Abstract 5593: Identification and characterization of an unusual monocyte subpopulation

Personalized immunotherapy for cancer has become an effective treatment choice in certain oncology indications. To successfully develop such therapeutic candidates requires a deep understanding of the biology including the interaction between a variety of hematopoietic cells, mesenchymal cells, and the microenvironment of bone marrow and peripheral blood. Flow cytometry offers unparalleled advantages in immunophenotyping, functional analysis, and receptor occupancy determination. For example, flow cytometry is capable of simultaneously analyzing millions of leucocytes and identifying populations of regulatory T cells, cytotoxic T cells, B cells, NK cells, monocytes, macrophages, and dendritic cells with multiple surface and intracellular biomarkers. Because of the diversity of clinical samples, variable protocols, and storage conditions, new challenges arise during the development and flow cytometric analysis of immunotherapeutic drug candidates. One of these challenges is the observation, upon flow cytometric analysis, of an unexpected pattern of immune cells. For example, monocytes are one of the important components in the immune system, but they are also readily modulated during an immune response. In our clinical studies, we found that some peripheral whole blood samples exhibit an extra population paralleling normal monocytes during flow cytometric analysis. To correctly categorize these cells, we applied a panel of 18 fluorochrome conjugated monoclonal antibodies, such as CD34, CD11c, CD1b, to define this uncharacterized cluster. Peripheral whole blood samples were maintained at ambient temperature as well as refrigerated at 4°C. The monocyte subpopulation was most evident in samples maintained at refrigerated temperature. We used a gating strategy to separate the two clusters, normal monocytes and the additional high forward scatter (FSC) monocyte subpopulation in FCS Express analysis software. Using custom designed data analysis templates, we performed side-by-side comparison of these two populations. The results indicated that the two clusters demonstrated a similar profile in this panel. In conclusion, we found that this extra cluster phenotyped as an altered subpopulation of monocytes, and was most evident following cold stimulation. The significance of this monocyte subpopulation remains unknown at the current time. The importance of panel design in flow cytometry was highlighted in this work. The results and subsequent bioinformatics proved informative in characterizing this unusual subpopulation by profiling an array of cellular biomarkers. Citation Format: Chengsen Xue, Joanne Cuomo, Lucie-Emily Rows, Kai Chen, Chrinstina D. Swenson, Thomas W. Mc Closkey. Identification and characterization of an unusual monocyte subpopulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5593.