Immunophenotyping with high-dimensional flow cytometry identifies Treg cell subsets associated with recurrence in papillary thyroid carcinoma

The activation of Treg cell subsets is critical for the prognosis of tumor patients; however, their heterogeneity and disease association in papillary thyroid carcinoma (PTC) need further investigation. We performed high-dimensional flow cytometry for immunophenotyping on thyroid tissues and matched peripheral blood samples from patients with multinodular goiters or PTC. We analyzed CD4(+) T cell and Treg cell phenotypes and compared the recurrence-free survival of PTC patients with different Treg cell subset characteristics using TCGA. Furthermore, PTC recurrent and non-recurrent group were compared by multiplex immunohistochemistry. High-dimensional flow cytometry and bioinformatics analysis revealed an enrichment of Tregs in tumors compared with multinodular goiters and peripheral blood specimens. Moreover, effector Tregs (e-Tregs) as well as FOXP3(+) non-Tregs were enriched in tumor samples, and the expression of CD39, PD-1, and CD103 increased on tumor Tregs. TCGA data analysis showed that individuals with CD39(hi) PD-1(lo)CD103(lo)e-Treg(hi) and CD39(lo)PD-1(lo)CD103(hi)e-Treg(hi) expression patterns had a high recurrence rate. According to the multiplex immunohistochemistry and analysis, compared with non-recurrent group, the proportion of high recurrence rate effector Treg clusters (CD39(+)PD-1(-)CD103(-) plus CD39(-)PD-1(-)CD103(+)) was increased in recurrent patients. Overall, our results highlight the potential of e-Treg subsets as future immunotherapy targets for PTC recurrence.