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.     

The 65-kDa heat-shock protein in the pathogenesis, prevention and therapy of autoimmune arthritis and diabetes mellitus in rats and mice

Conclusions hsp are molecules which are highly conserved from procaryotes to eukaryotes. At a first glance the immune system should treat these molecules as self. However, strong immune reactions to bacterial hsp are observed during infection in mammals.hsp65 plays a role in several autoimmune diseases in animal models. In AA in Lewis rats the involvement of hsp65 has been revealed by T cell clones which induce disease in naive recipients, or by T cell vaccination experiments. T cell clones which show in vivo activity have been used as tools in vitro to define epitopes involved in the disease process. In this manner mycobacterial hsp65 and its epitope peptide 180–188 were deduced for AA in Lewis rats. Similarily the epitope p277 was defined for diabetes in NOD mice.The role of hsp65 in several other autoimmune diseases was seen when animals were pretreated with hsp65 and found to be protected from subsequent induction of autoimmune disease. From the involvement of hsp65 in several different autoimmune diseases, it would appear that hsp65 is somehow a key factor in natural autoimmunity. At a fist glance this is surprising since mycobacterial hsp65 shows 50% amino acid homology with human hsp65, in other words it is half-self.Peptide epitopes, peptide 180–188 in AA in Lewis rats and p277 in IDDM in NOD mice, have been used for peptide vaccination, which represents another possibility for prevention of autoimmune disease. The immunological mechanism which leads to resistance from autoimmune disease involves hsp65 immunity and appears not to be associated with tolerance or non-responsiveness to hsp65, but seems to be due rather to modulation of naturally existing networks of idiotype-anti-idiotype T cells organized around hsp65 as the target antigen.     

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.     

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.     

Adjuvant arthritis and immunity to the mycobacterial 65 kDa heat shock protein.

The mycobacterial 65 kDa heat shock protein (HSP65) is of critical significance in the model of adjuvant arthritis (AA). Arthritogenic and protective T cell clones obtained from arthritic rats recognized the 180-188 sequence of HSP65. Previous reports have shown that administration of HSP65 prior to disease induction led to resistance to arthritis in the AA model and in several other models of experimental arthritis. Here, we report the development of immunity to HSP65 and the critical 180-188 epitope during the course of AA. Following Mycobacterium tuberculosis (MT) immunization both antibodies and T cell responses to HSP65 were detected. Proliferative responses to the 180-188 epitope were seen exclusively in the local draining lymph node cells at day 14 after immunization. The anatomical distribution and course of T cell responses to HSP65 and its 180-188 epitope are compatible with T cell regulated control of the disease. Although lower HSP65 antibody levels were observed in the animals with severe arthritis, in individual animals no evidence was obtained for a relationship between development of HSP65 humoral immunity and arthritis severity. Nevertheless, during disease exacerbation, elicited by HSP65 immunization during disease development, elevated T cell responses against HSP65 and its 180-188 epitope were found. In contrast, we obtained evidence that successful transfer of arthritis resistance to naive recipients depends on the transfer of HSP65 specific T cells. On the basis of these results, it seems that HSP65 plays a crucial role in the T cell regulatory events involved in both the induction of, and protection against, AA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Searching for the Cartilage-associated Mimicry Epitope in Adjuvant Arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis ( Mt ) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein ( Mt. hsp65 178-186 ). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65 178-186 specific T cells.     

Lysis of interferon-gamma activated Schwann cell by cross-reactive CD8+ alpha/beta T cells with specificity for the mycobacterial 65 kd heat shock protein.

Heat shock protein (hsp) 65 is a major T cell antigen of Mycobacterium leprae. The hsp 65 of M. leprae is nearly identical in M. bovis/M. tuberculosis (greater than 95% protein sequence homology) and surprisingly similar in man (65% protein sequence homology). Recently, we had provided evidence in a murine model that CD8+ T cells recognize and lyse Schwann cells presenting M. leprae antigen in the context of major histocompatibility (MHC) class I gene products. Because murine Schwann cells are class I negative, antigen presentation requires prior stimulation with interferon-gamma (IFN-gamma). CD8+ T cells were activated against tryptic fragments of mycobacterial hsp 65. These T cells recognized epitopes of hsp 65 which had been generated through the cytoplasmic class I processing pathway. They were also capable of lysing Schwann cells which had been activated by IFN-gamma and not primed with nominal hsp 65 peptides. In contrast, T cells activated against tryptic ova peptides only lysed Schwann cells which had been both stimulated with IFN-gamma and primed with ova peptides. Evidence is presented that class I (H-2D) restricted, CD8+ alpha/beta T lymphocytes with specificity for the mycobacterial hsp 65 recognize IFN-gamma-stimulated Schwann cells probably because they are specific for a(n) epitope(s) shared by the bacterial hsp and a host cognate. Activation of autoreactive T cells with specificity to shared epitopes could contribute to nerve damage in tuberculoid leprosy which is characterized by low to absent M. leprae in Schwann cells.     

Searching for the cartilage-associated mimicry epitope in adjuvant arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis (Mt) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein (Mt. hsp65(178-186)). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65(178-186) specific T cells.     

Recognition of exon-shuffled class II molecules by T helper cells

Exon shuffled I-A beta genes transfected into the B lymphoma cell line A20-2J were used to localize the epitope recognized by the monoclonal antibody 10.2.16 to the carboxy terminal portion of the beta 1 domain. In addition, several T helper cell hybrids were tested against these novel I-A molecules and the following observations were made: the beta 1 domain of A beta plays a dominant role in the restricted recognition by T helper cells; there appear to be multiple restriction epitopes on the I-A molecule; these epitopes can consist of conformational epitopes created by specific alpha and beta chains or consist of the polymorphic determinants encoded on the beta chain alone, and these novel I-A molecules serve as restriction elements in the antigen-specific recognition by T cells and in one case stimulate an alloreaction in the absence of antigen.     

Structural relationships between carrier epitopes and antigenic epitopes on hen egg-white lysozyme.

The spatial relationship for T-B cell cooperation between antigenic epitopes and carrier epitopes on the antigen molecule was studied. Two mono-DNP substituted derivatives of hen egg-white lysozyme (HEL), DNP1-33HEL and DNP1-96HEL were used as antigens; the former is dinitrophenylated only at lysine-33 and the latter only at lysine-96. Fragment peptides of HEL were used to induce specific T cells to the respective sites of the antigen. Adoptive cell-transfer experiments of site-specific T cells and DNP-primed B cells directly showed that multiple distinct carrier epitopes for T cells could help the antibody responses to the single antigenic epitope for B cells and that a single carrier epitope could help antibody responses to multiple antigenic epitopes. T cells primed with a synthetic peptide SP34-54 (corresponding to sequence 34-54 of HEL) co-operated with DNP-primed B cells on challenge with DNP1-96HEL, but not with DNP1-33HEL.     

Complexity of Human Immune Response Profiles for CD4+ T Cell Epitopes from the Diabetes Autoantigen GAD65

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplo-types characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.     

Complexity of human immune response profiles for CD4+ T cell epitopes from the diabetes autoantigen GAD65

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplotypes characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.     

Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.     

The effect of lipoylation on CD4 T-cell recognition of the 19,000 MW Mycobacterium tuberculosis antigen.

The mechanisms contributing to the dominant and degenerate recognition of the N-terminal region of the Mycobacterium tuberculosis 19,000 MW protein have been investigated. Using polyclonal and cloned T cells it was found that the apparently promiscuous response to the N-terminal peptide was due to the presence of multiple epitopes recognized in the context of different HLA determinants. This finding did not, however, explain the concentration of T-cell recognition on this part of the molecule. The 19,000 MW antigen is a lipoprotein, which raised the possibility that presence of a lipidation motif preceded by a signal peptide influenced the processing and presentation of the protein. Modified peptides covalently attached to lipid moieties were, therefore, tested on both polyclonal and cloned T cells. It was found that whilst lipoylation enhanced polyclonal T-cell recognition, the effect on cloned T cells was variable and depended on their epitope specificity and restricting HLA determinants. This suggested that whilst lipoylation may enhance some aspect of signalling for polyclonal T cells it does not affect the presentation of peptide to T-cell clones. The explanation for the immunodominance of the N-terminal region may, therefore, lie in some aspect of its processing.     

Stimulation of a memory B cell response does not require primed helper T cells

The use of universally immunogenic T cell epitopes, such as those identified in tetanus toxin or malaria circumsporozoite protein, could represent a major improvement in the development of synthetic vaccines. However, one limitation of this approach is the lack of T cell cross-reactivity between the vaccine and the pathogen. To determine whether the memory B cell response elicited by immunization with a synthetic peptide containing a B cell epitope linked to a T cell epitope can be restimulated by the same B cell epitope linked to different T cell epitope(s), we used a synthetic peptide which contains non-overlapping B and T cell determinants from hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV). The results of this study clearly show that primed T cells can increase the antibody response against a B cell epitope linked to the priming T cell determinant. However, the antibody response obtained was weaker than that obtained after two injections of the peptide containing both B and T cell epitopes, showing the important role played by memory B cells in secondary antibody responses. Moreover, a strong antibody response against the B cell epitope was elicited by boosting mice with the B cell epitope linked to a heterologous carrier, thus demonstrating that a strong B cell memory response can be revealed in the absence of primed T cells. These results therefore provide new important information for the design of synthetic or recombinant vaccines.     

Heat-shock protein T-cell epitopes trigger a spreading regulatory control in a diversified arthritogenic T-cell response

Summary: Adjuvant arthritis (AA) in Lewis rats is T-cell mediated and seems to depend on T cells recognising the 180–188 epitope of mycobacterial heat-shock protein (hsp) 60. Analysis of arthritogenic T-cell clone A2b has revealed a mimicry of this particular epitope with an articular cartilage-associated target T-cell epitope. Nasal administration of synthetic peptides covering this 180–188 sequence led to epitope-.specific tolerance and resistance to AA. Since this tolerisation protocol also inhibited avridine arthritis, one may conclude that this form of epitope-specific tolerance had effectuated a spreading tolerisation at the level of target antigens that included a diverse set of possible arthritis -associated antigens. In vitro anergised T cells exhibited suppressive activity in a co-culture system. As in this case - depending on the presence of the antigen of the anergic T cell – such T cells suppressed responder T cells of a different antigenic specificity, we postulated that anergic T cells may be responsible for a spreading of tolerance. It seemed that such spreading of tolerance was channelled through the antigen-presenting cells (APC) and was dependent on direct cell-cell contact. This and additional forms of spreading of tolerance could be responsible for specific nasal tolerance, causing inhibition of the development of an arthritogenic inflammatory response. This can be similarly che case for the arthritis protection that resulted from immunisation with hsps. Analysis of T-cell responses following hsp immunisations revealed that the arthritis inhibitory activity resided in T cells with specificity for a conserved part of microbial hsp60. The same T cells cross-responded to rat self-hsp60. Low level expression of the latter molecule on non-professional APC could possibly have induced a suppressive anergic state in these autoreactive cells. Thus, immunisation with microbial hsp would have led to an expansion of such T cells, leading to raised disease-suppressive potential when selectively trapped and activated in the inflamed self-hsp-overexpressing joint. Alternatively the cross-recognised self-hsp epitope could have the regulatory qualities of an altered peptide ligand or a partial agonist for T cells that see the microbial homologue as the full agonist.     

Cross-reactive mycobacterial and self hsp60 epitope recognition in I-A(g7) expressing NOD, NOD-asp and Biozzi AB/H mice

The highly conserved 60 kD endogenous heat shock protein (hsp60) has been suggested to be a target for T cell recognition in autoimmune diseases such as type I diabetes. We previously reported cross-recognition of both mycobacterial hsp60 (Mt60) and self hsp60 (m60) by Mt60 immunized NOD mice. To identify the epitopes involved, we generated T cell lines against m60 or its mycobacterial counterpart and tested these lines for recognition of complete sets of overlapping peptides spanning either hsp60 sequence. T cell lines responded to identical regions in the hsp60 proteins, regardless of their degree of conservation or I-A(g7) binding-affinity. Additionally, we determined whether a protective genetic background would affect the presence of hsp60 cross-reactive T cells in the peripheral repertoire by comparing epitope recognition in I-A(g7) expressing NOD, NOD-asp and Biozzi AB/H mice. Two out of five immunodominant murine peptides were able to induce proliferation in NOD and NOD-asp Mt60 T cell lines, but not in Biozzi AB/H T cell lines. Our results point out that Mt60 immunization not necessarily leads to proliferative T cells responding to endogenous hsp60 peptides in the context of diabetes-predisposing I-A(g7). Moreover, the capacity of T cells to respond to self hsp60 is not influenced by the presence of protective I-A(g7asp). Yet, proliferation of hsp60 autoreactive T cells is solely measured in combination with insulitis and as such serves as a surrogate marker for islet inflammation.