Tumor-Infiltrating Foxp3+T-Regulatory (Treg) Cells In Early-Stage Human Lung Cancer Exhibit Enhanced Suppressive Function When Compared To Blood Or Lymph Node (Ln) Treg Cells

Aim: Advances in immunotherapy require a detailed understanding of host immunity. Data from murine models indicate solid tumors of various types can recruit Tregs and/or promote iTreg development, and thereby curtail antitumor responses. However, clinical data have been restricted to either evaluation of Treg numbers in tumors, or studies of PBMC Treg in patients with cancers. Surprisingly, no studies of Treg function have ever been reported using Tregs isolated from tumors themselves. Methods: Using our recently reported technique, we isolated Tregs within 20 mins of resection from 13 lung cancer patients, stages Ia-IIIa, and 2 control non-cancer patients (diaphragmatic hernia and lung granuloma). The mean age of the cancer patients (10 males, 3 females) was 65.4±2.2 years; 7 patients had adenocarcinoma, 3 had squamous cell carcinoma, and the remainder had large cell neuroendocrine carcinoma, colon adenocarcinoma and high-grade carcinoma. The mean size of their tumors was 4.0±0.8 cm, and 1 of the 13 patients developed metastases during the follow-up period. We compared the suppressive function of Tregs isolated from each patient9s freshly resected tumor, LN and blood, using cryopreserved healthy donor PBMC aliquots as responders, and area-under-curve (AUC) analysis. We controlled for comparable CD4+ and FOXP3+ purity in all isolated Treg samples, and cell viability, excluding from further analysis all data from non-comparable FOXP3+ subsets. Thus, we were able to avoid artifacts arising from different cell conditions (e.g. use of autologous responder cells) or from unequal FOXP3+ content in Treg isolates. Results: Tumor-infiltrating Tregs had significantly enhanced suppressive function when compared with Tregs isolated from other locations in the same patient. Thus, tumor Tregs were significantly more suppressive than lung LN Tregs (p = 0.0003), and significantly more suppressive than blood Tregs (p = 0.03). Tregs isolated from LN in close proximity to tumors had comparable suppressive function to Tregs isolated from distant LN (p>0.05), and blood Tregs had comparable suppressive function to that of LN Tregs (p>0.05). Additional analyses showed that the enhanced suppressive function of tumor Treg was not due to Treg-induced killing/apoptosis of responder cells, cytokine and/or growth factor deprivation, or soluble factors produced by tumor Treg cells. Conclusions: We demonstrate for the first time that tumor-infiltrating human lung cancer Treg have significantly increased suppressive function. This finding is important for the development of effective anti-tumor therapy. While we are continuing to study the basis for such differences, our data suggest that studies of peripheral blood Tregs in cancer patients may be inadequate and inefficient approaches for understanding the fundamental mechanisms of limited host antitumor immune responses. Citation Format: Tatiana Akimova, Evgeniy B. Eruslanov, Pratik S. Bhojnagarwala, Jon G. Quatromoni, Jacqueline Morgen, Sunil Singhal, Steven M. Albelda, Wayne W. Hancock. Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2015-1291