TCR-based Immunotherapy Research Articles

Vβ18.1 + and Vα2.3 + T-cell subsets are associated with house dust mite allergy in human subjects

Background: The recognition of allergenic peptides by T cells through their T-cell receptor (TCR) represents a crucial step in the initiation of an allergen-specific immune response. In parallel to the superantigen-driven restricted expansion of Vβ subsets in autoimmune and infectious diseases, reports in animals and human subjects have shown a similar capacity of classical antigens. Objective: The study was performed to analyze the Vα/Vβ expression in house dust mite (HDM) allergy. Methods: The TCR repertoire of 15 subjects with HDM allergy, 22 atopic subjects without HDM allergy, and 19 nonatopic individuals, members of 2 extended and 4 nuclear families, was determined. By using flow cytometry, the expression of 22 Vβ and 3 Vα elements was analyzed in vivo and after in vitro allergen stimulation. Results: In comparison with nonatopic and atopic individuals without HDM allergy, freshly isolated PBMCs of individuals with HDM allergy showed a significantly higher frequency of Vβ18 + and Vα2.3 + T cells. Although members of all 3 groups had a similar lymphocyte proliferation response after in vitro stimulation with Der p 1 or Der p 1 peptide 101-131, a significant expansion of Vβ18 + and Vα2.3 + T cells in vitro occurred only in individuals with HDM allergy. Moreover, the degree of expansion correlated with the levels of allergen-specific IgE antibodies. No expansion of Vβ18 + and Vα2.3 + was observed after mitogen stimulation with PHA, indicating allergen specificity of the response. Conclusion: Our results strongly suggest restricted TCR Vα/Vβ gene use in HDM allergy and might be a step toward TCR-based immunotherapy. (J Allergy Clin Immunol 2002;109:517-23.)

Characterization of T cells that are activated after depletion of major encephalitogenic T cells in rat autoimmune encephalomyelitis

In rat experimental autoimmune encephalomyelitis (EAE), encephalitogenic T cells mainly use Vβ8.2 of TCR, but it is not clear whether Vβ8.2 is the sole encephalitogenic TCR. To address this issue, we examined the immunological status of Lewis rats that had been treated with anti-Vβ8.2 mAb and immunized with myelin basic protein (MBP). It was demonstrated that rats in which the majority of Vβ8.2+ T cells had been depleted developed clinical EAE and possessed newly expanded Vβ10. Analysis of T cell lines established from these animals revealed that T line cells responded to a minor epitope in the MBP molecule. However, treatment with a mixture of anti-Vβ8.2 and anti-Vβ10 mAbs reduced the clinical severity of EAE but did not induce complete suppression of the disease due to new activation of Vβ6. These findings suggest that rapid and frequent examination of the TCR repertoire is essential to identify pathogenic TCRs and to establish TCR-based immunotherapy for autoimmune diseases.

Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR.

The identification of TCRs of autoimmune disease-inducing T cells within a short period of time is a key factor for designing TCR-based immunotherapy during the course of the disease. In this study, we show that experimental autoimmune carditis-associated TCRs, Vbeta8.2 and Vbeta10, were determined by complementarity-determining region 3 (CDR3)-spectratyping analysis and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones. Immunotherapy targeting both Vbeta8.2 and Vbeta10 TCRs using mAbs and DNA vaccines significantly reduced the histological severity of experimental autoimmune carditis and completely suppressed the inflammation in some animals. Since depletion or suppression of one of two types of effector cells does not improve the severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary therapy for T cell-mediated autoimmune diseases. TCR-based immunotherapy after rapid identification of autoimmune disease-associated TCRs by CDR3 spectratyping can be applicable, not only to animal, but also to human autoimmune diseases whose pathomechanism is poorly understood.

Fine T cell receptor repertoire analysis of spinal cord T cells responding to the major and minor epitopes of myelin basic protein during rat autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis is a disease induced by neuroantigen-reactive T cells bearing particular types of T cell receptor (TCR). Although the nature of TCRs of encephalitogenic T cells has been partially delineated using encephalitogenic T cell clones established in vitro, the entire TCR repertoire formed in situ after immunization with neuroantigen remains unclear. In the present study, we immunized Lewis rats with myelin basic protein (MBP) and its fragment peptides and determined the TCR repertoire of spinal cord T cells formed after the immunization by CDR3 spectratyping. It was revealed that the oligoclonal expansion of Vβ2, Vβ8.2, and Vβ17 spectratypes was detectable after immunization with guinea pig MBP and its immunodominant epitope, the 68–88 sequence, whereas immunization with a peptide containing a minor epitope induced Vβ10 expansion. Immunization with rat MBP induced much broader TCR Vβ expansion (all of the above Vβs plus Vβ3). These findings suggest that TCRs activated by immunization with guinea pig MBP used as heteroclitic immunogen recognize autoantigen, rat MBP. Furthermore, the strategy used in this study gives insight into the pathogenesis of autoimmune disease and provides useful information for designing TCR-based immunotherapy. J. Neurosci. Res. 59:145–152, 2000 © 2000 Wiley-Liss, Inc.

Fine T cell receptor repertoire analysis of spinal cord T cells responding to the major and minor epitopes of myelin basic protein during rat autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis is a disease induced by neuroantigen-reactive T cells bearing particular types of T cell receptor (TCR). Although the nature of TCRs of encephalitogenic T cells has been partially delineated using encephalitogenic T cell clones established in vitro, the entire TCR repertoire formed in situ after immunization with neuroantigen remains unclear. In the present study, we immunized Lewis rats with myelin basic protein (MBP) and its fragment peptides and determined the TCR repertoire of spinal cord T cells formed after the immunization by CDR3 spectra-typing. It was revealed that the oligoclonal expansion of Vbeta2, Vbeta8.2, and Vbeta17 spectratypes was detectable after immunization with guinea pig MBP and its immunodominant epitope, the 68-88 sequence, whereas immunization with a peptide containing a minor epitope induced Vbeta10 expansion. Immunization with rat MBP induced much broader TCR Vbeta expansion (all of the above Vbetas plus Vbeta3). These findings suggest that TCRs activated by immunization with guinea pig MBP used as heteroclitic immunogen recognize autoantigen, rat MBP. Furthermore, the strategy used in this study gives insight into the pathogenesis of autoimmune disease and provides useful information for designing TCR-based immunotherapy.

Characterization of T cell receptor associated with the development of P2 peptide-induced autoimmune neuritis

To characterize experimental autoimmune neuritis (EAN)-inducing T cells in more detail, we performed CDR3 spectratyping analysis and found oligoclonal expansion of several Vβ spectratypes in nerve-infiltrating T cells. Vβ5 expansion was observed all the stages examined, whereas Vβ8.2 and Vβ17 expansion was mainly found at the peak and preclinical stages, respectively. Since Vβ5 expansion persists throughout the course of the disease, Vβ5 + T cells are judged to be the main effector cells. Vβ8.2 + and Vβ17 + T cells may also be pathogenic but are not the main effectors because expansion of these spectratypes was found at a limited period of time. Sequence analysis revealed that Vβ5, Vβ8.2 and Vβ17 spectratype-derived TCR clones possess their own dominant sequences in the CDR3 region with no homology among the clones. These findings suggest that polyclonally activated T cells are involved in the formation of the nerve lesion. Furthermore, vaccination with Vβ5 DNA, but not with Vβ10 DNA, suppressed the development of EAN significantly. Collectively, these findings indicate that determination of autoimmune disease-associated TCR by CDR3 spectratyping provides useful information for designing TCR-based immunotherapy for the disease.

Rat peripheral T-cell receptor Vβ repertoire in F344-to-B10 (rat-to-mouse) mixed xenogeneic chimeras

In previous studies, we demonstrated that T cell receptor (TCR) Vβ8.2 and Vβ10, both of which are frequently used by encephalitogenic T cells, spectratypes expand oligoclonally in spinal cord lesions of Lewis rats with experimental autoimmune encephalomyelitis (EAE) and that the DSSYEQYF and WDGSGNVLYF sequences are predominantly found in the complementarity-determining region 3 (CDR3) of spectratype-derived TCR clones. However, it is unknown whether these CDR3 sequences are used only by Vβ8.2- and Vβ10-positive T cells or by encephalitogenic T cells bearing Vβs other than these Vβs. The present study was undertaken to address this issue using a new approach, i.e. CDR3 spectratyping and subsequent DNA hybridization with several probes corresponding to various parts of the CDR3 region. Consequently, we found that probes specific for the Vβ8.2 spectratype-derived Dβ and Jβ2.7 hybridized only with the Vβ8.2 spectratype in acute EAE and with the Vβ8.2 and Vβ12 spectratypes in chronic relapsing EAE. Similarly, a probe specific for the Vβ10 spectratype-derived Dβ hybridized only with the Vβ10 spectratype in both acute and chronic relapsing EAE. In contrast, a probe specific for Jβ1.3 hybridized with several Vβ spectratypes including Vβ8.2 and Vβ10 only during the early stage of the disease. These findings suggest that T cells bearing a few Vβs with a limited number of the CDR3 sequences are activated in acute and chronic relapsing EAE induced in Lewis rats. Characterization of the CDR3 region of pathogenic TCR by this approach may be of value for the screening of autoimmune disease-associated TCR and for the development of TCR-based specific immunotherapy.

Persistent Expression of Experimental Autoimmune Encephalomyelitis (EAE)-Specific Vβ8.2 TCR Spectratype in the Central Nervous System of Rats with Chronic Relapsing EAE

AbstractMonitoring the TCR repertoire is indispensable for the assessment of T cell-associated autoimmune diseases and subsequent TCR-based immunotherapy. In the present study, we examined the TCR repertoire of spinal cord T cells of Lewis rats by CDR3 spectratyping during chronic relapsing experimental autoimmune encephalomyelitis (EAE) induced by immunization with spinal cord homogenate. It was found that Vβ8.2 spectratype with the shortest CDR3 expanded oligoclonally throughout the course of the disease. In addition, Vβ12 spectratype expansion was observed at the first and second attacks of EAE. Sequence analysis revealed that clones with the DSSYEQYF sequence, which is a representative sequence of myelin basic protein (MBP)-reactive T cell clones, constituted the predominant population in the Vβ8.2 family. Surprisingly, Vβ12 also used the identical amino acid sequence in the CDR3 region. These findings indicate that although infiltrating T cells in the central nervous system are activated polyclonally, the TCR repertoire remains unchanged throughout the course. Moreover, the finding that the predominant CDR3 amino acid sequence of Vβ8.2 and Vβ12 spectratypes is identical with that of MBP-induced EAE suggests that a single Ag in spinal cord homogenate, possibly MBP, is involved in disease development.

A new animal model for primary Sjögren's syndrome in NFS/sld mutant mice.

In this study, we report an established and characterized animal model for primary Sjögren's syndrome in NFS/sld mutant mice bearing an autosomal recessive gene with sublingual gland differentiation arrest. Significant inflammatory changes develop spontaneously in both the salivary and lacrimal glands of NFS/sld mutant mice thymectomized 3 days after birth without any immunization, whereas no significant inflammatory lesions were found in other organs or in nonthymectomized mice. This pathology resembles primary Sjögren's syndrome in humans, which involves the parotid, submandibular salivary, and lacrimal glands. A significantly higher incidence of autoimmune lesions was found in female mice, and the anti-salivary duct autoantibodies were detected in sera from mice with autoimmune lesions but not in control mice. The inflammatory infiltration into the salivary and lacrimal glands consisted mainly of CD3+ and CD4+ T cells, and a lesser proportion of CD8+ T cells and B220+ B cells during the course of disease. When the repertoire of TCR V beta genes transcribed and expressed within the inflammatory infiltrates was analyzed in mice with autoimmune lesions, a considerable preferential use of TCR V beta gene (V beta 8 predominant) was detected in these lesions from the onset of disease. Thus, we can propose a newly established animal model for primary Sjögren's syndrome developing in this mutant strain of mice. Moreover, the predominant patterns of TCR transcript expression in an animal model may be somewhat restricted, suggesting that TCR-based immunotherapy of Sjögren's syndrome is possible.

Immunotherapeutic strategies targeting rheumatoid synovial T-cell receptors by DNA inoculation.

Immunotherapy against autoreactive T-cell receptors (TCRs) has been reported to have promise in several animal models of autoimmune diseases. Facilitated DNA inoculation has many potential advantages as a modality for development of specific immune responses. Specifically, this technology is able to deliver exogenous antigens for processing via both the endogenous pathway, with subsequent presentation by class-I major histocompatibility (MHC) antigens, and the exogenous pathway, with subsequent presentation by class-II MHC antigens. This allows for induction of both arms of the cellular immune system. These cellular immune responses may be particularly important in targeting and controlling pathogenic cell populations. The application of this technology to the treatment of human autoimmune diseases depends on the availability of readily manipulated systems for the evaluation of specific interventions. Here we report the full length cloning and expression of TCRs from rheumatoid arthritis synovial tissue. These were developed by recombinant polymerase chain reaction, cloning and retroviral transduction into a TCR-alpha/beta-negative murine T-cell hybridoma. Reconstitution of CD3 expression was confirmed by flow cytometry. Similar constructs have been developed for TCR-based immunotherapy by facilitated inoculation of DNA intramuscularly. Preliminary analysis of immune responses in mice indicates that these constructs elicit anti-TCR responses. These studies indicate the ability to reconstitute expression of potentially autoreactive human TCRs in a model system wherein specific immune responses elicited against these TCRs by various immunogens can be evaluated.