The model reveals early inflammation-associated changes in the ve

The model reveals early inflammation-associated changes in the ventricle wall that can be determined by cardiac magnet resonance imaging (CMRI) and hence permits the evaluation of the central processes in the transition from autoimmune myocarditis to DCM. Furthermore, dissection of the Th-cell cytokine contribution to myocarditis and subsequent DCM revealed that IFN-γ signaling is critical for the development of myocarditis, whereas the concerted action of both IL-17A and IFN-γ is required for progression to DCM. Myhca614–629 peptide-specific T-cell hybridomas were generated using effector Th cells from mice suffering from peptide-induced EAM (Supporting Information Fig. 1A and B). Following sequence

identification of TCR variable regions (Supporting Information Fig. 1C) and cloning into TCR cassette vectors, linearized constructs were microinjected into fertilized oocytes to produce transgenic offsprings. Since the two TCR transgenic founder lines displayed almost identical transgenic TCR expression patterns, APO866 chemical structure all further analyses were continued with line 1 (designated as C.CB6-Tg(Tcra,Tcrb)562Biat, short TCR-M). Flow cytometric analysis revealed that TCR-M mice exhibit a strongly shifted CD4/CD8 lymphocyte ratio in secondary lymphoid organs (spleen and lymph nodes) and an increase of thymic CD4 single positive (SP) T cells with a strong reduction in double-positive (DP) thymocytes (Fig. 1A). Eighty to more than 95% of the peripheral CD4+ T cells and almost all thymic CD4 SP T cells expressed the transgenic TCR Vβ8 and Vα2 chains (Fig. 1B). To assess immune responsiveness of peripheral TCR-M T cells, splenocytes

PLEK2 were stimulated with different concentrations of myhca614–629 peptide and proliferation of CD4+ T cells was assessed. As shown in Fig. 1C, a concentration of 10 ng/mL (5 × 10−9 M) myhca614–629 peptide was sufficient to induce proliferation of TCR-M T cells. Furthermore, CD4+ TCR-M T cells responded to DCs loaded with cardiac myosin protein with vigorous proliferation (data not shown), suggesting that high avidity myhca614–629-specific T cells had seeded the periphery and that CD4+ TCR-M T cells had not been exposed to negative selection during their maturation in the thymus. Indeed, RT-PCR analysis from thymic tissue confirmed the selective lack of cardiac myosin alpha expression in BALB/c mice (Supporting Information Fig. 2), a finding that has been recently described for humans and transgenic NOD mice that express the human MHC class II molecule DQ8 [25]. The absence of central tolerance and the presence of high avidity myhca614–629-specific T cells in TCR-M mice precipitated spontaneous autoimmune myocarditis with first leukocyte infiltrations being detectable in hearts of TCR-M mice at 2 weeks of age (Fig. 2A).

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