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Human lung tumor FOXP3+ Tregs upregulate four “Treg-locking” transcription factors
Tatiana Akimova, … , Evgeniy B. Eruslanov, Wayne W. Hancock
Tatiana Akimova, … , Evgeniy B. Eruslanov, Wayne W. Hancock
Published August 17, 2017
Citation Information: JCI Insight. 2017;2(16):e94075. https://doi.org/10.1172/jci.insight.94075.
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Research Article Immunology

Human lung tumor FOXP3+ Tregs upregulate four “Treg-locking” transcription factors

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Abstract

Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non–small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these “Treg-locking” transcription factors was positively correlated with levels of FOXP3 mRNA, with highest correlations for EOS and SATB1. EOS had an additional, FOXP3 mRNA–independent, positive correlation with FOXP3 protein in tumor Tregs. Our study identifies distinctive features of intratumoral Tregs and suggests that targeting Treg-locking transcription factors, especially EOS, may be of clinical importance for antitumor Treg-based therapy.

Authors

Tatiana Akimova, Tianyi Zhang, Dmitri Negorev, Sunil Singhal, Jason Stadanlick, Abhishek Rao, Michael Annunziata, Matthew H. Levine, Ulf H. Beier, Joshua M. Diamond, Jason D. Christie, Steven M. Albelda, Evgeniy B. Eruslanov, Wayne W. Hancock

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Figure 1

Characteristics of peripheral blood Tregs in LC patients.

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Characteristics of peripheral blood Tregs in LC patients.
The frequency ...
The frequency of CD4+FOXP3+ Tregs was evaluated in (A) PBMCs and (B) CD4+ gated cells by flow cytometry in samples from LC (n = 33), LT (n = 27), and HD (n = 18) groups. Details of the Treg gating strategy are shown in Supplemental Figure 1. Expression of Treg-associated markers was evaluated in (C) PBMC Tregs from LC (n = 17) and LT (n = 14) patients, and (D) CTLA4 expression was evaluated in all PBMC T cell subsets of the same patients. Gating strategies for each marker are shown in Supplemental Figures 6, 7, 8, and 36. (E) CD4+CD25+ isolated Tregs were incubated with anti-CD3 stimulated CFSE-labeled donor PBMCs in 4-day suppression assay, and Treg-suppressive function was quantified as area under the curve (AUC). 11 LC, 8 HD, and 57 LT patient Treg samples were evaluated. (F) LC patients (n = 15) were divided into stage IA and stage IB–IIIA groups, and then percentages of Tregs in CD4+ subsets were compared preoperatively and postoperatively within each patient as the percentage of postoperative Tregs minus the percentage of preoperative Tregs. LC, lung cancer; HD, healthy donors; LT, patients with a lung disease pretransplant; Tx, transplant. The following statistics were used: (A, B, and E) Kruskal-Wallis test; (C) multiple t tests with false discovery rate set to 1%; (D) ANOVA with post-hoc Holm-Sidak’s multiple comparisons test; and (F) Mann Whitney test. *P < 0.05; ***P < 0.001.

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