Abstract A02: The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer
Authors: Eruslanov Evgeniy , Bhojnagarwala Pratik , Quatromoni Jon , O'Brien Shaun , Moon Edmund , Stephen Tom , Rao Abhishek , Garfall Alfred , Hancock Wayne , Conejo-Garcia Jose , Deshpande Charuhas , Feldman Michael , Singhal Sunil , Albelda Steven 
Published: October 2016

Abstract
To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer.
To address this question, freshly isolated tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of canonical TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). This subset of TANs was found in lung tumor tissue but not in adjacent uninvolved lung. We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter.
Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (&gt;10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. By contrast, mature CD11b+CD15+CD10+CD16hi neutrophils did not acquire the phenotype of hybrid TANs, when cultured under these conditions. In addition, we determined that IFN-γ and GM-CSF synergistically exerted APC promoting effects on immature neutrophils via the downregulation of the transcription factor, Ikaros. However, the development of APC-like hybrid neutrophils was profoundly inhibited under hypoxic conditions.
Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) produce APC cytokines such as TNF and IL-12 after stimulation, 2) stimulate antigen non-specific autologous T cell responses induced by plate-bound anti-CD3 antibodies 3) directly stimulate antigen-specific autologous memory T cell responses to virus-derived antigens, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex.
In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid TANs had enhanced ability to trigger and support T cell responses in direct cell-cell interactions. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction.
Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Shaun O'Brien, Edmund Moon, Tom Stephen, Abhishek Rao, Alfred Garfall, Wayne Hancock, Jose Conejo-Garcia, Charuhas Deshpande, Michael Feldman, Sunil Singhal, Steven Albelda. The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A02.