, and to a partially homologous peptide of the proteoglycan link protein.

Immune reactivity to the 65-kDa mycobacterial heat shock protein (hsp65) has been associated with arthritis in rats and humans. In this report we evaluated patients with juvenile rheumatoid arthritis for such immunity. A high proportion of affected children showed both antibody and T lymphocyte responses to hsp65 and to two related peptides: the nonapeptide 180-188 sequence of hsp65 and a partially homologous peptide of the cartilage proteoglycan link protein. The titer of circulating antibodies was generally higher in patients with clinically active disease. In contrast to the juvenile rheumatoid arthritis patients, patients with adult rheumatoid arthritis tended to have lower responses of their peripheral blood T lymphocytes to the whole hsp65 molecule. Moreover, the adult rheumatoid arthritis patients did not respond to the peptides. Thus, there appear to be immunological differences between juvenile and adult forms of rheumatoid arthritis related to hsp65 reactivity.     

T cell reactivity to an epitope of the mycobacterial 65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.     

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.