Because the circulating pathogenic T cells in a large pool of peripheral T cells may be very small, it is reasonable to assume that they seldom reach the detection threshold of flow cytometric analysis

Because the circulating pathogenic T cells in a large pool of peripheral T cells may be very small, it is reasonable to assume that they seldom reach the detection threshold of flow cytometric analysis. It has been demonstrated recently that interferon–inducible chemokines such as CXC chemokine ligand 10 (CXCL10) play an important role in the initial stage of autoimmune disorders involving endocrine gland [24]. distribution in those patients having disease longer than 5 years and requiring thyroid hormone replacement. Patients with Graves disease exhibited no skewing both in CD4+ and CD8+ T cells. These findings indicate that clonal expansion of CD8+ T cells in Hashimoto’s thyroiditis can be detected in peripheral blood and may support the role of CD8+ T cells in cell-mediated autoimmune attacks on the thyroid gland in Hashimoto’s thyroiditis. less than 005 were considered significant. Flow cytometric analysis for TCR V repertoire Three-colour immunofluorescence analysis was used to study TCR V repertoire distribution, as described previously [11]. Briefly, after washing twice in phosphate-buffered saline, peripheral blood samples were incubated with appropriate phycoerythrin (PE)-conjugated mAbs with specificity for TCR V 1-23 (Immunotech, Marseille, France), fluorescein isothiocyanate-conjugated anti-CD8 (Becton Dickinson) and R-PE-Cy5-conjugated anti-CD4 (Dako, Glostrup, Denmark) mAbs. After lysis of erythrocytes and washing, stained cells were analysed with a fluorescence activated cell sorter Calibur flow cytometer using CellQuest software (BD Bioscience, Tokyo, Japan). TCR V expression was represented as a percentage of CD4+ or CD8+ cells for each family. Three-dimensional graphic display of TCR diversity Qualitative alterations of TCR V repertoire obtained by CDR3 spectratyping were combined with the quantity of specific V+ CD4+ and CD8+ T cells for each V subfamily and plotted as landscape columns, as described previously [11,15]. Results Skewed CDR3 size pattern in Hashimoto’s thyroiditis but not Graves disease In healthy controls, the majority of V subfamilies exhibited a Gaussian curve with six peaks or more, reflecting a diverse TCR repertoire. Consistent with previous reports [16], CD8+ T cells exhibited a more skewed CDR3 profile than CD4+ T cells regardless of the presence of disease, probably because of age-related CD8+ T cell clonal expansion (Fig. 1aCc). All 25 different V segments were amplified from CD4+ and CD8+ T cells obtained from each patient’s sample. As shown in Fig. 1b and 1c, patients with Graves H3/l disease showed a diverse distribution in the majority of their V subfamilies both in CD4+ and CD8+ T cells. In contrast, the frequency of skewed TCR V subfamilies of patients with Hashimoto’s thyroiditis was significantly higher than that of Graves disease and healthy controls in CD8+ T cells but not Imeglimin CD4+ T cells. No evidence was found for preferential Imeglimin skewing of particular V subfamilies in patients with Hashimoto’s thyroiditis. Because none of the patients experienced medical and laboratory evidence of acute illness Imeglimin at the time of sample collection, we conclude that these alterations reflect a stable state of the TCR repertoire in these individuals. Open in a separate windowpane Fig. 1 CDR3 spectratyping of T cell receptor (TCR) V. (a) CDR3 size distribution. Each TCR V fragment was amplified from cDNA with one of 25 V-specific primers. The size distribution of polymerase chain reaction (PCR) products was determined by an automated sequencer and GeneScan software. Shown are the results of CDR3 size distribution in CD4+ and CD8+ T cells from individual 16 of Hashimoto’s thyroiditis. (b) CDR3 difficulty scores. Complexity scores for each TCR V were shown. (c) Rate of recurrence of skewed TCR V repertoire. Demonstrated are the mean (standard deviation) numbers of skewed TCR V from CD4+ and CD8+ T cells of the control and individuals with Graves disease and with Hashimoto’s thyroiditis. GD, Graves disease; HT, Hashimoto’s thyroiditis. * 005; ** 001. To elucidate further the characteristics of skewing of TCR V utilization in Hashimoto’s thyroiditis, those individuals were divided into different subgroups based on age, disease duration and levothyroxine requirement (Fig. 2aCc). Although clonal T cell development can be seen in healthy individuals with age [16], no difference was observed between individuals 14 years of age or younger and those older. In contrast, there were styles towards more skewing of CDR3 size distribution Imeglimin in CD8+ T cells from individuals having disease longer than 5 years.