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OBJECTIVE: To better understand the role of antigen (arthritogenic epitope)-specific T cells in the development of autoimmune arthritis. METHODS: A transgenic (Tg) mouse expressing the T cell receptor (TCR) V(alpha)1.1 and V(beta)4 chains specific for a dominant arthritogenic epitope (designated 5/4E8) of human cartilage proteoglycan (HuPG) aggrecan was generated. This TCR-Tg mouse strain was backcrossed into the PG-induced arthritis (PGIA)-susceptible BALB/c strain and tested for arthritis incidence and severity. RESULTS: CD4+ TCR-Tg T cells carried functionally active TCR specific for a dominant arthritogenic epitope of HuPG (5/4E8). T cells of naive TCR-Tg mice were in an activated stage, since the in vitro response to HuPG or to peptide stimulation induced interferon-gamma and interleukin-4 production. TCR-Tg mice uniformly, without exception, developed severe and progressive polyarthritis, even without adjuvant. Inflamed joints showed extensive cartilage degradation and bone erosions, similar to that seen in the arthritic joints of wild-type BALB/c mice with PGIA. Spleen cells from both naive and HuPG-immunized arthritic TCR-Tg mice could adoptively transfer arthritis when injected into syngeneic BALB/c.SCID recipient mice. CONCLUSION: TCR-Tg BALB/c mice display increased arthritis susceptibility and develop aggravated disease upon in vivo antigen stimulation. This model using TCR-Tg mice is a novel and valuable research tool for studying mechanisms of antigen (arthritogenic epitope)-driven regulation of arthritis and understanding how T cells recognize autoantigen in the joints. This type of mouse could also be used to develop new immunomodulatory strategies in T cell-mediated autoimmune diseases. [ABSTRACT FROM AUTHOR]

Background: The anti-inflammatory capacity of heat shock proteins (HSP) has been demonstrated in various animal models of inflammatory diseases and in patients. However, the mechanisms underlying this anti-inflammatory capacity are poorly understood. Therefore, the possible protective potential of HSP70 and its mechanisms were studied in proteoglycan (PG) induced arthritis (PGIA), a chronic and relapsing, T cell mediated murine model of arthritis., Methodology/principal Findings: HSP70 immunization, 10 days prior to disease induction with PG, inhibited arthritis both clinically and histologically. In addition, it significantly reduced PG-specific IgG2a but not IgG1 antibody production. Furthermore, IFN-gamma and IL-10 production upon in vitro restimulation with HSP70 was indicative of the induction of an HSP70-specific T cell response in HSP70 immunized mice. Remarkably, HSP70 treatment also modulated the PG-specific T cell response, as shown by the increased production of IL-10 and IFN-gamma upon in vitro PG restimulation. Moreover, it increased IL-10 mRNA expression in CD4+CD25+ cells. HSP70 vaccination did not suppress arthritis in IL-10(-/-) mice, indicating the crucial role of IL-10 in the protective effect., Conclusions/significance: In conclusion, a single mycobacterial HSP70 immunization can suppress inflammation and tissue damage in PGIA and results in an enhanced regulatory response as shown by the antigen-specific IL-10 production. Moreover, HSP70 induced protection is critically IL-10 dependent.

Systemic administration of agents that neutralize or antagonize Th1-mediated pro-inflammatory responses has been demonstrated to ameliorate inflammation in chronic autoimmune disease. However, systemic administration of such immunosuppressive biologicals causes serious side effects and has only limited success. To minimize these side effects, autoantigen-specific lymphocytes have been proposed as a carrier to deliver immunosuppressive agents to sites of inflammation. Here we studied the effects of primary cartilage proteoglycan-specific CD4+ T cells that were transduced using an efficient method of viral transduction with active genes encoding IL-1beta receptor antagonist, soluble TNF-alpha receptor-Ig, IL-4 or IL-10 in chronic proteoglycan-induced arthritis in mice. This is the first study describing such gene therapy using primary CD4+ T cells in a chronic arthritis. Moreover, the impact of proteoglycan-specific Th1, Th2 or naïve T cells was studied. Although proteoglycan-TCR transgenic CD4+ T cells can transfer arthritis to lymphopenic recipients, none of the proteoglycan-TCR transgenic T cell phenotypes that were tested induced worsening of arthritis in wild type hosts. Proteoglycan-specific T cells ameliorated arthritis when expressing the transduced IL-10 gene, and not when expressing the other transgenes/phenotypes. Although all of the tested biologicals can suppress in a wide range of different inflammatory disorders, especially IL-10 would therefore serve as a promising candidate to be used in cellular gene therapy for chronic arthritis.

Deficient T cell regulation can be mechanistically associated with development of chronic autoimmune diseases. Therefore, combining the regulatory properties of IL-10 and the specificity of autoreactive CD4(+) T cells through adoptive cellular gene transfer of IL-10 via autoantigen-specific CD4(+) T cells seems an attractive approach to correct such deficient T cell regulation that avoids the risks of nonspecific immunosuppressive drugs. In this study, we studied how cartilage proteoglycan-specific CD4(+) T cells transduced with an active IL-10 gene (T(IL-10)) may contribute to the amelioration of chronic and progressive proteoglycan-induced arthritis in BALB/c mice. TCR-transgenic proteoglycan-specific T(IL-10) cells ameliorated arthritis, whereas T(IL-10) cells with specificity for OVA had no effect, showing the impact of Ag-specific targeting of inflammation. Furthermore, proteoglycan-specific T(IL-10) cells suppressed autoreactive proinflammatory T and B cells, as T(IL-10) cells caused a reduced expression of IL-2, TNF-alpha, and IL-17 and a diminished proteoglycan-specific IgG2a Ab response. Moreover, proteoglycan-specific T(IL-10) cells promoted IL-10 expression in recipients but did not ameliorate arthritis in IL-10-deficient mice, indicating that T(IL-10) cells suppress inflammation by propagating the endogenous regulatory IL-10 response in treated recipients. This is the first demonstration that such targeted suppression of proinflammatory lymphocyte responses in chronic autoimmunity by IL-10-transduced T cells specific for a natural Ag can occur via the endogenous regulatory IL-10 response.

Proteoglycan (PG)-induced arthritis (PGIA), a murine model for rheumatoid arthritis (RA), is driven by antigen (PG)-specific T and B cell activation. In order to analyze the pathogenic role of antigen-specific T cells in the development of autoimmune arthritis, we have generated a transgenic (Tg) mouse. The CD4(+) T cells of this TCR-5/4E8-Tg line express a functional T cell receptor (TCR) composed of the Valpha1.1 and Vbeta4 chains with specificity for the dominant arthritogenic T cell epitope of human cartilage PG. Adoptive transfer of naive TCR-5/4E8-Tg cells induced arthritis with severe clinical symptoms in syngeneic immunodeficient BALB/c.RAG2(-/-) mice. In vivo activation of TCR-5/4E8-Tg CD4(+)Vbeta4(+) cells with cartilage PG seemed to be critical for arthritis induction. Arthritis never developed after transfer of naive wild-type cells. The arthritis was characterized as a chronic progressive disease with intermittent spontaneous exacerbations and remissions. Inflamed joints showed extensive cartilage damage and bone erosions leading to massive ankylosis in peripheral joints. These PG epitope-specific TCR-5/4E8-Tg mice can be valuable research tools for studying antigen-driven T cell regulation in arthritis, and migration of T cells to the joints. In addition the model may be used for the development of immune modulating strategies in T cell-mediated autoimmune diseases.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.

Monoclonal antibody (mAb) U36 was developed for the treatment of minimal residual disease of head and neck squamous cell carcinoma (HNSCC). The mAb-U36-defined antigen was characterized by cDNA cloning, and was shown to be identical to the keratinocyte-specific CD44 splice variant epican. The epitope recognized by mAb U36 was shown to be located in the v6 domain. Two amino acids within the epitope appeared to differ from the sequences that have been described in literature. The sequence of the epitope appeared to contain glutamic acid at position 367 and lysine at position 374, while valine and arginine respectively have been described before. Interestingly, another anti-CD44v6 antibody with possible clinical application, VFF18, recognizes an epitope in the same area. With respect to the applicability of these antibodies for tumor targeting, this variation might have an influence on antibody-antigen interaction and mAb accumulation in the tumor. Furthermore, this observation raised the question whether the different epitopes are related to the malignant behavior of tumor cells. In this paper we determine the relative affinity of mAb U36 for the variant epitope sequences by tumor cell binding assays using synthetic peptides for competition. The presence of glutamic acid instead of valine at position 367 caused strong competition. Further evaluation showed that the published valine variant does not exist in vivo, and is the result of a sequencing artefact. The effect of substitution of lysine for arginine at position 374 had no effect on the binding of mAb U36 to the cells. This amino acid variation was shown to be due to allelic polymorphism. There was no trend towards allelic imbalance in tumor cells as compared to normal cells.