Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD451 leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68 /CD11b1 /Gr1 hi ). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.Keywords: interstitial macrophages; alveolar macrophages; monocytes; myeloid dendritic cells; neutrophilsThe heterogeneous population of myeloid cells in the lungs is important for maintaining homeostasis and regulating inflammation, injury, and remodeling. These cells are thought to originate from bone marrow-derived precursors, and they differentiate into mature cells with a variety of functional properties, depending on environmental cues. Multiple studies have demonstrated functional and phenotypic differences between myeloid cell subsets, including monocytes, myeloid dendritic cells, macrophages, and neutrophils. However, the specific identification and quantification of subsets of these cells in tissues are complex, and a comprehensive scheme for isolating these cells from the lungs is not readily available. To date, studies of lung myeloid cells have been limited by the inconsistent use of cellular markers and a lack of clearly defined signatures for these cells, resulting in difficulties comparing studies from different laboratories and generalizing findings across different model systems. Therefore, we undertook a study to identify and quantify myeloid cell populations comprehensively in the lungs, using flow cytometry.Based on the recent explosion of information regarding the mechanisms that underpin innate immune responses, a clear need is apparent for better strategies to identify and study myeloid cell subpopulations in the lungs. The traditional method for the identification and quantification of lung myeloid cells involves the manual counting of cells obtained from the airways by bronchoalveolar lavage (BAL). This methodology normally yields a predominance of alveolar macrophages, because these are the primary immune cells in the airspaces. Interstitial macrophages, however, comprise a substantial portion of the lung myeloid cell population (1), and may have dif...

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/ sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206 2 macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.Keywords: alveolar macrophages; interstitial-associated macrophages; interstitial macrophages; interstitial lung disease Clinical RelevanceFlow cytometry is an important method that allows for delineation of specific cell components of immune responses and disease states. A flow cytometry panel for myeloid cells in human lung samples (bronchoalveolar lavage and lung tissue) has not been performed previously. Here we develop a single flow cytometry panel that allows for the accurate identification of cellular components in human blood, bronchoalveolar lavage, and lung tissue.

Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.

To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to monotherapies. BALB/c mice bearing orthotopic EMT-6 mammary tumors and μMt mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen-reactive IFNγ-producing CD8 T cells was evaluated by ELISpot assay. NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor-bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8 T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8 T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy. These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors. .