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Updated: Aug 22, 2026
Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry
Published on: January 7, 2019
Functional lymphocyte subset assessment of the Th1/Th2 profile in patients with autoimmune thyroiditis by
1Endocrinology Unit, CHR-St. Joseph/Warquignies Medical Center, Mons, Belgium. colin@isto.ucl.ac.be
Background:
Hashimoto's thyroiditis (HT) results from a parenchymal infiltration by Th1 T cell clones that ultimately may cause tissue destruction. We analysed here whether the quantitative assessment of the cytokine profile in peripheral lymphocytes could help for the evaluation of patients with HT.
Methods:
We added to a flow cytometric evaluation of lymphocyte subpopulations, an assay to identify specific Th1 and Th2 functional subsets. Whole blood diluted in RPMI was activated with PMA and ionomycin for 4h at 37 degrees C in the presence of brefeldin. Immunophenotyping of samples was performed with PerCP-Cy5.5-conjugated CD3, and APC-conjugated CD4 antibodies. After staining for surface antigens, red cells were lysed and white cells were permeabilized. Intracellular cytokines were detected with FITC-conjugated INFgamma (Th1) and PE-conjugated IL-4 (Th2) monoclonal antibodies (mabs).
Results:
Twenty-three consecutive patients were selected based on the detection of anti TPO ab concentration equal or higher than 600 UI/L. They were compared to 17 healthy control subjects (with undetectable TPO abs). The lymphocyte count and the proportion of the different lymphocyte subsets (T cells, B cells, NK cells, T-CD4+, T-CD8+, Th1 CD3, Th2 CD3, Th1 CD4, Th2 CD4 cells) were alike when both groups were compared. A significant difference appeared when the Th1/Th2 ratio measured on CD3 T cells was considered. This ratio was significantly increased in HT patients when compared to controls (24.91+/-2.9 vs. 15.5+/-1.4 (mean +/- SEM); p <0.05). When the same analysis was performed on CD4 T cells, the Th1/Th2 ratio was again higher in HT patients, although without reaching significance (13.5+/-1.9 vs. 8.8+/-0.6; p >0.05).
Conclusions:
Our data indicate that the Th1 context analysed in peripheral lymphocytes is dominant in HT patients. Flowcytometry could be used as a diagnostic tool to better understand the pathogeny and the outcome of destructive autoimmune thyroiditis.
