Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases

For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.     

Isolation of antigen-specific T cell clones from nonresponder mice

The mechanisms responsible for major histocompatibility complex (MHC)-linked unresponsiveness are still poorly understood. Here we examine the cellular events that follow when B10. A mice are immunized with cow insulin, an antigen to which they make no apparent immunologic response. Despite the fact that there is no detectable antibody or T cell proliferative response to cow insulin, we have been able to clone out responding T cells after priming and restimulating in vitro with this "nonimmunogenic" antigen. These cells are L3T4+, and co-recognize specific antigen and class II MHC gene products. The data demonstrate that "nonresponder" mice to cow insulin have both the capacity to present antigen and T cells capable of recognizing that antigen. The diversity within this population was investigated by analyzing various parameters of cellular activation. These include fine specificity of both antigen and MHC recognition, as well as recognition of allogeneic MHC and M1s determinants. In addition, the antigen-presenting cell requirements were studied. The results demonstrate that this population comprise a surprisingly heterogeneous group in terms of its repertoire of receptors.     

Analysis of T cell clones in rheumatoid arthritis

Conclusions While it is apparent that much more needs to be learnt about T cells in RA, it seems evident that because of their central role in the immunoregulation their study will provide a lot of valuable information concerning the pathogenesis and perhaps even the aetiology of RA. Two areas of particular importance are the investigation of epitopes recognised on autoreactive T cells, e. g., on collagen type 11, but also on other T cells, and those expressing the receptor. Knowing which cells are regulating the production of mediators in the RA joint and which mediators are of fundamental importance in perpetuation of the disease process may permit the development of new therapeutic strategies.Abbreviations AMLR Autologous mixed lymphocyte reaction - BSF-2 B-cell stimulatory factor-2, also known as IFN2 or IL-6 - GM-CSF granulocyte macrophage colony stimulating factor - IFN interferon- - IFN interferon- - IL-1 interleukin-1 - IL-2 interleukin-2 - LT lymphotoxin or TNF - PBL peripheral blood lymphoid cells - RA rheumatoid arthritis - TFC thyroid follicular cells - TGF transforming growth factor - TNF tumour necrosis factor     

Restricted appearance of self-reactive clones into T cell receptor intermediate cells in neonatally thymectomized mice with autoimmune disease

Neonatally thymectomized (NTx) mice fall victim to such autoimmune diseases as gastritis and pancreatitis with aging. Self-reactive T cell clones are known to be consistently generated through TCR intermediate (i.e. TCR^int) cell differentiation in normal mice (i.e. via the extrathymic pathways and an alternative intrathymic pathway). It was investigated whether the generation of such clones in NTx mice follows this rule or whether they are generated by default via mainstream T cell differentiation in the thymus. The majority of T cells generated in NTx mice were TCR^int cells in all organs tested. In contrast to athymic mice, which carry only TCR^int cells with aging, a leaky appearance of high TCR (i.e. TCR^hi) cells emerged in the lymph nodes and other organs of NTx mice. Self-reactive clones estimated by anti-Vβ monoclonal antibodies in conjunction with the Mls system were confined to TCR^int cells in all tested organs, including a target organ, the stomach, in NTx mice. A leaky population of TCR^hi cells did not contain a significant number of self-reactive clones. Moreover, such self-reactive clones among TCR^int cells in NTx mice with autoimmune disease were shown to be nonanergic in the proliferation assay. The present results suggest that the generation of self-reactive clones is totally due to TCR^int cell differentiation, although it is still undetermined whether the expanding TCR^int cell population is generated via the extrathymic pathway or an alternative intrathymic pathway. It is shown here not to be due to a failure of the TCR^hi cell-differentiation pathway in NTx mice.     

High incidence of spontaneous autoimmune thyroiditis in immunocompetent self-reactive human T cell receptor transgenic mice

Autoantigen-specific TCR transgenic mice allow us to assess the role of T cells in autoimmunity. We have recently generated humanized TAZ10 transgenic mice expressing the human TCR specific for the immunodominant epitope of thyroid peroxidase (TPO). We have shown that these transgenic mice do not undergo tolerance in vivo and that on Rag deficient background they are susceptible to spontaneous autoimmune thyroiditis. Here we show that, in contrast to other transgenic models of autoimmunity, almost all TCR(+)Rag1+ (T+R+) T cells are activated in vivo leading to the development of spontaneous autoimmune thyroiditis. In these mice, disease is also accompanied by a significant reduction of CD4+CD25hi regulatory T cells. These data indicate that the pathogenic activity of the self-reactive TCR can circumvent the regulatory function operated by the non-transgenic T cells that are normally present in T+R+ mice, leading to autoimmunity.     

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.     

Distinct epitopes are recognized by cytolytic T lymphocyte clones on the same class I molecule: direct demonstration using DNA-transfected targets and long-term cytolytic T cell clones

By producing long-term, stable, cytolytic T lymphocyte clones and utilizing targets expressing only a single gene product derived from the stimulator mouse strain, we have been able to directly demonstrate that T cells recognize distinct epitopes expressed on a single H-2 molecule. These multiple determinants are distinguishable by inhibition patterns with monoclonal antibodies (mAb). When two T cell clones, P-2.14 and P-2.17, are tested on an L cell transfected with the Dp gene (lambda 12a), the T cells kill the transfected targets as well as blasts derived from Dp mouse strains. mAb 7-16.10 inhibits recognition and killing of Dp targets by both P-2.14 and P-2.17. This mAb recognizes a private specificity H-2.m22. Interestingly mAb 11-20.3 which also recognizes the H-2.m22 specificity inhibits clone P-2.14 but not P-2.17. The mAb 7-16.10, however, competes with 11-20.3 for binding to the surface of L cells expressing the Dp gene. Thus the two T cells must recognize an overlapping specificity. Other mAb which bind to the H-2Dp molecule are unable to inhibit either of these two cytolytic T cell clones. Paradoxically, any of the mAb when tested individually are sufficient to inhibit the polyclonal response derived from in vitro mixed lymphocyte culture. Therefore, by using targets expressing only a single H-2 molecule derived by DNA-mediated gene transfer and cytolytic T cell clones we have been able to directly demonstrate the presence of multiple epitopes on a single molecule and define their inhibition with mAb directed to that same molecule.     

自身反应性T细胞系及克隆的建立与TCR使用情况分析

目的：体外建立可长期生长的自身反应性T细胞系克隆，并分析其T细胞受体的使用情况。方法：用狼疮小鼠BXSB自身脾脏作为刺激细胞，在体外建立长期生长的自身反应性T细胞系（ATL）通过有限稀释法进行克隆，应用锚定PCR方法分析其特异的T细胞受体应用。结果：建立了H－2^b限制性的自身反应T细胞系和2个T细胞克隆。T细胞系主要应用TRBV2S1、TRAV5S1和TRAV1021，而2个T细胞克隆均为TRBV2S1和TRAV10S1。分析其表型为CD3^ CD4^ CD28^ 。结论：从狼疮小鼠获得了自身反应性T细胞系及克隆，其T细胞受体利用具有一定的局限性。     

Regulatory T cells protect from autoimmune arthritis during pregnancy

Pregnancy frequently has a beneficial effect on the autoimmune disease Rheumatoid Arthritis, ranging from improvement in clinical symptoms to complete remission. Despite decades of study, a mechanistic explanation remains elusive. Here, we demonstrate that an analogous pregnancy-induced remission can be observed in a mouse model of arthritis. We demonstrate that during pregnancy mice are protected from collagen-induced arthritis, but are still capable of launching normal immune responses to influenza infections. We examine the role of regulatory T (T(R)) cells in this beneficial effect. T(R) cells are essential for many aspects of immune tolerance, including the suppression of autoimmune responses. Remarkably, transfer of regulatory T cells from pregnant 'protected' mice was sufficient to confer protection to non-pregnant mice. These results suggest that regulatory T cells are responsible for the pregnancy-induced amelioration of arthritis.     

Demyelination induced by T cell lines and clones specific for myelin basic protein in mice

Neuropathological studies were performed in order to investigate experimental allergic encephalomyelitis induced by long-term cultured T cell lines and clones specific for myelin basic protein, which were established from SJL/J and DDD/1 mice. All antigen-activated T line or clone cells induced similar disease in euthymic and athymic mice with a common I-A haplotype. The lesion was characterized by perivascular and parenchymal infiltration of mononuclear cells with abundant polymorphonuclear cells located mainly in the lower spinal cord. Axons were severely affected and decreased in number. However, demyelination was present in all cases and was especially marked when recipient mice were: given whole-body X-ray irradiation, I-A compatible other strains, or were congenitally athymic. Topographically, demyelinated axons were most prominent in the root exit and entry zones of the lower spinal cord. Repeated transfer or relapse did not seem to be the factor responsible for enhancing demyelination. We conclude that: inflammation with axonal damage is the main feature of murine experimental allergic encephalomyelitis induced by myelin basic protein-specific T cell lines and clones, demyelination definitely occurs under certain conditions and in certain areas especially at the root exit and entry zones in nude mice, and a single T cell clone induces experimental allergic encephalomyelitis lesions associated with demyelination without the aid of interaction with another recipient-derived T cell population.     

T cell response to myoglobin: a comparison of T cell clones in high-responder and low-responder mice

Mice carrying the H-2b haplotype (e.g., inbred strains C57BL/6 and C57BL/10) are low responders to sperm whale myoglobin when tested in the T cell proliferation assay. Their response is improved by the removal of the Ly-2+ cells from the lymph node population, but it still remains significantly lower than that of cells cells from high-responder strains (e.g., DBA/2, H-2d). To determine whether T cells from the low and high-responder mice recognize the same or different epitopes on the immunizing antigen, we obtained sets of T cell clones from both strains and tested them against peptides representing different regions of the myoglobin molecule, as well as against myoglobins from species other than the sperm whale. Four types of T cell clones were obtained from the DBA/2 mice: 3 types responded to the peptide 107-120 (9 clones altogether), and 1 type responded to the peptide 133-149 (4 clones altogether). The 3 types responding to the peptide 107-120 could be distinguished by their response to horse myoglobin or by the restriction of the response (Ad vs. Ed). Similarly, 5 types of T cell clones were obtained from the C57BL/6 mice: 2 types responded to the peptide 10-22 (1 type, but not the other, responded to horse myoglobin); 1 type responded to the peptide 133-149; and 2 types did not respond to any of the peptides used (1 type, but not the other, responded to dog myoglobin). All 5 types (13 clones altogether) were presumably Ab restricted. These results demonstrate the diversity of epitopes in single antigenic regions and show equivalent heterogeneity of T cell repertoires in high and low responder mice. Attempts to demonstrate specific T cell suppression in the low responder mice failed; only partial, nonspecific suppression was observed.     

Relapsing arthritis induced by cell transfer from collagen-immunised mice

Collagen-induced arthritis is a well-established model of chronic inflammatory arthritis. We here introduce a development of this model which combines the benefits of adoptive transfer and sequential relapse. DBA/1 x B10.Q F1 hybrid mice were immunised with bovine type II collagen, and those which developed a sufficiently high level of arthritis served as donors of spleen cells transferred into BALB/c SCID hosts. After boosting with 500 microg collagen, the development of host arthritis was monitored over a period of up to 256 days, during which up to three successive peaks were detected. In comparison with bovine collagen, mouse collagen used for boosting induced a lower initial peak but higher relapses. As expected, the transferred disease was more uniform than the freshly induced one. Previous information suggests that a shifting cytokine balance between protective and aggressive T cells may account for the relapse and remission. This study provides a model of relapsing polyarthritis, obtained with normal immunocytes boosted with a well-defined protein antigen in animals not themselves treated with adjuvant. As such it is relevant to the etiology of inflammatory arthritis in man, and, if further developed, could be of value for testing new therapeutic strategies.     

T cell receptor usage by HLA-DQw8-specific T cell clones.

To investigate whether T cells recognizing the same HLA molecule may demonstrate homology in parts of their TCR, five different HLA-DQw8-specific T lymphocyte clones (TLC) were studied. The TCR alpha and -beta genes of four alloreactive, HLA-DQw8-specific and one antigen-specific TLC were sequenced. All TLC used different V alpha and V beta genes. However, four of the TLC shared a certain CDR1 beta motif and all five used either J beta 2.3 or -2.5. In addition, two used the same J alpha. The results indicate a possible preferential usage of certain TCR structures by T cells specific for DQw8.     

Generation and maintenance of autoantigen-specific CD8(+) T cell clones isolated from NOD mice.

The non-obese diabetic (NOD) mouse develops insulin dependent diabetes mellitus (IDDM) spontaneously with a higher incidence in females than in males. There are many similarities to the human disease, making it an ideal model. Our group is examining the role that CD4(+) and CD8(+) T cells play in IDDM in the NOD mouse, as it is known that both T cell subsets are required for onset of disease. Although IDDM has an autoimmune etiology, the initial triggering event is unknown and the autoantigen involved has not been identified. This investigation focussed on one of the potential autoantigens involved, the enzyme glutamic acid decarboxylase (GAD). We raised GAD peptide-specific CD8(+) T cells by immunising NOD mice with the GAD peptide alongside an irrelevant peptide that induced a CD4(+) T cell response. In order to maintain these peptide specific T cells in vitro and generate clones, it was found that antibodies specific to CD4(+) and MHC class II molecules needed to be included in the culture medium. This paper outlines the methods we employed to generate and maintain these CD8(+) T cells in vitro.     

Peptide competition at the level of MHC-binding sites using T cell clones from a rheumatoid arthritis patient.

The inhibition of antigen presentation in rheumatoid arthritis by blocking peptide binding to MHC at the antigen presenting cell (APC) level was investigated using various synthetic peptides derived from the 65 kDa mycobacterial protein. Human T cell clones from tuberculosis and rheumatoid arthritis patients were stimulated with peptides in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). Two peptides (residues 65-85 and 412-426) were found to be able to bind to the HLA-DR1 protein. Cross-competition was observed between these peptides when APCs were cultured with a suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptides and T cell clones from tuberculosis patients as responder cells. These T cell clones responded not only to the peptides but also to the native protein. In other experiments, we used T cell clones from a rheumatoid arthritis patient to demonstrate the blocking of MHC-binding sites by adding the p412-426 in the recognition of DR1 restricted T cell clone specific to p65-85; MHC binding was not observed using a control peptide (residues 198-217). This approach has permitted the identification of MHC-specific blockers. Further experimentation is required to determine the particular amino acids involved in MHC binding. Our data support the idea that modulation of antigen presentation by peptide competition could be a useful tool for immunotherapy in autoimmune diseases.     

Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease

Targeted mutation of TGFbeta1 in mice demonstrated that TGFbeta1 is one of the key negative regulators of immune homeostasis, as its absence leads to activation of a self-targeted immune response. Nevertheless, because of the highly pleiotropic properties of TGFbeta and the presence of TGFbeta receptors on most cell types, its biologic role in the regulation of immune homeostasis is not yet understood. To limit the consequences of TGFbeta effects to a single cell type, we developed a transgenic approach to abrogate the TGFbeta response in key immune cells. Specifically, we expressed a dominant-negative TGFbeta receptor type II under a T cell-specific promoter and created a mouse model where signaling by TGFbeta is blocked specifically in T cells. Using this transgenic model, we show that T cell homeostasis requires TGFbeta signaling in T cells.     

Islet-infiltrating T cell clones from non-obese diabetic mice that promote or prevent accelerated onset diabetes

In humans and non-obese diabetic mice (NOD), insulin-dependent diabetes mellitus (IDDM) results from a spontaneous T cell-dependent autoimmune destruction of the insulin-producing pancreatic beta cells. Previous data suggest that a delicate balance between autoaggressive T cells and suppressor-type immune phenomena determine whether expression of autoimmunity is limited to insulitis or progresses to IDDM. To resolve the cellular basis of this intricate network of pathogenic CD4+ and CD8+ T cells and the role of T cells in suppressive immune phenomena. T cell clones were propagated directly from islets of NOD mice at the onset of insulitis. Insofar as insulitis, but not IDDM, is universal in NOD mice, we have screened for the in vivo effects of the islet-infiltrating T cell clones upon expression of IDDM, not insulitis. A CD4+ T cell clone, IS-3S7D, proliferates in response to islet antigen(s) and its transfer into prediabetic NOD mice promotes the rapid onset of IDDM. An interleukin 2 (IL 2)-dependent noncytolytic, V beta 11+ CD8+. T cell clones IS-2.15, prevents an accelerated onset diabetes in two distinct models. The present study, which documents the presence of CD4+ diabetogenic T cell clones and CD8+ T cell clones that dampen autoimmunity, gives tangible evidence that opposing autoimmune processes may determine whether an autoimmune-prone host develops frank disease.     

Sendai virus-specific T cell clones II. Induction of interferon production by Sendai virus-specific T helper cell clones

Interferon (IFN) was detected upon co-culture of cloned Sendai virus (SV)-specific T lymphocyte with SV-presenting syngeneic stimulator cells. The antiviral activity was defined as IFN-alpha, beta. The T cell clones, upon contact with antigen-presenting stimulator cells, stimulated adherent cells present in the stimulator cell population to secrete IFN. Induction of IFN production was independent from interleukin 2 production by the T cell clones.