Abstract 1013: Location matters: unique functional and transcriptional, but not flow cytometric, characteristics of intratumoral FOXP3+ Tregs vs. Tregs from other sites of patients with lung tumors
Authors: Hancock Wayne W. , Akimova Tatiana , Zhang Tianyi , Eruslanov Evgeniy , Singhal Sunil , Albelda Steven M. 
Published: July 2017

Abstract
FOXP3+ Tregs are considered important to limiting antitumor immunity but are rarely characterized clinically. We studied 227 samples from 66 lung cancer patients using blood, lymph node (LN), tumor and distant lung samples, with a mean age of 67.8±1.1 years, 67% males, 62% adenocarcinoma, 31% squamous cell carcinoma, 7% miscellaneous tumors, mean tumor size of 3.4±0.3 cm, and 21% rate of metastases. In addition to quantitating FOXP3+CD4+ Tregs, we evaluated their expression of 35 markers by flow cytometry: CD15s, CD25, CD26, CD27, CD39, CD40L, CD45RA/RO, CD62L, CD69, CD101, CD120b, CD161, CCR4, CTLA4, GARP, GITR, Helios, HLA-DR, ICOS, LAP, neuropilin, PD-1, TIGIT, Tim3, CCR4, CCR5, CCR7, CXCR3, CXCR4 and CCR8, and tested Treg suppressive function. We used PrimeFlow to evaluate mRNA expression of target genes in 100% pure human Treg cells, gated on CD4+FOXP3+ cells. FOXP3+ Tregs (%) in the CD4 cels of tumors and LN were significantly increased (p&lt;0.0001 Kruskal-Wallis) compared to other sites; tumors 19.2±8.5%, LN 14.7±8.5%, healthy PBMC donors 7.2±2.5%, PBMC lung cancer 7.2±2.7%, and lungs 6.6±3.2%. Tregs of lung tumors were remarkably suppressive vs. all other sites (p&lt;0.0001 vs. PBMC & LN, p=0.0106 vs. lung Tregs). None of the 35 markers evaluated by flow cytometry were statistically significantly different for tumor Tregs vs. other sites. However, PrimeFlow showed tumor Tregs, but not FOXP3- T cells or Tregs from other sites, had upregulated mRNA expression of 4 transcription factors (TF): Eos, Irf4, Satb1 and Gata1. These 4 TFs plus LEF1 were recently described as a quintet of Treg self-locking signature TF; expression of any 2 TF plus FOXP3 promoted full Treg gene expression and function. Tumor Tregs also showed significant upregulation of FOXP3 mRNA and protein. Tumor and lung Tregs expressed more Eos, Irf4, Satb1 and Gata1 mRNA, and much higher levels of FOXP3 protein per cell, while in PBMC, LNs and healthy donor Tregs upregulation of TFs did not correspond with increase of FOXP3 protein, indicating the “Treg self-locking signature” is subject to regulation by local factors. Indeed, cultured tumor Tregs downregulated FOXP3 protein, mRNA and TF expression, while PBMC and LN Treg incubated in tumor-conditioned media upregulated FOXP3 mRNA and protein, and Treg TF expression, moving toward the tumor Treg-like phenotype. We found significantly increased numbers and suppressive function of FOXP3+ Tregs within lung tumors vs. other sites in the same patients. In addition, while large-scale flow cytometric studies were not useful in identifying key features of tumor Tregs vs. Tregs at other sites, PrimeFlow showed that tumor Treg have a unique phenotype with upregulated expression of FOXP3 mRNA and protein, as well as Eos, Irf4, Satb1 and Gata1 mRNAs. These features appear to be malleable and associated with features of the local tumor microenvironment.
Citation Format: Wayne W. Hancock, Tatiana Akimova, Tianyi Zhang, Evgeniy Eruslanov, Sunil Singhal, Steven M. Albelda. Location matters: unique functional and transcriptional, but not flow cytometric, characteristics of intratumoral FOXP3+ Tregs vs. Tregs from other sites of patients with lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1013. doi:10.1158/1538-7445.AM2017-1013