Induction of experimental autoimmune arthritis by a public epitope of the T cell receptor variable alpha domain of an arthritogenic T cell clone

T cell receptor (TCR) peptide immunizations have been demonstrated to protect against experimental autoimmune diseases. These findings have led to clinical trials employing TCR peptides in multiple sclerosis and rheumatoid arthritis patients. Previously, we identified a strongly immunogenic region of the TCR alpha chain of an arthritogenic T cell clone (AV11 66-80). In this report, we show that rats immunized with AV11 66-80 developed arthritis with clinical symptoms and histology similar to adjuvant arthritis (AA). Transfer of this disease into naive rats using AV11 66-80-specific T cells proved the T cell-mediated character of the disease. The AV11 66-80 arthritic rats developed resistance to Mycobacterium tuberculosis-induced AA, indicating that both forms of arthritis depended on similar regulatory mechanisms. This first demonstration of TCR peptide-induced arthritis, together with an earlier report on a polymorphism in this very same AV11 66-80 region involved in arthritis resistance in mice, suggests a central role of the public epitope AV11 66-80 in the control of autoimmune arthritis. Although TCR peptide immunizations can be exploited to prevent experimental autoimmunity, caution should be taken in the induction of TCR peptide-specific T cells for immunotherapy to avoid adverse effects as shown here.     

Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the alpha/beta T cell receptor.

Adjuvant arthritis (AA) in rats is an experimentally induced autoimmune disease mediated by T lymphocytes specific for Mycobacterium tuberculosis. We raised the question whether T cells carrying the gamma/delta T cell receptor (TcR), reactive or not to mycobacterial antigens, are involved in the pathogenesis of AA. For this purpose, T cells bearing the TcR alpha/beta were depleted from circulation by treatment with a monoclonal antibody against the rat TcR alpha/beta (R73). This treatment efficiently suppressed existing disease. Even more efficient was pretreatment with R73 from birth, which prevented AA induction completely. In these alpha/beta+ T cell-depleted animals an elevated level of alpha/beta- T cells (about 15% vs. 1% in normal rats) was evident, which was not significantly increased by Mycobacterium tuberculosis injection. We found no positive evidence that gamma/delta + T cells do contribute to AA induction. Moreover, treatment with an anti-TcR alpha/beta monoclonal antibody may be very efficient treatment of T cell-mediated autoimmune diseases.     

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12

Abstract: We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.     

Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.     

Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis

Heat shock proteins (hsp) are highly conserved, immune-dominant microbial proteins, whose expression is increased at sites of inflammation. In the experimental model of adjuvant arthritis (AA) immune responses to hsp determine the outcome of disease. AA can be transferred with a single T cell clone specific for a sequence of mycobacterial hsp65 (Mhsp65). Immunization with whole Mhsp65 on the other hand, protects in virtually all forms of experimental arthritis, including AA. This protective effect seems the consequence of the induction of a T cell response directed against self-hsp60. A similar protective effect of self-hsp60-specific T cells seems present in patients with a spontaneous remitting form of juvenile idiopathic arthritis. Next to hsp60, other hsp have similar protective effects in arthritis, while other conserved microbial proteins lack such capacity. Nasal administration of hsp60 peptides induces IL-10-driven regulatory T cells that are highly effective in suppressing arthritis. Thus hsp60, or peptides derived from hsp60, are suitable candidates for immune therapy in chronic arthritis.     

The suppressive effect of an antibody to the alpha beta cell receptor in rat adjuvant arthritis: studies on optimal treatment protocols

An antibody (R73) to the alpha beta T cell receptor (TCR) was able to dramatically suppress adjuvant arthritis (AA) in rats. The efficacy of R73 treatment was investigated with regard to antibody dosage, injection route and timing of injections. R73 was equally effective in reducing joint swelling throughout a wide dose range (80 to 2000 microgram/dose) when given on day 15, 18, and 21 after arthritis induction. Both i.p. and i.v. injection were able to suppress the pre-existing joint swelling to the same extent. However, R73 was only effective when given before or at the peak of joint swelling which occurred between day 18 to 24. Synovial membrane hyperplasia, mononuclear cell infiltration as well as cartilage and bone destruction were markedly reduced after therapy. The effect of R73 was associated with depletion of alpha beta+ T cells from the circulation even at low antibody doses. Only few alpha beta+ T cells were found in the pannus tissue. Late treatment on day 27, 30 and 33 after arthritis induction did not influence clinical scoring of the disease and histological examination thereafter did not show any difference between treated animals and controls. We conclude that antibody therapy directed at the TCR seems to be very effective even in pre-existing autoimmune diseases if the relevant T cell population is affected. However, inflammation and joint destruction may reach a state at which anti-TCR treatment is no longer effective.     

Anti-immunoglobulin antibody detection in adjuvant arthritis by an ELISA technique

Sprague-Dawley rats, injected in the hind paw with heat-killed mycobacteria dispersed in oil, develop a severe polyarthritis. In this paper, we detected and quantified by a micro-ELISA technique autoantibodies against immunoglobulins in rats with adjuvant arthritis. Increased total anti-immunoglobulin antibodies levels were found from 3 days after induction and remained elevated until day 42. IgG anti-immunoglobulin antibodies in arthritic animals were significantly elevated during days 35-42. These results show that alterations in the humoral immune response (synthesis of anti-immunoglobulin antibodies) are present in adjuvant arthritis as they are in human rheumatoid arthritis.     

Triggering of the phosphoinositide transduction pathway by a monoclonal antibody specific for the human gamma/delta T cell receptor

We have developed a monoclonal antibody (mAb), termed anti-TigammaA, which recognizes an antigenic determinant carried by a variable segment of the T cell receptor (TcR) gamma chain. This determinant, encoded by the V gamma 9 gene, is expressed on approximately 3% of peripheral blood lymphocytes. In the present study, we have found that binding of anti-TigammaA mAb to its specific ligand results in the triggering of the phosphatidylinositol (PI) cycle-related metabolic process. Indeed, an increased labeling of both phosphatidic acid and PI, related to an enhanced turnover of PI cycle-dependent phospholipids, was observed following exposure of 32P orthophosphoric acid-labeled cells to anti-TigammaA mAb. In addition, there was a rapid rise in intracellular free calcium concentrations. Similar experiments have been performed previously on CD3+ TcR alpha/beta- -cells with an anti-CD3 mAB. They predicted that signals produced by the interaction between the second TcR and its ligand(s) would be transmitted via the PI cycle-linked intracellular second messengers. We confirm this hypothesis in an experimental system where stimulation occurs directly through the gamma/delta receptor structure.     

T cell receptor genes in rheumatoid arthritis

Conclusion Rheumatoid arthritis is an oft debilitating chronic disease of an autoimmune nature. Although the putative antigen remains unknown, the recent elucidation of the structure and functional relationships of the trimolecular complex governing the immune response to antigen, facilitates the development of novel approaches to the treatment of RA and enhances our understanding of the etiopathogenesis of this disorder and autoimmunity in general. The characterization of immune active T cell populations, particularly at inflammatory sites such as the synovium, with respect to their expressed and genomic TCR repetoire, may permit the identification of crucial genetic components which contribute either directly or indirectly to the susceptibility and/or the development of RA.Abbreviations CIA collagen-induced arthritis - IL-2 interleukin-2 - MHC major histocompatibility complex - RA rheumatoid arthiritis - RFLP restriction fragment length polymorphism - TCR T cell receptor - RR relative risk     

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.     

Generation and characterization of transgenic mice expressing a T-cell receptor specific for the tumour-associated antigen MDM2

T-cell-based antigen-specific immunotherapy targeting tumour-associated antigens offers the potential for cancer immunotherapy. However, the majority of identified tumour-associated antigens are also expressed at low levels in normal tissues and mechanisms of tolerance induction are likely to affect the quality of T-cell responses to such antigens. In this study a T-cell receptor transgenic model was developed to determine the magnitude of T-cell tolerance to the tumour-associated antigen murine double minute-2 (MDM2), a widely expressed protein that is found at elevated levels in many tumours. The analysis of transgenic mice showed that thymic deletion was responsible for purging large numbers of MDM2-specific T cells from the repertoire. However, some T cells with specificity for MDM2 were able to escape thymic deletion and persisted in the peripheral T-cell pool. Functional analysis revealed that these T cells displayed defects in antigen-driven expansion. This functional impairment of the MDM2-specific T cells was maintained following adoptive transfer of the T cells into hosts that were unable to present the T-cell-receptor-recognized antigen. This study demonstrates that thymic deletion and the functional impairment of T cells present in the periphery both operate to establish T-cell tolerance to the tumour-associated antigen MDM2. Furthermore, the tolerant phenotype was stable and did not require continuous MDM2 peptide presentation in normal tissues.     

Prevention of collagen induced arthritis in mice by treatment with an antibody directed against the T cell receptor alpha beta framework.

Collagen induced arthritis (CIA) is an animal model of inflammatory polyarthritis. Type II collagen is the major matrix protein of hyaline cartilage. Susceptibility to CIA is linked to the Major Histocompatibility Complex Class II genes but the presence of T cells expressing specific variable beta (V beta) chain of their T cell receptor (TCR) is also required. Pretreatment with the monoclonal antibody H57-597 directed against the TCR alpha beta framework prevented the onset of arthritis in the majority of animals. The depletion of the T cell population did not lead to any apparent health problems. These experiments demonstrate the important role of the alpha/beta T cell and its receptors in the CIA model. Further, anti-TCR alpha beta antibodies may be of value in the therapy of autoreactive disorders.     

Inhibition of T cell proliferation specific for acetylcholine receptor epitopes related to myasthenia gravis with antibody to T cell receptor or with competitive synthetic polymers.

Long-term T cell lines and clones of C3H.SW origin specific to synthetic peptides representing immunogenic epitopes of the human aetylcholine receptor alpha-subunit were established. Using these lines and clones, it was possible to characterize the T cell recognition process of myasthenic epitopes. Testing a panel of N-and/or C-terminal truncated peptides it could be demonstrated that the deletion of the two C-terminal amino acids of peptides p195-212 and p259-271 resulted in a loss or reduction of the stimulatory capacity of the peptides towards the specific T cell lines. In contrast, no substantial effect on the stimulation of the line could be observed by shortening peptide p195-212 by up to five amino acids at the N-terminal end. The proliferation of T cell lines and clones specific to peptide p195-212 was inhibited by a mAb directed against the V beta 8 region of the T cell receptor. Furthermore, it was possible to block the peptide-specific proliferative responses of the lines and clones by the I-Ab restricted synthetic polypeptide (T, G)-A--L but not by the I-Ak restricted polypeptide antigen (H,G)-A--L. Similarly, p195-212 inhibited the proliferative response of the TCSW259-271 T cell line and p259-271 inhibited the specific proliferative response of the TCSW195-212 line. Moreover, the C-terminal shortened peptides inhibited significantly the in vitro stimulation of the T cell lines by the immunogenic peptides p195-212 and p259-271. The inhibition by the synthetic peptides or by (T,G)-A--L may be due to competitive blockade of the MHC binding site for the T cell line stimulating AChR peptides.     

Activation of resting T lymphocytes by a monoclonal antibody directed against an allotypic determinant on the T cell receptor

The murine monoclonal antibody F23.1 reacts with an allotypic determinant on the beta chain of the T cell receptor expressed by approximately 20% of T helper and cytotoxic T lymphocytes of most common mouse strains. This IgG2a antibody, either in soluble form or covalently coupled to Sepharose beads, can activate resting T cells from naive animals to proliferate. Interestingly, under all conditions of activation, the antibody can only induce proliferation if exogenous lymphokines in the form of Con A supernatant are provided. Thus, it is unlike most lectins and anti-T3 antibodies in this regard. Furthermore, under all conditions of culture, F23.1 activates preferentially the Lyt-2+ subset of T cells. This is the case even in the presence of accessory cells. Further evidence is provided that two soluble lymphokines, different from IL2, are required to initiate IL2-dependent growth and to allow the expression of lytic activity.     

Generation and characterization of a neutralizing monoclonal antibody against erythroid cell stimulating factor

Erythroid cell stimulating factor (ESF) is present in mouse serum and has been reported to function in concert with erythropoietin (EPO) in the formation of erythroid cells in in vitro culture systems. We report here the generation and characterization of a monoclonal antibody (MAb) directed against ESF, with potent anti-ESF-neutralizing activity. A hybridoma-producing MAb to ESF was selected following enzyme-linked immunosorbent assay (ELISA)-based screening of 270 colonies obtained from a fusion of immunized mouse splenocytes with NS1 myeloma cells. Western blot analyses of mouse serum using this antibody specifically detected a single protein (approximate molecular weight of 60 kDa and 120 kDa, under reducing and nonreducing conditions, respectively) corresponding to ESF, with no reactivity to EPO. Furthermore, this MAb demonstrated reactivity to a protein similar in molecular mass, across species, showing reactivity in sera obtained from human, horse, goat, guinea pig, rabbit, and rat. Immuno-chemical characterization demonstrated this antibody to be of IgG3 isotype, bearing kappa light chains. Injection of this monoclonal anti-ESF antibody to exhypoxic polycythemic mice at 6 and 24 h after EPO injection significantly reduced 59Fe incorporation into red blood cells, demonstrating its ability to neutralize in vivo erythropoiesis in our mouse model system. Thus, this novel erythroid cell-specific MAb will be an invaluable tool for further delineating the physiological role of ESF in in vivo erythropoiesis.     

Production and characterization of monoclonal antibodies specific for the murine T cell receptor ζ chain

The T cell receptor (TCR) comprises an antigen-specific β heterodimer non-covalently associated with the CD3 γδε and TCR ζ subunits. Both the CD3 and TCR ζ subunits are proposed to be responsible for the intracellular signal-transduction events. We report here the production of eight monoclonal antibodies (mAbs) that bind in an ELISA assay to a 113 amino acid synthetic peptide corresponding to the cytoplasmic domain of TCR ζ. Western blot analysis of anti-CD8 precipitates of lysates of transfectants expressing chimeric CD8/ζ constructs encoding increasing COOH-terminal truncations of TCR ζ indicates that four of these mAbs recognized the region of TCR ζ chain comprising the last 29 COOH-terminal residues. Thus, this region of TCR ζ may encode an immunodominant epitope. Furthermore, one of these mAbs, G3, is capable of precipitating both non-phosphorylated and tyrosine phosphorylated TCR ζ. The G3 mAb should be useful for elucidiating the structural and signalling characteristics of the TCR ζ chain.     

Down-regulation of the T cell receptor by a mitogenic anti-Thy-1 antibody

Antigen-specific unresponsiveness lasting at least 2 weeks can be induced in a T cell clone by 24-h pretreatment with mitogenic anti-T cell receptor antibodies. In this report the relationship is explored between the antigen-specific unresponsiveness and activation pathways triggered via the T cell receptor and Thy-1: the latter pathway is dependent on the former. A mitogenic anti-Thy-1 antibody (KT16) made the T cell clone unresponsive to specific antigen and to an anti-T cell receptor antibody coupled to Sepharose. The unresponsiveness lasted for at least 7 days. However, cells made unresponsive to specific antigen in these ways (the T cell receptor and Thy-1) could be activated by both interleukin 2 and KT16. KT16 down-modulated the T cell receptor immediately after the pretreatment, but not on day 7 after the pretreatment. These facts indicate that the state of the unresponsiveness was caused by blocking transduc-tion of an activation signal triggered by the T cell receptor to an activation pathway shared by the T cell receptor and Thy-1.     

Generation and characterization of a monoclonal antibody specific for human herpesvirus 6 variant A immediate-early 2 protein.

Two variants (A and B) of human herpesvirus 6 (HHV-6) can be isolated from humans, with each variant having unique biological properties. HHV-6 variant typing is mainly done following amplification of viral genomic DNA followed by restriction endonuclease digestion. Our objective was to generate a monoclonal antibody (mAb) that would allow us to discriminate between variants A and B of HHV-6. BALB/c mice were immunized with a recombinant glutathione-S-transferase protein fused to the immediate-early (IE) 2 protein from HHV-6 variant A. Following splenocytes fusion, one IgG1 kappa light chain mAb (P6H8) was isolated and found to react specifically with variant A IE2 protein in immunofluorescence and western blot assays. The P6H8 antibody represents a useful tool for both fundamental research and clinical applications allowing for the discrimination of infections caused by HHV-6 variants A or B.     

Production and characterization of an anti-idiotypic antibody specific for a monoclonal antibody to glycoprotein D of herpes simplex virus

A monoclonal antibody specific for glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was used to prepare an anti-idiotypic antibody in rabbits. After removal of antibody reactivity to constant region determinants by absorption with polyclonal mouse immunoglobulins and a monoclonal antibody of the same subclass as the anti-gD monoclonal, the anti-idiotypic (anti-id) antibody reacted specifically with anti-gD. Using an ELISA inhibition assay with immunoaffinity-purified gD, the anti-id D reagent inhibited the binding of anti-gD to gD, suggesting that anti-id D mimics an epitope of gD by binding the antigen-combining site of anti-gD. Immunization of mice with anti-id D could prime splenocytes in vivo to proliferate in response to HSV antigen stimulation in vitro. The possibility that anti-id D could act similarly to gD and stimulate an immune response to HSV when administered in vivo is discussed.