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.