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

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

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 3

Assessment of intratumoral iTreg conversion.

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Assessment of intratumoral iTreg conversion. (A) Helios and (B) neuropil...
(A) Helios and (B) neuropilin expression was evaluated in CD4+FOXP3+ Tregs in different samples, including at least 6 (Helios) or 4 (Neuropilin) samples/group; all comparisons for Helios expression were not significant, and the single significant result for Neuropilin is indicated. (C) LC PBMCs, LNs, tumors, and distant lung cells were CD25 depleted, and FOXP3+ expression was evaluated in aliquots of starting populations (top row, FOXP3 in CD4+ gated cells). Cells were stimulated with CD3/28 mAbs plus TGF-β and IL-2 for 7 days, and FOXP3+ expression was evaluated as a percentage of in vitro–converted iTregs (bottom row, FOXP3 in CD4+ gated cells); 1 representative experiment of 4 is shown. (D) Demethylation of FOXP3 at the TSDR region was evaluated in CD4+CD25+ Tregs. Suppressive function of the same cells was confirmed in suppression assays, and FOXP3+ expression in isolated cells was evaluated by flow cytometry. Ratios between the percentage of FOXP3+ cells and the percentage of TSDR-demethylated cells, FOXP3/TSDR, were counted in all groups: healthy donors (n = 4), LC PBMCs (n = 4), LN (n = 4), tumors (n = 8), and distant lungs (n = 5). To decrease variability, 2–3 technical replicates of TSDR demethylation obtained from the same isolated DNA sample were included, as detailed in Supplemental Figure 4B. (E) FOXP3/TSDR ratios were evaluated as described above in Tregs from two groups of LT patients of different ages: the 6 youngest male LT patients, aged 30–44, mean ± SEM 38.5 ± 2.4 years, and 8 males aged 55–73, mean ± SEM 64.9 ± 2.4 years. Gating strategies are shown in Supplemental Figures 1, 12, and 37. The following statistics were used: (A and B) Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons test and (E) Mann Whitney test. *P < 0.05.